Aptamers Engineered Nanocarriers for Cancer Therapy 0323858813, 9780323858816

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Aptamers Engineered Nanocarriers for Cancer Therapy Edited by

Prashant Kesharwani Assistant Professor, Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

Woodhead Publishing is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2023 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-85881-6 For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Matthew Deans Acquisitions Editor: Sabrina Webber Editorial Project Manager: Joshua Mearns Production Project Manager: Prasanna Kalyanaraman Cover Designer: Miles Hitchen Typeset by TNQ Technologies

Contributors

Khalil Abnous Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Mohammed A.S. Abourehab Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt Barbara Adinolfi Istituto di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Italy Sara Davari Ahranjani Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran Abdulfattah Al-Kadash Jordan

Cell Therapy Center, The University of Jordan, Amman,

Bayan Abu Al-Ragheb Jordan

Cell Therapy Center, The University of Jordan, Amman,

Mohammad Sarwar Alam Department of Chemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, Delhi, India Walhan Alhaer Cell Therapy Center, The University of Jordan, Amman, Jordan Mahnaz Alipour Polymer Composite Research Laboratory, Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran Atul Anand Animal Disease Control Program, Department of Animal Husbandry and Dairying, Ministry of Fisheries, Animal Husbandry & Dairying, Governemnt of India, New Delhi, India Peyman Asadi Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran Mohammad Banazadeh Pharmaceutical Sciences and Cosmetic Products Research Center, Kerman University of Medical Sciences, Kerman, Iran; Students Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran

xii

Contributors

Mahmood Barani Medical Mycology and Bacteriology Research Center, Kerman University of Medical Sciences, Kerman, Iran Jaleh Barar Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran; Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Payam Bayat Faculty of Medicine, Department of Immunology and Allergy, Immunology Research Center, BuAli Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran Behzad Behnam Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; Faculty of Pharmacy, Students Research Committee, Kerman University of Medical Sciences, Kerman, Iran; Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran; Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran Magdolna Casian Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania Cecilia Cristea Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania Carla Cruz CICS-UBIdCentro de Investigaç~ao em Ciências da Saude, Universidade da Beira Interior, Covilh~a, Portugal Rambabu Dandela Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India Sapna Devi Division of Veterinary Medicine, Faculty of Veterinary Science & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu (SKUAST-J), R.S. Pura, Jammu, India Ezaldeen Esawi

Cell Therapy Center, The University of Jordan, Amman, Jordan

Morteza Eskandani Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Aida Gholoobi Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Metabolic Syndrome Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Ambra Giannetti Istituto di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Italy Lopamudra Giri Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India

Contributors

xiii

Elahe Gozali Department of Health Information Technology, School of Allied Medical Sciences, Urmia University of Medical Sciences, Urmia, Iran Mukesh Kumar Gupta Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Farnaz Hosseini Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran Oana Hosu Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania Said I. Ismail Qatar Genome Project, Qatar Foundation, Doha, Qatar; Department of Biochemistry and Physiology, Medical School, The University of Jordan, Amman, Jordan Ananya Kar Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India Harsimran Kaur Department of Pharmaceutics, Delhi Pharmaceutical Sciences and Research University, New Delhi, Delhi, India Gowtham Kenguva Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India Prashant Kesharwani Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India Mehrdad Khatami Noncommunicable Diseases Research Center, Bam University of Medical Sciences, Bam, Iran; Faculty of Medical Sciences, Department of Medical Biotechnology, Tarbiat Modares University, Tehran, Iran Zainab Lafi

Faculty of Pharmacy, The Middle East University, Amman, Jordan

Ismail Sami Mahmoud Faculty of Applied Medical Sciences, Department of Medical Laboratory Sciences, The Hashemite University, Zarqa, Jordan Muhammed Majeed

Sabinsa Corporation, East Windsor, NJ, United States

Atena Mansouri Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran André Miranda CICS-UBIdCentro de Investigaç~ao em Ciências da Saude, Universidade da Beira Interior, Covilh~a, Portugal Mohammad Mohajeri Department of Pharmacology and Toxicology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran

xiv

Contributors

Seyedeh Alia Moosavian Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran Hamdi Nsairat Faculty of Pharmacy, Pharmacological and Diagnostic Research Center, Al-Ahliyya Amman University, Amman, Jordan Yadollah Omidi Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL, United States Hossein Omidian Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL, United States Kumar Pranay Department of Biochemistry, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India Alexandra Pusta Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania Rajkumar Rajendram Department of Medicine, King Abdulaziz Medical City, King Abdulaziz International Medical Research Center, Ministry of National Guard e Health Affairs, Riyadh, Saudi Arabia; College of Medicine, King Saud Bin Abdulaziz University of Health Sciences, Riyadh, Saudi Arabia Smruti Rekha Rout Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India Pratikshya Sa Laboratory of Nanomedicine, Institute of Life Sciences, Bhubaneswar, Odisha, India; Regional Centre for Biotechnology, Faridabad, Haryana, India Amirhossein Sahebkar Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; School of Medicine, The University of Western Australia, Perth, WA, Australia; Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; Faculty of Medicine, Department of Medical Biotechnology and Nanotechnology, Mashhad University of Medical Sciences, Mashhad, Iran Sanjeeb Kumar Sahoo Laboratory of Nanomedicine, Institute of Life Sciences, Bhubaneswar, Odisha, India Fatemeh Salahpour-Anarjan Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran Kiarash Saleki Student Research Committee, Babol University of Medical Sciences, Babol, Iran; USERN Office, Babol University of Medical Sciences, Babol, Iran Tiago Santos CICS-UBIdCentro de Investigaç~ao em Ciências da Saude, Universidade da Beira Interior, Covilh~a, Portugal

Contributors

xv

Thozhukat Sathyapalan Department of Academic Diabetes, Endocrinology and Metabolism, Hull York Medical School, University of Hull, Hull, United Kingdom Mahsa Shahriari Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Neelesh Sharma Division of Veterinary Medicine, Faculty of Veterinary Science & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu (SKUAST-J), R.S. Pura, Jammu, India Afsana Sheikh Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India Vanshikha Singh Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India Seyed Mohammad Taghdisi Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Yong Teng Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, United States Mihaela Tertis Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania Sara Tombelli Istituto di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Italy Somayeh Vandghanooni Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Rezvan Yazdian-Robati Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Fatemeh Yazdian Faculty of New Sciences and Technologies, Department of Life Science Engineering, University of Tehran, Tehran, Iran Fatemeh Zahedipour Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Faculty of Medicine, Department of Medical Biotechnology and Nanotechnology, Mashhad University of Medical Sciences, Mashhad, Iran Faraz Zare

Faculty of Medicine, Aalborg University, Aalborg, Denmark

Cell-SELEX technology for aptamer selection

1

Gowtham Kenguva 1 , Smruti Rekha Rout 1 , Lopamudra Giri 1 , Amirhossein Sahebkar 2, 4, 5 , Prashant Kesharwani 3,6 and Rambabu Dandela 1 1 Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India; 2Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 3Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 4Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 5Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; 6University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India

1.1

Introduction

Aptamers are oligonucleotides (short form of RNA or DNA) having multiple applications in different fields like diagnostics, forensics, food science, research, drug discovery, and gene testing and are specially used for drug delivery carrier agents due to their special property affinity and target specificity [1]. The compounds with high affinity for the target molecules, which are found in a huge combinatorial nucleic acid database, are known as Aptamers. They are very specific to the target molecules as well as they have some other qualities such as robustness which opposes the reduction conditions as well as the heat denaturation [2e6]. When compared to the antibodies, aptamers offer a different set of benefits, such as the preparation of chemicals is easy, compact size, lower molecular weight, nonimmunogenicity, and simplicity of alteration and ligation to nanoparticles for therapeutic, diagnosis, and imaging applications [7]. Developing aptamers for the biomolecular target on the cell membrane in their natural shape, like as proteins, molecular biomarkers, and receptors, has evolved as a potential helpful technique to develop effective disease-specific probes [8]. A live infectious organism, African trypanosomes also known as Trypanosoma brucei, was used in the first cell-SELEX study. The findings revealed the discovery of three classifications of high attraction RNA aptamers which are particular for the parasite’s infectious bloodstream lifespan series phase. The aptamers are chosen to target the parasite’s flagellar pocket, but none of them attach to the VSG protein, that is among the most common polypeptides on the Trypanosoma interface [9]. Aptamers have been shown in several investigations to not only attach to the cell external but also to be carried intracellularly.

Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00019-1 Copyright © 2023 Elsevier Ltd. All rights reserved.

2

Aptamers Engineered Nanocarriers for Cancer Therapy

Aptamers are discovered using a continuous selectioneamplification process known as selective generation of ligands by exponential enrich. In the process, cultivation of target molecule with a solo stranded nucleic acid pool which have 1014e1015 variations of randomized 30e100-nt sequenced takes place. After that, the variants having required binding activity are redeemed, then the enriched library is amplified by reverse transcriptionepolymerase chain reaction (PCR). The single-stranded pool is then produced by deleting the template-strand, which can be done in-vitro as well, where PCR product is generated. The whole procedure is repeated more than 20 times to identify aptamers [2]. The SELEX procedure has been established in 1930 that is selection of RNA molecules that bind to certain proteins in vitro [10]. RNA ligands to bacteriophage T4 DNA polymerase, systematic development of ligands through exponential enrichment, since then the various aptamers had been created against different targets, which includes both small chemical molecules and large molecules like proteins (multidomain). Various SELEX process has been developed by scientist which is compatible with the requirement and makes the whole selection procedure highly efficient. Capillary electrophoresis-SELEX, microfluidic SELEX, rapid-SELEX, and cell-SELEX are some of these methods. All of the previous SELEX approaches, with the exception of cell-SELEX, rely on target information for aptamer choice, and mostly aptamers found against pure protein targets fails to identify the same protein in its native form. To achieve highest aptamers, the protein must be kept in a homogeneous, naturally folded condition. SELEX has been done directly against pure plasma membranes in order to choice aptamers under more physiological settings [7]. In the year 1998, Morris and Jensen were the first to use the cellbased SELEX approach by which the membranes of human red blood cells were used as a complex mixture to identify aptamers that provides an in vitro procedure for isolating high affinitive aptamers, especially against a compound combination of potential target compounds. Contrasting to the other SELEX approaches, cell-SELEX chooses an aptamer in contradiction of the entire cell, ensuring that molecular targets at cell membrane are in their natural condition and represent its normal folding structure [8]. Incubation, partitioning, and amplification are the primary processes of cell-SELEX, which are comparable to standard SELEX (Fig. 1.1). Positive and negative collections are used in the cell-SELEX process to develop aptamers that can fight cancer selectively. -ve selection is required to eliminate motifs that attach to normal cells while also improving the selectivity of potential aptamers. To begin, with the aptamer, a single-stranded oligonucleotide collection with a wide variety of randomly sequenced is generated and cultured with goal cells. Following washing, the DNA fragments attached to the marked cell surface are extracted from the cells through heating cell-ssDNA complexes at high temperatures and centrifugation. And then the restored pool and the negative control cells are kept for the incubation and after that all the ssDNA which binds with the negative control cells are separated. Utilizing a biotinylated reversing primer, unbinded motifs are enlarged by PCR, resulting in the absorption of a particular binder to the target. For capturing the separated biotinylated antisense strand as well as unlabeled ssDNA streptavidin

Cell-SELEX technology for aptamer selection

3

Figure 1.1 The cell-SELEX systematic evolution of ligands by exponential enrichment technique is used to select aptamers [69].

magnetic beads are utilized. At the end, the enrich pool is then sequenced as well as the DNA(representative) aptamers are selected for the required characterization [11]. There are various rewards of Cell-SELEX, which are as follows: 1. Cell-SELEX technology can generate aptamers, which has the potential not just to particularly identify cells, but also distinguish among the molecular signatures of various cell kinds without foreknowledge of their molecular property. 2. Many molecular variations can be found on the surfaces of different cell types, especially membrane-assured proteins. Such particles could be goal for cell-SELEX. As a result of effective selections, numerous aptamers which opposes the various targets can be developed. 3. Aptamer probes can straightforwardly differentiate the cognate target, as aptamers binds to target molecule in their native state, forming a true molecular identity of diseased cells. 4. Aptamers aid in the discovery of new biological markers. Modification at the molecular scale in cells is associated with both advanced physiological and pathological methodologies. However, the reason of these modifications has not been determined, cell-based SELEX permits the generation of these oligonucleotides that identify unspecified biological markers.

Because of this precedence, the cell-SELEX technique is nowadays has been utilized all over the world and novel cell-selective aptamers. The utilization of the aptamers as aiming moieties has resulted in the growth in a plethora of nanotools for effective diagnostics and therapy of cancerous cells [12].

1.2

Cell-SELEX technology

Cell-SELEX technique differs from traditional SELEX procedures. Beneficial and harmful selection are required by Cell-SELEX in order to select an aptamer with

4

Aptamers Engineered Nanocarriers for Cancer Therapy

higher effect and sensitivity. A variety of living cells has been investigated as targets for SELEX using live cells, or Cell-SELEX, due to various appealing aspects of the process (Table 1.1). Many aptamers have been created against a wide range of targets since the SELEX technique was established, from tiny chemical compounds to huge multidomain proteins. Although SELEX is usually done with a highly pure target molecule, a theoretical work [19] suggests that complicated heterogeneous targets could potentially be used to generate particular aptamers. The discovery of aptamers against red blood cell ghosts, followed by the discovery of aptamers against live African trypanosomes, has experimentally verified this hypothesis [9]. Target cell-specific aptamers can be created using a combination of a negative selection step that eliminates variations that attach to nontarget cells.

Table 1.1 Various living cells investigated as targets for SELEX using live cells or cell-SELEX. S. No.

Cells that are targeted

1.

Vital Burkitt lymphoma cells

DNA

2.

Human leukemia B cells

DNA

3.

PC12 cells

20 -fluoroRNA

Construction

Approach of selection FACS-SELEX methods were used 10 times to find the required aptamers. Instead of binding, selection is based on intracellular absorption.

Uses parental PC12 cells for selection.

Results

References

Fluorescenceactivated cell sorting is a reliable, efficient, and fast method. DNA motifs that have been discovered are readily transferred into cells and improve cell activation An antagonistic aptamer that binds to RET (receptor tyrosine kinase) on the cell surface has been discovered.

[13]

[14]

[15]

Cell-SELEX technology for aptamer selection

5

Table 1.1 Various living cells investigated as targets for SELEX using live cells or cellSELEX.dcont’d S. No.

Cells that are targeted

4.

Trypanosoma cruzi

20 -fPy-RNA

5.

Trypanosoma brucei

20 -fPy-RNA

6.

Trypanosoma brucei

RNA

1.2.1

Construction

Approach of selection The addition of an excess amount of the matrix molecule elutes variations attached to the cells. Selection targets included purified proteins from two VSG variants or cells producing them. Effective selection for trypanosomes in the live, bloodstream stage

Results

References

Aptamers that prevent parasite entrance have been discovered.

[16]

An aptamer that binds to a wide range of variant surface glycoprotein variations has been discovered. One aptamer identified a transferrin receptor subunit and the attached aptamer became internalized quickly.

[17]

[18]

Challenges associated with the technique

Numerous novel SELEX approaches show that the ability aptamers under various binding circumstances have been presented in various studies that have its own set of advantages and limitations. As a result, selecting an appropriate technique for clinical applications is not simple [20]. The therapeutic utility of aptamers is hampered by the fact that many sequences acquired using traditional aptamer methods are incorrect. In other cases, the detection performance is nonspecific or poor. Clinicopathological research is a term that refers to research that focuses on the Cell-SELEX is one of the methods that can be used. The most effective is the innate

6

Aptamers Engineered Nanocarriers for Cancer Therapy

system of the protein aims a method for developing proper probes able of detecting in both in vivo and in vitro settings [21]. The inadequacy of the long selection method is the two key drawbacks of Cell-SELEX. To circumvent these difficulties in a study, aptamers against aspartate-hydroxylase (ASPH), an identified tumor marker, were created as part of altered Cell-SELEX was used (Fig. 1.2). To detect oligos that are not specific for ASPH, five successive cycles of counter choice using sequences assured to negative cells were run in conjunction with the usual Cell-SELEX. Each successful attained family’s representative was submitted after high-throughput sequencing to flow cytometry confirmation, accompanied by fluorescent immunostaining of histopathological sections [22]. It is important to mention that just a few technologies based on aptamers are in use. Rather than putting effort and energy into isolating new aptamers for practical application, the mainly the importance is based on existing aptamers, for designing new tactics. According to Geoffrey Baird, the major challenges in the area of aptamer are referred to problem of “thrombin.” Other factors contributing to aptamer problems include (i) in vivo, nuclease breakdown and renal clearance resulting in a considerable short half-life (ii) due to inadequate SELEX procedures, the current process of isolating novel aptamers is time-consuming and monotonous (iii) SELEX comes with a hefty price tag (iv) limited ability of polymerases to tolerate modified and artificial nucleic acids [23e25]. Limitations of selection of Aptamer by SELEX method: a. Selectivity: Multianalyte identification aptasensors for proteins as well as other, small molecules have also been developed due to selectivity difficulties. A sensitive biosensor for adenosine and Lysine was recently demonstrated employing [Ru (NH3)6]3þ as AuNP amplification and signal transducer, proving that a bifunctional aptamer can attach to two different goals [26].

Figure 1.2 Schematic representation of counter-SELEX can be used in conjunction with cellSELEX to address the shortcomings of the negative selection step [22].

Cell-SELEX technology for aptamer selection

7

b. Sensitivity: The concentration of a target analyte in the medium of detection determines sensitivity required for analyte. Electrochemical approaches are having a lot of success, especially with aptamers, which have demonstrated rising potential and advantages in terms of stability and affinity. However, these tactics have a sensitivity which is lower. The use of homogeneous solutions in an electrochemical aptamer-based adenosine triphosphate assay was published in research work to boost sensitivity [27]. c. Degradation: Nucleases rapidly degrade aptamers (particularly RNA aptamers), which is a severe problem. Based on the oligonucleotide content and conformational structure, the typical duration for the degradation of oligonucleotides in the cells blood can take some minutes [28].

1.2.2

Advantages and limitation associated with technique

The introduction of cell-based screening technologies has substantially increased screening targets and broadened the scope of aptamer usage. Cell-SELEX has been the go-to technology for designing aptamers to detect specific biomarkers on cancer cell surfaces for diagnostic and therapeutic purposes. To create membrane protein, aptamers against specific illnesses using typical protein aptamers, prior knowledge of protein targets is required. Moreover, a sufficient lot of high recombinant membrane proteins would be required. Hence, Cell-SELEX addresses the challenges of purifying recombinant membrane proteins [29]. Membrane proteins generated in vitro expression systems have low solubility and yield, which limits their applicability. Many new types of aptamers have been discovered using variations of the SELEX technique, but the aptamer field still faces many challenges before its full potential is realized in the scientific community. As previously indicated, many researchers attempted to tweak the selection strategy in order to optimize the pace at which aptamers can be produced. (a) Nucleases rapidly degrade aptamers (particularly RNA aptamers), which is a severe problem. Depending on the oligonucleotide content and conformational structure, in the blood and cells, oligonucleotide degradation takes anything from a few minutes to several minutes. A number of strategies for shielding aptamers from nuclease degradation have been explored. SELEX using modified oligonucleotides is one of the traditional ways for generating nuclease-resistant aptamers [28]. Most aptamers in analytical studies are chemically altered by replacing the position of 20 with an amino, O-methyl group or fluoro, locking nucleic acids, and capping the end of 30 with overturned thymidine to increase nuclease resistance while simultaneously enhancing binding affinity [30e32]. (b) The Cell-SELEX method also allows for aptamer selection without knowing the target. However, cell-SELEX, like all other approaches, has drawbacks. For example, the inclusion of dead cells in an aptamer production suspension resulted in nonspecific absorption or binding of oligonucleotides by these cells, which harmed the entire selection process [33]. In most circumstances, aptamer production necessitates the presence of purified target molecules. A target’s type and purity are also important factors in aptamer selection. SELEX targets are usually obtained by expressing them in prokaryotic or eukaryotic cells and then purifying them using chromatography. Aptamers developed contrary to targets produced

8

Aptamers Engineered Nanocarriers for Cancer Therapy

in prokaryotic systems don’t always bond with the similar receptor in case of cells of eukaryotic [34]. (c) Separating target-bound sequences from sequences that have no affinity for the target is an important step in the SELEX technique. By selectively adsorbing the target and any associated aptamer sequences on a matrix, protein targets can be partitioned. Nitrocellulose filters, for example, are a cost-effective and convenient matrix for this purpose due to their nucleic acid penetrability and capacity to retain proteins through adsorption by hydrophobic. Cell partitioning can be accomplished using centrifugation, fluorescence-activated cell sorting, or a gentle wash of adhering cells [35]. (d) Aptamer cross-reactivity can be a hurdle to practical deployment because to the potentially harmful effects of aptamer contact with other proteins. Aptamers with particular target recognition can also attach to entities with comparable structures. Four of the aptamers designed to suppress DNA polymerase can also bind to and inhibit DNA polymerase from another family. In a SELEX-negative selection stage, this challenge can be overcome by choosing structurally similar molecules [36]. (e) In order to gain a better understanding of the relationship between pool enrichment and aptamer affinities, novel bioinformatics device that can process huge-scale data as well as secondary assembly predictions are urgently needed. Despite the fact that aptamers have a long way to go from the lab to the market, this proves to be a fascinating and interesting topic that is well value the time and effort [37].

1.3

Various cell-SELEX methods

To increase the efficiency and selectivity of aptamer screening, many cell-SELEX approaches have been developed in recent years. SELEX techniques to aptamer selection employ different procedures and instruments, but they all work on the same basic premise of amplification iterative selection to generate oligonucleotides with high target specificity. FACS is a sophisticated flow cytometry apparatus that uses fluorescence and scattering characteristics to distinguish cells with attached aptamer in the solution from cells with loose aptamer in the solution. In a study, Mayer et al. used a FACS system to develop a way that simultaneously identifies, isolates, and eliminates the unbound aptamer species from the cell population. A thorough approach for selecting aptamers that target the cell subpopulations of interest is broadly classified in appropriate scheme. To distinguish and discrete those subpopulations of cells that have both unbound and bound aptamers at the same time, a fluorescence-activated cell sorting device is used. Because undefined binding of nucleotides to cells with reduced membrane integrity or their unselective uptake by dead cells is more common than with other cell-selection approaches, this method provides fewer false-positives [35]. DNA aptamers particular to epithelial cell adhesion molecule (EpCAM) overexpressed on the surface of Hep G2 cells and mouse embryonic stem cells were successfully isolated using this approach. EpCAM is overexpressed in the majority of malignancies and in the clinic, and the product is fully utilized for diagnosis, therapy, prognosis, and imaging. As a result, the EpCAM in contrast to aptamer could be employed in a variety of clinical settings in addition to its use as a stem cell marker [38].

Cell-SELEX technology for aptamer selection

9

This study used a novel selection technique to find “cell-internalizing RNA” and “cell-type specific,” ligands like aptamers that can deliver therapeutic short involving RNAs to HER2-positive breast cancer cells. SiRNAs targeting the antiapoptotic gene Bcl-2 were covalently attached to the most selective and internalizable RNA aptamers. When given to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(þ)-cells and silence Bcl-2 gene expression. Importantly, decreasing Bcl-2 makes these cells more susceptible to chemotherapy (cisplatin), providing a novel treatment option for breast tumors with HER2(þ) status. As a result, a novel cell-based selection mechanism for identifying RNA aptamers that internalize the cell the therapeutic siRNAs to HER2-expressing breast cancer cells has been discovered [39]. A combination aptamer is incubated with live targeted cells for a period of time at 37 C in the SELEX cell-internalization process. After incubation, the unbound and bound aptamers are washed using stringent conditions of buffer cells. For following round of selection, the internalized sequences are retrieved and amplified using RTPCR. The final aptamer sequences can be determined using both upcoming sequencing and bioinformatics [40]. Aptamers with this internalization property can be utilized to deliver siRNA and intracellular medicines via aptamer-mediated siRNA delivery, which could be useful for disease prognosis [41]. Another variation of cell SELEX involves developing aptamers that bind to the cell’s target using a combination of cellSELEX and 3-D cell culture approach. The advantage of 3D cell cultures over 2D culture models is that they imitate the native biological microenvironment in which cells grow and evolve, potentially boosting basic research and drug development. Three-dimensional cell cultures are created using magnetic levitation skill with twodimensional cell culture [42]. Magnetic levitation of cells in the form of a gold hydrogel, filamentous bacteriophage, and magnetic iron oxide nanoparticles results in a three-dimensional tissue culture was reported in a study. By spatially changing the magnetic field, the shape of the cell mass can be altered, permitting multicellular cluster of many cell kinds in coculture. Protein expression profiles in magnetically levitated human glioblastoma cells matched those in xenografts of human tumors. These findings show that lifted up three-dimensional growth with magnetized phage-based hydrogels prove to better replicates in vivo protein expression and may be used in long-term studies and is practicable [43]. In research, it was suggested that a microfluidic system that uses to quickly screen aptamers, researchers used the systematic evolution of ligands by enhancement of SELEX approach that are unique to cancer stem-like cells. The technology uses magnetic bead-based techniques to identify DNA aptamers and has a number of benefits, including a rapid, automatic screening procedure, and reduced cell and reagent usage. By combining a microfluidic control module, a temperature control module and a magnetic bead-based aptamer extraction module, the entire Cell-SELEX procedure can be completed in less time. Hence, this microfluidic device is effective and uses less trial quantities than the standard Cell-SELEX procedure [44]. The complete aptamer selection process may be over in a less amount of time on a single chip, and it has been shown to be effective in discovering aptamers that are unique for tumor stem-like cells. In a study, a group of scientist developed an microfluidic scheme for the long-term choosing of aptamers with high affinity and precision for cancerous cells [45]. A microfluidic device was

10

Aptamers Engineered Nanocarriers for Cancer Therapy

designed that can automatically identify cholangiocarcinoma cells-specific aptamers. On-chip, the created arrangement could undertake based on cell systematic development of ligands using a Cell-SELEX technique. This automated technique might be tweaked to find aptamer to act against various cancer cells, allowing for earlier detection and, potentially, a better prediction [46]. Ligand-guided selection (LIGS) is a variation of the cell-SELEX technique that selects aptamers that bind to specific cell surface according to interest. LIGS is a new approach for picking aptamers targeting epitopes on extracellular receptors that leverages Ab binding as a ligand. Using preexisting molecular and cellular interactions, this basic method might be enhanced and employed as a specific screening platform in phage displays, small molecular libraries, and peptide libraries to locate artificial molecules toward active sections of macromolecules as a model [47]. Using a huge multimodal single-stranded oligonucleotide pool and numerous rounds of division and replication, Cell-SELEX selects DNA ligands against whole cells with varying DNA-binding affinities and specificities. LIGS exploits the division stage by introducing a secondary, before high-affinity monoclonal antibody ligand, which outcompetes and elutes particular aptamers near to the antibody’s attaching target, rather than the cell. In conclusion, it can describe that specific aptamers may be detected in their endogenous state using an antibody against a single domain of a multidomain protein complex with no post- or pre-SELEX protein [48]. In a study, there is development of a novel form of the SELEX technology called cross-over SELEX to improve selection efficiency and prevent creating aptamers against biomarkers or chemically stated on target cells. Here in this investigation, they have changed the classic selection of aptamer by introducing a choice of functional step that selects for RNA molecules that bind the target receptor and are absorbed by cells. A number of aptamers were shown to be selective for the animal receptor, which was immediately endocytosed by cells and shared a same core structure [49]. Hence, Cell-SELEX technology is an appealing option for isolating aptamers against cell surface targets in their instinctive configuration without any prior knowledge of the targets. This technology encourages the ability to recognize the aptamer in its conformation or ordinary state, that aids in bringing the aptamer to life which further, brings the aptamer nearer to achieve its clinical potential. Cell-SELEX is a promising technology for creating novel pharmaceuticals, in addition to aptamer selection [7].

1.4

Aptamers generated by cell-SELEX technology

To identify aptamers in vitro, SELEX against a pure protein is typically utilized. If the protein that is aimed at undertakes a stable shape, using pure proteins as targets has the benefit of allowing for easy selective enrichment throughout the selection procedure. To overcome selective aptamers, demerits come to nonnative protein conformations, an approach known as cell-SELEX has been devised that utilizes living cells as aptamer selection targets. This technique does not require knowledge of protein complexes, unlike protein-based SELEX. Purifying target proteins via techniques that disturb their native structure is also unneeded. Throughout the selection process, all surfaces of cell stay in their natural context, preserve their innate foldaway structure,

Cell-SELEX technology for aptamer selection

11

and may have posttranslational changes. As a result, aptamers chosen from complete living entities can easily target’s inherent folded surface. This has a lot of scientific research opportunity as well as the creation of specific to living cell diagnostics in addition to therapies. It’s important to think about both target and nontarget cells before creating aptamers against complete live cells. Genetic differences between these two nearly linked cellular components, such as tumor and normal cells, determine the amount of SELEX cycles needed and the overall success. Negative cells are used in a counterselection step to avoid aptamer enrichment for numerous nonspecific proteins, hence increasing the specificity of the aptamers. To evaluate enrichment by flow cytometry, the sense strand of the primer is tagged with a fluorophore, and sensible strands are differentiated from antisense by combining biotin to the reversible primer for streptavidin-biotin contact to distinguish sense strands from antisense [8]. Cell-SELEX technology has evolved over time as aptamer technology has been used to tumor cell identification and treatment, and the variety of targets has grown. To increase the achievement rate of aptamer transmission, a range of innovative screening approaches based on cell-SELEX have appeared [2].

1.4.1

FACS-SELEX

Mayer et al. developed a thorough procedure for selecting aptamers that target specific cell subpopulations using fluorescence-activated cell sorting (FACS). The mark cells are treated with a fluorescently tagged aptamer library in this procedure. A sensitive, high-throughput and efficient, approach is employed to differentiate and separate cells that have stuck to aptamers at the same time using a FACS equipment. The aptamers are purified, eluted, and replicated after that. The SELEX method relies on the integrity of the cell membrane. As a result, those cells that are undergoing necrosis or apoptosis which may take up nonspecifically bound nucleic acids during the SELEX procedure must be removed.

1.4.2

TECS-SELEX

Ohuchi et al. created TECS-SELEX; an innovative SELEX approach that uses a cell-external to select the target that has shown to be recombinant protein. On Chinese hamster ovary cells, RNA aptamers against the transforming growth factor (TGF-) type III receptor were expressed and were isolated using this approach. In vitro, one of the RNA aptamers competed with TGF- for binding to the cell surface receptor and had a separation constant in the 1 nm array. The development of TECS-SELEX has provided a valuable, unique method for isolating aptamers against any cell external protein of interest, which is particularly helpful after the pure protein aim is difficult to obtain. Aptamer selection by SELEX approach bought a revolution in treating cancer. Cell-based SELEX’s able to monitor tiny molecular gap between different phenotypic conditions of or else identical cells. These oligonucleotides have been discovered that distinguish tumor vessels from normal brain vessels, cancer cells from normal cells of the same origin, multiple hematological cancer lineages, and cells expressing a human oncogene, and the field is rapidly increasing. In a study, researchers developed RNA

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Aptamers Engineered Nanocarriers for Cancer Therapy

ligands that are resistant to nucleases that recognized cells that are highly metastatic and have a high affinity and selectivity, limiting their migratory and invasive abilities. Aptamers were created using isogenic cell lines with identical tumorigenic potential but opposite metastatic aggressiveness, as well as a cell-based subtractive SELEX method. E37 and E10, two aptamers that bind selectively to the metastatically aggressive cell line and changed the phosphorylation of multiple tyrosine kinases, were discovered [50]. A SELEX approach was created for discovering aptamers that differentiate between molecular marks produced by two rigorously isogenic cancer cell lines that differ solely in their in vivo metastatic potential, according to a study. Most intriguingly, some of these aptamers stop cells from invading or migrating in vitro. Many domains of molecular medicine can benefit from these aptamers. Various cell types can be differentiated using cell-based aptamers, even if the target molecules are unknown beforehand. Aptamers can be used to select and identify sick cells in the laboratory for clinical use diagnostics by combining with various other ways such as nanotechnology and microfluidic technology. Additionally, aptamer molecular target identification is a unique biomarker-finding tool. Due to their great specificity and simplicity of chemical modification, cell-specific aptamers can also be easily modified for medication administration and targeted therapy. With the rapid progress of microchips, microfluidic devices, nanotechnology, RNAi, and other modern ways, cell-SELEX will surely change how we evaluate, heal, and prevent illness [51].

1.4.3

3D cell-SELEX

In order to simulate the tissue milieu in vitro, 3D cell culture, it is considered as the novel technique for selecting specific nucleic acid ligands across spheroid cells. Magnetic levitation is used to conduct 3D cell culture (MLM). During the incubation time, this magnetism is attached to the surface of the culture plate to promote magnetic levitation and the formation of suspended in the air spheroid cells. Compared to 2Dcell culture, the approach allows for more homogenous exposure of extracellular regions of membrane proteins to aptamers and improved cell viability interaction between aptamers. It was coupled with a negative selection against a nontumor cell line during the first round of 3D cell-SELEX [52].

1.4.4

Cell-internalization SELEX

Cell-internalization SELEX is a screening system that relies on cells to approach for identifying and characterizing RNA aptamers for siRNA that are cell-internalizing medication delivery into target cells’ cytoplasm. The internalized RNA sequences are recovered for amplification, while surface-bound RNAs are discarded in the continuous selection in the innovative selection technique. As a result, it enhances the RNAs that the target cell internalizes. Cellular uptake is the most significant barrier to RNAibased therapies. This challenge is being addressed by using a delivery strategy, cell-internalizing RNA aptamers in combination with siRNAs [40]. Aptamers that internalize cells with the ability to express certain cell surfaces were studied widely. Aptamers were coupled covalently to siRNAs, which further targets

Cell-SELEX technology for aptamer selection

13

the antiapoptotic gene Bcl-2 to precisely identify and internalize HER2-expressing cells. The HER2 aptamer-Bcl-2 siRNA conjugates silence Bcl-2 gene expression and target HER2(þ) cells, when administered to HER2-expressing breast cancer cells. Furthermore, suppressing Bcl-2 makes these cells more susceptible to chemotherapy, indicating a new treatment option for HER2-positive breast cancer [39,53].

1.4.5

Hybrid-SELEX

Due to on the cells of interest, off-target component is coexpressed. In traditional cellSELEX, aptamer enrichment efficiency is low. Hicke et al. used a hybrid-SELEX technique to develop Tenascin-C-specific RNA aptamers in 2001, combining cell-based SELEX with isolated protein-based SELEX [54]. Purified proteins as well as cells with the surfaces possessing same protein served as goals in hybrid-SELEX. A crossover SELEX experiment was utilized to enrich TN-C aptamer representation in the cell aptamer pools after a predetermined number of rounds of cell-SELEX by performing two more rounds of assortment with purified mark protein. When comparing two versions of crossover choice on isolated target protein enhanced the affinity of the cell-SELEX result pool, according to the screening data by 50-fold. In hybrid-SELEX, the first cell-SELEX technique intended to choose aptamers against a target on the cell external in its native state. The cell aptamer pool lacked high-affinity aptamers, thus extra stages of selection with purified destination were used to improve them [55].

1.5

Cell-SELEX technique for aptamer selection development and applications

Despite of knowing the measurements of cell-surface biomarkers, aptamers created using cell-SELEX can distinguish them uniquely. As a result, aptamers may be used as a medication carrier or as aptamer-functionalized nanomaterials that aid in the discharge of pharmaceuticals in particular target locations, allowing for medication targeted delivery. More significantly, aptamers have fewer off-target toxic impacts due to greater goal of physical interaction capabilities. Membrane antigen specific to the prostate is a good prostate tumor cell marker that is associated with prostate cancer cells. Researchers employed the pure protein as the goal in a study and discovered an aptamer named as A10 that might detect Prostrate specific membrane antigen’s (PSMA) extracellular domain [56]. For active targeting, in a particular study created a bioconjugate comprising docetaxel (DOX) enclosed nanomaterials and PLGA-bPEG coupled to aptamers A10 of RNA. The effectiveness for delivery systems of DOX to prostate cancerous cells was tested and discovered that the conjugation is proficient of attaching to tumor cells with overexpression of PSMA and then being immersed by the tumor cells to cause damage of the cell and attain treatment strategies. In the nude mouse model, the targeted medication also showed strong antitumor activity and lower toxicity in vivo than DOX alone [57,58].

14

Aptamers Engineered Nanocarriers for Cancer Therapy

Furthermore, aptamer-based delivery of medications such as DOX, daunorubicin, docetaxel, and toxins such as gelonin are reported that various photosensitizer treatment drugs, as well as a variety of short interfering RNAs, have been used. There are a number of studies highlighted the potential for nanoparticle-aptamer bioconjugates to be used as cancer therapeutics. Aptamer-based delivery may allow medications to overcome a variety of biological barriers, including endothelial and epithelial barriers, and enhance drug transfer to intracellular areas of action, improving therapeutic safety and efficacy [59]. However, aptamers with effective focusing capability to cancer tissues and cells not only represents a potential means to transport drugs, but also have the probable to be employed as an anticancer agent in tumor therapy. Several aptamers are studied in number of laboratories to conduct various experiments related to malignancies. AS1411 is the most commonly studied aptamer for cancer treatment. AS1411 is a DNA strand which attaches to the nucleolin external domain, and is involved in cell survival, growth, and proliferation. AS1411 has recently been evaluated in a number of cancer cell types, including renal cell carcinoma, ovarian, breast, prostate, lung, pancreatic, colon, and cervical cancers [60]. In the preclinical stage, further anticancer aptamers are being investigated. These aptamers share several anticancer mechanisms in common. Blocking signaling pathways by blocking phosphatases, kinases, carboxypeptidases, and other enzymes, for instance, RET aptamer D4, can impede downstream activation and signaling for tumor growth. Not only did Aptamer D4 recognize the RET’s extracellular domain, also supress downstream signaling and hindered RET phosphorylation, as well as following cellular and molecular activities [61]. Antitumor aptamers might be useful diagnostic tools in treatment for cancer. Another option is to bind to proteins linked to tumor progression. Many cancer treatment aptamers under development today work by blocking target functional protein molecules. Cells’ migratory capacity, for example, is controlled by the CD44 protein. Anti-CD44 DNA aptamers can bind to CD44 protein, preventing breast cancer cells from migrating. In further research, the aptamers’ potential therapeutic efficacy will be assessed in vivo [62]. In future investigations, the aptamers’ potential therapeutic efficacy will be assessed in vivo. Meanwhile, further these nucleotides with potential tumor suppress performance in preclinical research are predictable to be tested in clinical trials in the coming years, paving the framework for the growth of aptamer-based cancer treatment. Based on cell-SELEX technology, these synthesized entities have been used in biomarker identification and detection, therapy, and a variety of other domains [63]. Aptamers can be used as diagnostic equipment because of their strong affinity for their substrates. Aspects of aptamers, such as their ease of conjugation and labeling, allow these to be easily combined with other novel technologies to improve their diagnostic capabilities, such as endogenous nucleic acid detection, microfluidic cell separation, flow cytometry, or nanomaterials sensing. Bruno et al. created the very first aptamer used as a screening tool. To identify anthrax spores, they used an aptamer that was chosen against Bacillus anthracis spores. Aptamers have been used extensively in the diagnosis of ophthalmology, cardiovascular illnesses, and cancer diseases to date. Aptamers, which are based on cell-SELEX technology, have been widely employed in the diagnosis and characterization of biomarkers, cancer imaging, cancer therapy, and other fields [64].

Cell-SELEX technology for aptamer selection

15

As we have discussed above the applications and need to develop aptamers but before using aptamers in further study, it is necessary to describe their binding properties. Understanding important aptamer binding features including as affinity, kinetics and buffer sensitivity aids in aptamer design. To assess aptamer-target binding affinity, there have been various biophysical instruments or techniques created. Isothermal Titration Calorimetry (ITC) is a technique that uses thermodynamic principles. By measuring the heat generated at time of the creation of an aptamer-target complex, ITC is mostly utilized to evaluate the stoichiometry, affinity, and thermodynamic characteristics of molecular interactions [65]. Surface plasmon resonance (SPR) technique is a label-free, elevated, real-time platform for characterizing aptamer and target affinity and kinetic properties. Usually, the aptamer is immobilized on the sensor chip’s surface, and then varied nontethered analytes levels are pumped through. It is found to bind and then described by recording the alterations in the refractive index caused by the creation of the binding compound [66]. Flow cytometry is a laser-based method for detecting the adhesive affinity of aptamers and targets. It is commonly used to detect the binding qualities of aptamers and whole cellular units [67]. Different morphological SELEX methods have been emerged as a result of the evolution of SELEX technique since the previous 2 decades. Traditional SELEX is used to select the majority of aptamers against purified proteins. However, evidence indicates that aptamers designed to recognize isolated membrane proteins do not recognize their targets in living cell lines. Cell-SELEX could create aptamers against a certain target cell line in order to distinguish it from others. As a result, cell-SELEX is utilized to identify aptamers to use in the diagnosis and treatment of a variety of illnesses, particularly cancer. As a result, in clinical practice, of the use of probes has a distinct benefit over single biomarker-based arrangements, offering significantly additional data for effective illness diagnosis and prediction [68]. There are various applications of aptamers despite of limitations associated with it (Fig. 1.3).

Figure 1.3 Schematic diagram of different applications of aptamers.

16

1.6

Aptamers Engineered Nanocarriers for Cancer Therapy

Conclusion

SELEX technique is a unique method in that it selects aptamers from entire cells deprived of knowing which proteins are present on the cell surface. The SELEX technique applied aptamer selection is a great way to bind to cells which are ill, especially cancer cells. Importantly, technology of cell-SELEX paves the way for the creation of tumor which target specific, subpopulations of cellular units in diverse composite combinations of cells. This method could be a primary step towards the utilization of aptamers for individualized diagnostic because it will be accessible to laboratories to access the precise targeting agents. Aptamers have been created for cancer detection and therapy resulting from cell-based selection is still in its early phases. Before it can be implemented, it must overcome a number of obstacles. To begin, additional aptamers must be screened in order to develop and create aptamers based on nucleic acid that can be used as chemical antibodies in a wider range of diseases. Although great effort has gone into establishing automated, innovative, or high-throughput schemes SELEX procedures, cell-based selection remains challenging to achieve. Tumor cells come in a variety of species (including subtypes) and exhibit a wide range of molecular features. SELEX technology still requires more screening research in order to truly screen the aptamer molecular impression, which could indicate different phases of the ailment and diverse subtypes of cancer.

Acknowledgment Rambabu Dandela thanks DST-SERB for Ramanujan fellowship (SB/S2/RJN-075/2016), Core research grant (CRG/2018/000782) and ICT-IOC start-up grant. The authors acknowledge ICTIOC Bhubaneswar for providing necessary support.

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[43] G.R. Souza, J.R. Molina, R.M. Raphael, M.G. Ozawa, D.J. Stark, C.S. Levin, L.F. Bronk, J.S. Ananta, J. Mandelin, M.M. Georgescu, J.A. Bankson, J.G. Gelovani, T.C. Killian, W. Arap, R. Pasqualini, Three-dimensional tissue culture based on magnetic cell levitation, Nature Nanotechnology 5 (4) (2010) 291e296. [44] C.H. Weng, I.S. Hsieh, L.Y. Hung, H.I. Lin, S.C. Shiesh, Y.L. Chen, G.B. Lee, An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers, Microfluidics and Nanofluidics 14 (3e4) (2013) 753e765. [45] L.Y. Hung, C.H. Wang, Y.J. Che, C.Y. Fu, H.Y. Chang, K. Wang, G.B. Lee, Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing on-chip cell-SELEX, Scientific Reports 5 (1) (2015) 1e12. [46] P. Gopinathan, L.Y. Hung, C.H. Wang, N.J. Chiang, Y.C. Wang, Y.S. Shan, G.B. Lee, Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform, Biomicrofluidics 11 (4) (2017). [47] H.E. Zumrut, M.N. Ara, M. Fraile, G. Maio, P. Mallikaratchy, Ligand-guided selection of target-specific aptamers: a screening technology for identifying specific aptamers against cell-surface proteins, Nucleic Acid Therapeutics 26 (3) (2016) 190e198. [48] H.E. Zumrut, M.N. Ara, G.E. Maio, N.A. Van, S. Batool, P.R. Mallikaratchy, Ligandguided selection of aptamers against T-cell receptor-cluster of differentiation 3 (TCRCD3) expressed on Jurkat. E6 cells, Analytical Biochemistry 512 (2016) 1e7. [49] S.E. Wilner, B. Wengerter, K. Maier, M.D.L. Borba Magalh~aes, D.S. Del Amo, S. Pai, F. Opazo, S.O. Rizzoli, A. Yan, M. Levy, An RNA alternative to human transferrin: a new tool for targeting human cells, Molecular TherapydNucleic Acids 1 (5) (2012) e21. [50] E. Zueva, L.I. Rubio, F. Ducongé, B. Tavitian, Metastasis-focused cell-based SELEX generates aptamers inhibiting cell migration and invasion, International Journal of Cancer 128 (4) (2011) 797e804. [51] M. Ye, J. Hu, M. Peng, J. Liu, J. Liu, H. Liu, X. Zhao, W. Tan, Generating aptamers by cell-SELEX for applications in molecular medicine, International Journal of Molecular Sciences 13 (3) (2012) 3341e3353. [52] A.G. Souza, K. Marangoni, P.T. Fujimura, P.T. Alves, M.J. Silva, V.A.F. Bastos, L.R. Goulart, V.A. Goulart, 3D cell-SELEX: development of RNA aptamers as molecular probes for PC-3 tumor cell line, Experimental Cell Research 341 (2) (2016) 147e156. [53] Y.Z. Huang, F.J. Hernandez, B. Gu, K.R. Stockdale, K. Nanapaneni, T.E. Scheetz, M.A. Behlke, A.S. Peek, T. Bair, P.H. Giangrande, J.O. McNamara, RNA aptamer-based functional ligands of the neurotrophin receptor, TrkB, Molecular Pharmacology 82 (4) (2012) 623e635. [54] B.J. Hicke, C. Marion, Y.F. Chang, T. Gould, C.K. Lynott, D. Parma, P.G. Schmidt, S. Warren, Tenascin-C aptamers are generated using tumor cells and purified protein, Journal of Biological Chemistry 276 (52) (2001) 48644e48654. [55] A. Boltz, B. Piater, L. Toleikis, R. Guenther, H. Kolmar, B. Hock, Bi-specific aptamers mediating tumor cell lysis, Journal of Biological Chemistry 286 (24) (2011) 21896e21905. [56] S. Lupold, B. Hicke, Y. Lin, D.C.-C. Research, Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostatespecific membrane antigen, AACR 62 (2002) 4029e4033. [57] O.C. Farokhzad, S. Jon, A. Khademhosseini, T.N.T. Tran, D.A. LaVan, R. Langer, Nanoparticle-aptamer bioconjugates A new approach for targeting prostate cancer cells, Cancer Research 64 (21) (2004) 7668e7672. [58] O.C. Farokhzad, J. Cheng, B.A. Teply, I. Sherifi, S. Jon, P.W. Kantoff, J.P. Richie, R. Langer, Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo,

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Aptamers in biosensing: biological characteristics and applications

2

Ambra Giannetti * , Barbara Adinolfi * and Sara Tombelli Istituto di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Italy

2.1

Introduction

In recent years, the use of biosensors is enormously increased and their application is extended in many different fields, such as environmental monitoring, food safety and biomedical application. Therefore, the global biosensors market size is also rapidly increasing as reported by H. Yoo et al. [1], who estimated a growth starting from US$ 21.2 billion in 2019 going to US$ 31.5 billion by 2024. Nevertheless, even the most sophisticated biosensors still lack sensitivity and selectivity for some target detection. For example, some cases, such as early diagnosis of diseases, require a very low limit of detection capability since the biomarkers are present in blood just in traces (pg - ng mL
1 range) [2]. In some other cases, the available bioreceptors (usually antibodies, enzymes, nucleic acids, and even whole cells) could need an enhancement of their binding affinity to target molecules, but those are difficult to be physically or chemically modified, enormously limiting the improvement of biosensors development [3e5]. Beyond the just mentioned traditional requirements of high sensitivity and selectivity for biosensors, other interesting aspects could be included, such as a real-time detection ability. Aptamers can fulfill all those requirements with their high programmability given by the chemical synthesis, which allows the tailoring to a wide range of targets, and their structure-switching as a consequence of target binding enables the analyte detection even in complex mixtures and possibly in real-time format; not to overlook the fact that the cost for aptamer production is competitive compared to the other bioreceptors. Among all the other bioreceptors, antibodies have had a profound impact on biology and medicine, and they are still an important component of the biotechnology industry. Nevertheless, they are included in the list of the bioreceptors, which have significant limitations. The main difficulty is related to the recognition of small molecules, which have limited surface area to bind, then the binding affinity is not capable to reach nanomolar (nM) values. A huge step forward has been made by the monoclonal

*

These authors equally contributed to the work.

Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00008-7 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Aptamers Engineered Nanocarriers for Cancer Therapy

antibodies (mAbs), but they still present issues in the selection, selectivity, preparation, stability, cross-reactivity, high costs of production, etc. [6]. Excellent substitutes for those bioreceptors are the aptamers, which are small oligonucleotide molecules with the capability to mimic antibodies and exhibit high binding affinity, with dissociation constants typically in the nM and even picomolar (pM) range, and high selectivity toward their targets. In fact, even if aptamers have a molecular weight much lower than antibodies, they are capable to fold in a complex tertiary structure with sufficient recognition surface areas to compete with the binding affinities of antibodies, with the ability to differentiate between isoforms [7]. Due to their important characteristics, aptamers are currently proposed for applications in diagnostics [8], therapeutic field [9], cell imaging [10], drug delivery [11] and biomarker discovery fields [12]. In this chapter, starting from their biochemical characteristics, we will give an overview of some of the most challenging and modern applications of aptamers in biosensing, focusing, in particular, on aptamer-based exosome isolation and detection and intracellular sensing.

2.2

Biochemical characteristics of aptamers exploited for biosensing

A strong point of aptamers is that their performances, both in terms of affinity and stability, can be modulated synthetically by chemical modification of the nucleotides, optimizing their recognition sequence as well as by altering their secondary structures. The most common applied modifications are, for example, the 20 position of the ribose sugar (for RNA), modification of the phosphate backbone [13], sugars and/or the bases [14,15], end-capping at the 30 or 50 termini [16] and locked nucleic acids (LNA), containing an intramolecular 20 -O to 40 -C methylene bridge [17]. Herman T. et al. [7] reported the fact that just the selection process with which aptamers are synthesized is based on the ability to bind ligand molecules with high affinity and specificity. In fact, there are studies of high-resolution threedimensional structures of complexes formed by aptamers with their specific ligands, in which it is evident that the conformation assumed within the complex follows precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. The above-mentioned characteristics of aptamers, make them suitable for the development of biosensors, which are named aptasensors if the biorecognition element is indeed the aptamer. Until now, many aptasensors have been developed using different signal transducers (fluorescent, electrochemical, colorimetric, chemiluminescent, surface-enhanced Raman scattering (SERS), etc.), anyhow, some working principles have analogous elements and three of them have emerged more frequently in the literature: structure-switching, enzyme-assisted recycling, and split aptamer-based modes [18e20].

Aptamers in biosensing: biological characteristics and applications

2.2.1

23

Structure-switching method

In this method, the aptamer is labeled with a fluorophore and a complementary sequence is labeled with a quencher. Before the interaction with the target molecule, the fluorescence is quenched due to the proximity of the quencher, but, as a consequence of the interaction between the aptamer and the target, there is a fluorescence enhancement. This example is schematized in Fig. 2.1 where the aptamer for thrombin is in a quenched state and only after the interaction with thrombin does the structure change from the quenching state into a G-quartet structure, which leads to fluorescence enhancement [21]. The fluorophore/quencher pair can also be replaced by other types of signal transduction, such as gold nanoparticles [22], carbon dots [23], quantum dots [24], etc.

2.2.2

Enzyme-assisted recycling method

This method, known also as signal amplification design, is very attractive due to its high sensitivity [25]. In fact, different types of nucleases (polymerases, exonucleases, deoxyribonucleases, and endonucleases) can be used to construct aptasensors and their property is to trigger several rounds of target recycling, giving the possibility to get a signal from the biosensor even starting from a small number of target molecules. For example, Ning et al. [26] developed an amplified aptamer-based sensor for adenosine triphosphate (ATP) detection using deoxyribonuclease I (DNase I) and graphene oxide (GO). The scheme of this aptasensor is reported in Fig. 2.2. In particular, they designed Figure 2.1 Schematic illustration showing thrombininduced structure change of the aptamer from quenching-state into G-quartet structure, which leads to fluorescence enhancement: (a) hairpin design and (b) duplex design. Reprinted with permission from Ref. [21].

24

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.2 Schematic illustration of the GO-based fluorescent aptasensor for ATP detection by using DNase I-mediated target cyclic amplification. Reprinted with permission from Ref. [26].

a DNA probe formed by two partially complementary sequences, the capture probe and the signal probe (fluorescently labeled with carboxyfluorescein). In the absence of ATP, the DNA probe is adsorbed onto the GO surface via p-stacking interactions, provoking the quenching of the fluorophore and the protection of the probe from nuclease cleavage. In presence of ATP, it binds to the aptamer resulting in the dissociation from the GO. In these conditions, DNase I can degrade the aptamer, releasing the ATP and the fluorophore, inducing an increase in the fluorescence signal.

2.2.3

Split aptamer-based method

The split aptamer design is based on the fact that the target can bind two oligonucleotidic fragments with high affinity, inducing the formation of a sandwich structure (fragment1-target- fragment2). Numerous aptasensors based on sandwich assays have been then designed by splitting the aptamer into two fragments. Bai et al. [27] developed an aptasensor based on fluorescence resonance energy transfer (FRET) for the determination of 19-nortestosterone. The 17b-estradiol aptamer was split into two pieces (P1 and P2, respectively). P1 was labeled with a quencher (Black Hole Quencher, BHQ), and P2 with a fluorophore (6-Carboxyfluorescein, 6FAM). In the absence of the target, P1 and P2 were far away from each other showing a strong fluorescence. In presence of the target, P1 and P2 were going to form the sandwich format bringing in close proximity the fluorophore and the quencher inducing a fluorescence decrease. Fig. 2.3 shows a schematic representation of the split aptamer-based method.

2.3

Aptamer-based biosensing systems for the detection of exosomes

Extracellular vesicles (EVs) are increasingly studied to understand their role under physiological and pathological conditions [28]. Among EVs, exosomes have been

Aptamers in biosensing: biological characteristics and applications

25

Figure 2.3 Schematic illustration of homogeneous detection of 19- nortestosterone using a split aptamer-based sandwich-format fluorescence resonance energy transfer (FRET) assay. Reprinted with permission from Ref. [27].

indicated as important targets for diagnosis and therapy not only for cancer [29], but also for other pathological conditions, such as cardiovascular diseases [30] or diabetes [31]. Exosomes are widely present in body fluids and they have the fundamental characteristics of carrying all the information related to their cell of origin representing an important source of disease biomarkers [32]. These EVs have, in general, a diameter ranging from 30 to 150 nm, they are produced by the endocytosis process of cells and they contain substances deriving from the donor cells, such as lipids, microRNAs, proteins, enzymes and others [33]. There is a strong interest in the development of reliable methods to detect exosomes due to their minimally invasive character as biomarkers, which can be exploited in liquid biopsies for the early detection of several diseases, cancer above all [34]. Biosensor technology is being increasingly applied to the detection of exosomes, in order to overcome some of the major challenges in the analysis of these EVs, such as their difficult isolation [35] or the necessary high sensitivity [36]. Among the different biosensors for exosome detection, aptamer-based biosensors have received noticeable attention in recent years sustained by the selection of a high number of aptamers for exosome-related molecules [37]. As reported in Fig. 2.4, in the last 5 years the number of publications related to aptamers for exosomes and to aptasensors for exosomes, has seen a 6e7 times increase (data from Scopus), demonstrating the huge increase of the interest in this field of the scientific community. Proteins are the key markers used for exosome capture and/or detection: among these, CD63 is the most considered protein in this kind of biosensing strategy [38]. CD63 is a cellular transmembrane protein and, together with the other tetraspanins

26

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.4 Number of publications related to aptamers for exosomes and to aptasensors for exosomes in the last 5 years (data from Scopus).

CD81 and CD9, has been found abundant in EVs. In addition, it has been supposed that only those EVs actually carrying CD63 alone or in combination with the other tetraspanins, are endosome-derived exosomes while those missing CD63 did not form in endosomes [39]. Due to this peculiarity, aptamers for this protein have been selected and used in exosome detection and isolation [38]. A plethora of aptamer-based sensors has been developed by using the CD63 specific aptamer as reported in Table 2.1. Due to the high sensitivity requested for the detection of exosomes, especially in the early stages of diseases, different amplification strategies have been coupled to the specificity of aptamers in these aptasensors; this aspect is also taken into account in Table 2.1. In particular, an interesting amplification method has been used in combination with the CD63 aptamer to capture and detect exosomes via a regenerable electrochemical biosensor [40]. In this work, two modified probes have been used to specifically capture exosomes: one probe is modified with cholesterol, which binds to the lipid bilayer of exosomes and the other has the CD63 aptamer at the 50 end to recognize CD63 on the exosome membrane. The simultaneous capture of exosomes by the two probes, having also at the 30 end a Pb2þ dependent DNAzyme tail sequence, generates an amplification system, i.e., DNA walker, for amplified signal production with a final regeneration step, as illustrated in Fig. 2.5. The different steps involved in the amplification and detection process are: - exosomes are captured by the two probes and the binding brings the tail sequences into close proximity; - in presence of Pb2þ, the Pb2þ-dependent DNAzyme cleaves the track DNA hybridized to the two probes, releasing an intermediate DNA (T); - at this point, the dissociated initial probes can further hybridize with the track DNA, activating the DNA walker, which ends in the production of a large amount of T strands;

Aptamer target

Detection method

Amplification strategy and/or nanomaterial

Sample type

CD63

Electrochemical

DNA walker

CD63

Electrochemical

CD63

Capacitance

Black phosphorus nanosheets þ ferrocene-doped metal
organic frameworks MoS2

Fetal bovine serum (FBS) Serum from patients

CD63 PTK-7 CD63 EpCAM CD63 EpCAM CD63 EpCAM CD63, EpCAM HER2 MUC1 CD63 PSMA PTK-7 PSMA, HER2 AFP

SPR Fluorescence Electrochemical Electrochemical FRET Fluorescence

LOD (particles/ mL)

Ref.

1.6  104

[40]

100

[41]

2.2  103

[42]

AuNPs Structure switching aptamer Hybridisation chain reaction DNA walker Quantum dots/AuNP Afterglow semiconducting polyelectrolyte nanocomplex

Undiluted human serum Serum from patients Serum from patients Serum from patients Serum from patients Serum from patients Plasma and cell culture medium

5.0  103 3.4  108 500 1.3  104 13 0.24 mg/mL

[43] [44] [45] [46] [47] [48]

FRET

Ti3C2 MXene nanosheets

Serum

1.4  103

[49]

SERS

SERS probes þ magnetic beads

Serum from patients

35  103

[50]

Aptamers in biosensing: biological characteristics and applications

Table 2.1 Recent aptamer-based biosensing systems for the isolation and detection of exosomes.

27

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Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.5 Schematic illustration of the working mechanism of the regenerable electrochemical biosensor based on the DNA walker. A) DNA walker generation after exosome binding; B) electrochemical detection via hybridization with an hairpin DNA probe. Reprinted with permission from Ref. [40]. - these T strands are used in an on-off-on electrochemical system composed of a hairpin DNA (H) with an electroactive methylene blue (MB) tag at 50 terminal on the electrode: the sensor is the “on” state without T strands and in the “off” state when the T strands hybridize with H taking MB far away from the electrode surface; - finally, regeneration can be performed via the hydrolysis of the dsDNA by Lambda exonuclease, restoring the initial “on” state.

Other nanomaterials were used in the work, such as porous nano-carbon, which was deposited on the surface of glassy carbon electrodes to enhance the interfacial electron transfer. By this method a detection limit of 1.6  104 particles/mL was obtained and, more importantly, it was possible to discriminate the difference in concentration among exosomes secreted by different cancer cells and by normal cells. Other electrochemical aptasensors have recently appeared in the literature for the detection of exosomes by using the antiCD63 aptamer for their capture: Sun et al. [41], with the aim of developing still a sensitive but also simple aptasensor system, constructed a platform by assembling black phosphorus nanosheets (BPNSs) and ferrocene (Fc)-doped metal
organic frameworks (ZIF-67) on indium tin oxide (ITO) slices and using an MB-labeled antiCD63 aptamer. This dual signal and the self-calibrating sensor were based on the detection of the signal of MB, which

Aptamers in biosensing: biological characteristics and applications

29

decreased upon the recognition of exosomes by the labeled aptamer and on the use of the stable signal of Fc taken as reference. Interestingly, the sensor was able to discriminate between exosomes captured from the plasma of healthy individuals and from breast cancer patients due to the higher expression of CD63 in exosomes derived from cancer cells. An excellent detection limit of 100 particles/mL was achieved. In order to reduce the time and cost of analysis, an electrical biosensor system based on a capacitive method was recently realized for the detection of exosomes by the use of the antiCD63 aptamer [42]. The use of the capacitive method allowed the detection of exosomes in undiluted serum due to the freedom from the used buffer, in contrast to the more matrix-sensitive electrochemical methods; in addition, short analysis time was achieved thanks to the possibility of detecting exosomes without sample pretreatment. The sensor was composed of an interdigitated microgap electrode (IDMGE)/ printed circuit board (PCB) system modified with an antiCD63 aptamer coupled to molybdenum disulfide (MoS2) nanoparticles to enhance electrical sensitivity: a limit of detection (LOD) in undiluted human serum of 2.2  103 particles/mL was achieved. The same antiCD63 aptamer has been also used for the development of biosensors based on optical methods: in particular, the aptamer was fixed onto surface plasmon resonance (SPR) chips and exosomes were detected by the use of a double amplification procedure based on the multiple binding of gold nanoparticles (AuNPs) coated with the antiCD63 aptamer having a tail of 30 thymines and, subsequently, with AuNPs coated with a fragment of 30 adenines [43]. A good LOD of 5.0  103 particles/mL was achieved and analysis of fetal bovine serum (FBS) samples spiked with exosomes from cancer cells was achieved with a good comparison with Western blot methods. Other single-protein targeting detection methods for exosomes have focused on different proteins, such as protein tyrosine kinase-7 (PTK-7): this protein, also known as colon carcinoma kinase-4 (CCK-4), plays an important role in carcinogenesis [51] and correlates with cancer progression to metastasis [52]. A fluorescence-based aptasensor has been developed [44] for the detection of exosomes from cancer cells by the use of a structure-switching anti-PTK-7 aptamer and N-methylmesoporphyrin IX as an intercalating dye. Very interestingly, in several works the detection of exosomes via their capture by the anti-CD63 aptamer has been combined with their specific recognition with an aptamer for epithelial cell adhesion molecule (EpCAM): this combination allowed not only to detect the number of exosomes but also to select those specifically released from solid tumor cancer cells [46]. EpCAM is a transmembrane glycoprotein which has been recognized as a specific marker for solid tumors and it has been also demonstrated to be a valuable target for therapy to inhibit cell signaling promoting tumor growth [53]. The combination of antiCD63 and antiEpCAM aptamers has been successfully exploited for the realization of an electrochemical biosensor based on microelectrodes and hybridization chain reaction (HCR) amplification [45]. An excellent LOD of 500 particles/mL has been reached with this method thanks to the high electron transfer efficiency of microelectrodes and to the HCR amplification strategy. The scheme of the sensor procedure is illustrated in Fig. 2.6: the antiEpCAM aptamer is combined with an initiator long strand, which can hybridize with two other probes

30

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.6 Scheme of the realization of an electrochemical biosensor based on microelectrodes and hybridization chain reaction (HCR) amplification. (a) Procedure for preparing biotinlabeled HCR exosomes; (b) top, modification of microelectrodes with CD63; (b) bottom, picture of a fabricated electrochemical micro-aptasensor with CD63-modified electrodes and a microfluidic chamber; (c) working mechanism of the electrochemical micro-aptasensor. Reproduced from Ref. [45] under Creative Commons CC BY license.

labeled with biotin. The binding of the aptamer to the EpCAM-expressing exosomes results in vesicles bearing multiple biotin molecules on their surface; these exosomes are then captured on the surface of microelectrodes functionalized with the antiCD63 aptamer and the electrochemical signal is generated by the addition of streptavidinlabelled horseradish peroxidase (HRP) and 5,50 -Tetramethylbenzidine (TMB)/H2O2 substrate. In addition to the excellent LOD achieved, this double-aptamer sensor was able to differentiate samples from early-stage lung cancer patients to those from late-stage patients. Exceptional sensitivity was achieved by Zhu et al. [47] by employing the two aptamers, antiCD63 and antiEpCAM, in an exosome detection method based on the combination of magnetic beads and quantum dots (QDs) by using FRET as an optical mechanism for sensing. In particular, as illustrated in Fig. 2.7, FRET was obtained by the combination of QDs on magnetic beads and AuNPs carrying a DNA fragment complementary to the aptamer: in absence of specific exosomes, fluorescence emission is inhibited by the close proximity to QDs of the AuNPs, whereas, upon binding of

Aptamers in biosensing: biological characteristics and applications

31

Figure 2.7 Working principle of the FRET-based aptasensor for exosomes exploiting the combination of QDs on magnetic beads and AuNPs carrying a DNA fragment complementary to the aptamer. Reprinted with permission from Ref. [47].

exosomes to the aptamer, the DNA with AuNPs is released and fluorescence of QDs is restored. The use of QDs was presented to have different advantages, such as broad absorption spectra and stronger photoluminescence performance and, actually, with this sensing approach, a remarkable sensitivity of 13 particles/mL was achieved together with discrimination between exosomes from healthy patients and from cancer patients. With the aim of identifying different cancer cells, several groups have developed biosensing systems able to specifically recognize different proteins on the membrane of exosomes creating a sort of fingerprint to recognize the exosome parental cell.

32

Aptamers Engineered Nanocarriers for Cancer Therapy

Among these, by using a particular fluorescence-based approach and four different aptamers, it was possible to identify exosomes secreted by five different cell types [48]: aptamers specific for CD63, EpCAM, human epidermal growth factor receptor 2 (HER2), and mucin 1 (MUC1) were used to recognize exosomes produced by HeLa (cervical cancer cells), chondrocytes (benign cells), MCF-7 (breast cancer cells), SKOV3 (ovarian cancer cells), and HepG2 (liver cancer cells) by creating a heat map with the expression levels of the four proteins (Fig. 2.8). The detection approach was based on the use of an afterglow semiconducting polyelectrolyte nanocomplex, which has the optical characteristic of emitting long-living luminescence even after the

Figure 2.8 Differentiation of cancer exosomes using afterglow semiconducting polyelectrolyte nanocomplex coupled to aptamers. (a) Cell release of exosomes possessing different biomarkers. (b) Heat map of the expression levels of four biomarkers (CD63, EpCAM, HER2, MUC1) on exosomes from 5 cell lines (Chondrocyte, HeLa, MCF-7, SKOV3, HepG2). (c) Proof-of-concept demonstration of distinguishing benign and cancer-cell-secreted exosomes. (d) Western blot analysis of the HER2 expression in the 5 cell lines. Reprinted with permission from Ref. [48].

Aptamers in biosensing: biological characteristics and applications

33

cessation of light excitation [54]. Fluorescence and afterglow emission depend on the quenching by the BHQ-2-tagged aptamer, which happens only in absence of the exosome; when exosomes bind to the aptamer, this is released from the nanocomplex and fluorescence emission is restored. Similar results were obtained [49] by an optical method based on the use of aptamers for CD63, PTK-7 and prostate-specific membrane antigen (PSMA) to discriminate exosomes secreted by B16 cells, MCF-7 (breast cancer cells), OVCAR-3 (ovarian cancer cells), and HepG2 (liver cancer cells). The detection method was based on the quenching of the free aptamer labeled with the Cy3 dye by Ti3C2 MXene nanosheets and the subsequent emission of fluorescence upon aptamer binding to the target protein causing its release from the nanosheets. A multiplex aptasensor was realized based on SERS [50]: bimetallic SERS-active nanotags (goldesilveresilver coreeshelleshell nanotrepangs (GSSNTs)) were decorated with DNA probes complementary to three different aptamers specific for PSMA, HER2 and alpha-fetoprotein (AFP), all proteins present on the membrane of exosomes. In absence of exosomes, the aptamer, fixed onto magnetic beads, is hybridized to the complementary DNA probe; when bound to the specific exosome, the aptamer separates from the DNA probe linked to GSSNTs, which remains in solution after separation of the exosome-bound aptamer via magnetic attraction. In this way, the SERS-active GSSNTs are released and SERS signals were attenuated. Multiplexing was obtained by encoding the GSSNTs with different Raman reporter molecules, 2mercaptopyridine, 4-aminothiophenol and 4-nitrothiophenol and by immobilizing onto each encoded GSSNTs a probe complementary to one of the three chosen aptamers.

2.4

Aptamer-based intracellular biosensing

In recent years, several reviews have highlighted the high specificity and affinity of aptamers for different targets, including proteins, small molecules, ions, bacteria, viruses and cancer cells as well as their potential use in vivo [18]. In this section, the focus will be on aptamer-based intracellular biosensing coupled with the use of aptamers for targeted therapy. Before getting to the heart of the topic of this section, which is intracellular biosensing by aptamers, some examples of the use of aptamers as therapeutics will be also described.

2.4.1

Aptamers as direct therapeutics for cancer

The most important aptamer used for cancer treatment is AS1411, an unmodified guanosine-rich 26-mer DNA sequence, which is the first aptamer to be entered in clinical trials for cancer treatment [55]. AS1411 specifically binds nucleolin, a protein highly expressed on the surface of many cancer cells and related to cell growth, proliferation, and survival. After binding to nucleolin, AS1411 is internalized into the cells and the aptamer-nucleolin complex leads to the inhibition of DNA replication [56].

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Soundararajan et al. [57] further discovered that AS1411 could significantly reduce the stability of B-cell lymphoma-2 (BCL-2) mRNA and promote apoptosis. Girvan et al. [58] found that AS1411 could inhibit cell proliferation by blocking the nuclear factor kappa B (NF-kB) signaling pathway, so determining the arrest of the cell cycle in the S phase. Although some potential mechanisms of AS1411 action have not been fully elucidated, AS1411 still represents a huge innovative strategy for inhibiting the proliferation of tumors. Another aptamer used in oncology is the RNA aptamer A9g against PSMA and developed by Dassie et al. [59]. PSMA is a transmembrane protein overexpressed in prostate cancer (PC) cells and, for this reason, it is considered a potential biomarker for the targeted therapy of PCs. A9g is able to inhibit the enzymatic activity of PSMA, diminishing PC cell migration/ invasion in vitro. In 2013, Mahlknecht et al. [60] selected a 42-nt (trimeric version) DNA aptamer targeting human HER2, a member of the epidermal growth factor receptor (EGFR) family, a receptor tyrosine kinase expressed in a variety of cancers, including breast and gastric tumors. The authors demonstrated that this aptamer is able to improve the internalization of HER2 owing to the formation of multimolecular complexes at the cell surface. It has been demonstrated that, after treatment with the trimeric aptamer, the growth of the gastric cancer cells is inhibited by accelerating the lysosomal degradation of the target protein. Moreover, the tumor volume in HER2positive immunocompromised mice is greatly reduced by intraperitoneal injection of the trimeric aptamer. In addition to the tumor-specific aptamers above mentioned, other aptamers against different types of cancer cell biomarkers have been developed and tested. For example, AX102 aptamer targeting platelet-derived growth factor (PDGF)-B chain [61], Gint4.T targeting platelet-derived growth factor receptor-b (PDGFR-b) [62], and NOX-A12 targeting CXC chemokine ligand 12 (CXCL12) [63]; they act as anticancer agents by inhibiting cell migration and proliferation, inducing cell differentiation, impeding tumor growth in vivo, or downregulating signaling pathways associated with tumor activation. In addition to aptamers directed against specific tumor markers, another strategy proposed to counteract tumor proliferation aims to stimulate the immune response. In this field, an important target is represented by 4-1BB, recognized as a major costimulatory receptor that can promote the survival and expansion of activated T cells, implicated in promoting protective antitumor immune responses [64]. In this field, McNamara et al. [64] selected aptamers targeting 4-1BB to enhance CD8þT cell proliferation and cytolytic activity, aiming to inhibit tumor growth in mice. This strategy provides a possible new avenue for cancer therapy, manipulating the immune system, by aptamers.

2.4.2

Aptamers as direct therapeutics for other diseases

Aptamers have been used as therapeutic agents toward targets specific to other human pathologies. Pegaptanib, the first therapeutic aptamer approved by the US FDA as an

Aptamers in biosensing: biological characteristics and applications

35

antiangiogenic medicine, is a 27-nt RNA aptamer that specifically binds to VascularEndothelial Growth Factor (VEGF). This aptamer can reduce neovascular age-related macular degeneration (AMD) by blocking intraocular blood vessel growth [65]. Another aptamer used for the anemia treatment is called NOX-H94: it is a 44-nt RNA aptamer that specifically binds to hepcidin. Hepcidin is a protein that in humans is encoded by the hepcidin antimicrobial peptide gene and represents a key regulator of the entry of iron into the circulation in mammals. During conditions in which the hepcidin level is abnormally high, such as inflammation, serum iron falls due to iron trapping within macrophages and liver cells decrease gut iron absorption. This typically leads to anemia. The bond between NOX-H94 and hepcidin blocks hepcidinregulated ferroportin degradation and this effect makes this aptamer a potential therapeutic agent for the treatment of anemia [66]. ARC1779 is a 49-nt DNA/RNA aptamer that binds specifically to von Willebrand factor (vWF), a blood glycoprotein involved in hemostasis and, more specifically, in the platelet adhesion. It is deficient and/or defective in vW disease and it is involved in many diseases, including thrombotic thrombocytopenic purpura, Heyde’s syndrome and hemolyticeuremic syndrome. Increased plasma levels of this factor are found in many cardiovascular, neoplastic, metabolic (e.g., diabetes), and connective tissue diseases. The interaction between ARC1779 and vWF inhibits the interaction between the vWF A1-domain and the platelet receptor GPIb, so reducing vWF-dependent platelet activation and pathological thrombosis [67]. All these pieces of evidence support the idea that aptamers could represent a new alternative for the targeted-specific treatment of some human diseases.

2.4.3

Aptamer-drug conjugate systems for targeted therapy

Aptamers can be used, in addition to the direct treatment of several diseases, also for targeted drug delivery. In oncology, the direct use of cytotoxic drugs is limited by their side effects on normal cells and low maximum tolerated dosage owing to the off-target effects. Owing to the high specificity of aptamers, for example, the preparation of aptamercytotoxic drug complexes via noncovalent or covalent conjugation methods can allow cytotoxic drug targeted delivery, which will increase the accumulation rate of the drug in target cells and reduce the adverse effects. Bagalkot et al. [68] proposed the targeted delivery of Doxorubicin (Dox) to PC cells by employing the PSMA aptamer A10. Dox is an anticancer drug that can inhibit DNA replication and transcription by inserting itself into the double-stranded CG sequences of DNA and RNA. It is used for the treatment of many cancer types, such as cell acute lymphoblastic leukemia, breast cancer, and malignant lymphomas [69]. Dox can lead to cardiotoxicity, including dilated cardiomyopathy and congestive heart failure [70] and, for these reasons, it is useful to develop a targeted Dox-delivery system PSMA aptamer A10. Dox molecules are noncovalently intercalated in the double-stranded region of A10 and form an aptamer-Dox complex. When this complex is used to treat cancer cells, the conjugate is internalized into PC cells by specific binding between A10 and PSMA, followed by the intracellular release of Dox [68].

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Aptamers Engineered Nanocarriers for Cancer Therapy

Another strategy, described by Huang et al. [71], uses a construct consisting in DoxDNA aptamer sgc8c, with a hydrazone linker for the specific killing of target cells. The aptamer sgc8c is able to recognize PTK-7 highly expressed in T-cell acute lymphoblastic leukemia (TALL) cells. The Dox-sgc8c conjugate can be efficiently internalized by TALL cells via binding to PTK-7. After internalization, Dox can be released from the conjugate in lysosomes (pH 4.5e5.5) owing to the pH-sensitive covalent linkage between Dox and sgc8c. The attractive properties of aptamers make them prominent candidates for the targeted delivery of several other molecules/drugs, such as siRNA. Aptamer-siRNA conjugates were used, for example, to treat AIDS. Zhou and colleagues [72] developed aptamer-siRNA chimeras for the cell type-specific delivery of siRNAs in an HIV-1 infected RAG-hu mouse model. All the above examples clearly highlight the importance of aptamers as new tools to enable the precise recognition of cellular elements. A further step is the use of aptamers as agents able to follow, even in real-time, intracellular events, making them tools useful for intracellular biosensing when coupled, for example, to fluorophores.

2.4.4

Aptamers for intracellular biosensing

Aptamers can be used for extracellular and intracellular detection of several molecules, such as ATP, a crucial indicator for cellular energy status and viability. Recent studies on COVID-19 virus suggested that infection causes ATP deficit and release at the early stage of the disease and also in disease complications. Liu et al. [73] realized a sensitive and selective fluorescence signal-on probe for ATP detection. The probe is composed of two elements, a 30 BHQ attached ATP aptamer (ssDNA) and a 50 Cy5 labeled reporter strand. The strands hybridize and present the fluorophore near the quencher when ATP is absent, leading to the fluorescence off state. When ATP incorporates into the molecule complex, a conformational change of the aptamer occurs allowing fluorescent signal emission.

2.4.5

Aptamer-nanomaterial conjugated systems for intracellular biosensing and drug delivery

Drug nanodelivery systems involving both aptamers and nanomaterials are also noteworthy. In some of these systems, the optical properties of drugs and nanomaterials are used to perform therapy and sensing at the same time. In this context, Bagalkot and colleagues [74] developed a novel QD-aptamer conjugate to be loaded with Dox for the targeted delivery of the drug to PC cells based on the mechanism of binary (Bi)FRET (Fig. 2.9). As shown in Fig. 2.9a, the conjugate consists of three components: QDs that act as fluorescent imaging vehicles, the A10 PSMA aptamer covalently linked to QDs, which serves as cellular recognition element and Dox-carrying vehicle and Dox, which intercalates into double-stranded CG sequences of the aptamer as a therapeutic agent. In this conjugate, the green fluorescence of the QD was quenched by Dox, while the

Aptamers in biosensing: biological characteristics and applications

37

Figure 2.9 Illustration of QD-Apt(Dox) Bi-FRET system. (a) The QD was functionalized with A10 PSMA aptamer followed by the intercalation of Dox, leading to quenching of both QD and Dox fluorescence through a Bi-FRET mechanism. (b) Specific uptake of QD-Apt(Dox) into target PC cell through PSMA mediate endocytosis. The Dox release from the QDApt(Dox) conjugates determined the recovery of fluorescence from both QD and Dox. Reprinted with permission from Ref. [74].

red fluorescence of Dox was at the same time quenched by a double-stranded RNA aptamer. After endocytic uptake of the conjugate into the target cells expressing the PSMA protein, Dox is gradually released from the conjugate, leading to the simultaneous visualization of Dox (red) and QD (green) fluorescence. Hence, this multifunctional delivery system not only delivers the drug Dox to the targeted cells but also allows imaging of the delivered Dox and of the fluorescence of QDs. Another example of aptamer-QD conjugate is described by Savla et al. [75]. In this work, a pH-responsive QD-mucin one aptamer-Dox (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer is described. In this conjugate, both the MUC1 aptamer and Dox are linked to the QDs. Dox is linked to the QDs via a pHsensitive hydrazone bond, which allows drug release in an acidic environment inside cancer cells. The nanocomplex obtained determines the quenching of QD fluorescence due to the close proximity between QDs and Dox. After the conjugate internalization into cancer cells, Dox is released from the conjugate, with the restoration of QD and Dox fluorescence. Moreover, in vivo studies have shown that, 1 hour after intraperitoneal administration, the construct QD-MUC1, compared to the unmodified QD, showed higher accumulation in the tumor area and lower accumulation in other studied organs with respect to the unmodified QD. Liu et al. [76] reported a graphene quantum dot (GQD) based aptasensor able to specifically detect ultrasmall amounts of cytokine molecules intracellularly. Graphene QDs modified with cytokine aptamers (Ap-GQDs) and epitope modified GQDs (EpGQDs) were prepared. The aggregation between Ap-GQDs and Ep-GQDs determines

38

Aptamers Engineered Nanocarriers for Cancer Therapy

the quenching of the fluorescence. After incubation of the cytokine-secreting cells with the conjugates of Ap-GQDs and Ep-GQDs, the binding of cytokines with the aptamers results in the disaggregation of Ap-GQDs and Ep-GQDs, and in the recovery of fluorescence. These conjugates were used as nanosensors for monitoring intracellular cytokine interferon g (IFN-g) secretion with very high sensitivity (2 pg mL
1). Other interesting nanomaterials used in association with aptamers to favor the internalization of drugs are represented by AuNPs. Luo et al. [77] developed an sgc8c aptamer/hairpin DNA-AuNP conjugate for the targeted delivery of Dox for TALL treatment. The DNA aptamer is used as a recognition ligand for PTK-7; it is assembled onto the surface of AuNPs and the hairpin DNA on the AuNP surface is used for loading the anticancer drug Dox. After the conjugate is taken up into the target cells, Dox is released from the Dox-loaded drug carrier with the assistance of photoenergy and local photothermal heating response induced by illumination with a continuouswave (CW) laser, so emitting fluorescent signal (Fig. 2.10). An innovative strategy to improve the therapeutic efficacy and reduce the development of drug resistance is described by Shiao et al. [78]. The authors realized an aptamer-functionalized AuNP to codeliver two different anticancer drugs. In this construct, the AS1411 aptamers are linked to AuNPs via strong goldethiol linkages, and then the potent inhibitor of human telomerase, TMPyP4, and Dox are physically inserted into the AS1411-conjugated AuNPs, leading to the formation of the T/D:dsNPs conjugate. After the conjugate penetration into tumor cells, such as HeLa and Dox-resistant MCF-7R cell lines, reactive oxygen species induced by TMPyP4 molecules are generated when illuminated at 632 nm, resulting in cell damage. At the same time, Dox is released from the conjugate during the photodynamic reaction, leading to an increase in cancer cell damage and fluorescence emission (Fig. 2.11).

Figure 2.10 Dox release from Dox:apt/hp-AuNP nanocomplexes induced by light inside targeted cancer cells. Upon the nanocomplex internalization, laser could assist the release of fluorescent Dox molecules from the Dox-loaded drug carrier. Reprinted with permission from Ref. [77].

Aptamers in biosensing: biological characteristics and applications

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Figure 2.11 Scheme of the co-drugs delivery system based on aptamer-functionalized AuNPs. The aptamers were conjugated to AuNPs followed by the intercalation of Dox and TMPyP4. Light induced the release of Dox (fluorescent signal) and TMPyP4. Reprinted with permission from Ref. [78].

Yang et al. [79] designed a new ratiometric fluorescent nanoprobe, termed aptamerbased FRET nanoflare, for sensing Kþ ions in living cells. The aptamer-based FRET nanoflares consist of AuNPs, short single-stranded oligonucleotides and dualfluorophore-labeled G-quadruplex sequences, in which FAM acts as a donor (D) and carboxy tetramethylrhodamine (TAMRA) as acceptor (A). The complementary oligonucleotides are designed to bind with the G-quadruplex sequences (flares) and immobilized on the AuNP surface via an AueS bond. In the absence of target Kþ, the G-quadruplex sequences are captured by binding with the complementary strands, separating the donor (FAM) from the acceptor (TAMRA), and inducing a low FRET efficiency. In this open state, only the fluorescence of donors can be detected. In the presence of target Kþ, the flares are gradually displaced from the complementary strands, subsequently forming G-quadruplex structures that bring the donor and acceptor into close proximity and result in high FRET efficiency. In this closed state, the fluorescence of the acceptor can be detected. Thus, the fluorescence emission ratio of acceptor to the donor (A/D) can be used as a signal for the detection of target Kþ (Fig. 2.12). Hwang and coworkers [80] reported a cancer-specific multimodal imaging probe consisting of cobalteferrite nanoparticles protected by a silica shell and coated by fluorescent rhodamine. The authors demonstrated that the AS1411 aptamer-multimodal nanoparticle system not only enabled the targeted fluorescence imaging of nucleolin-expressing C6 cells but also allowed magnetic resonance imaging (MRI) in vivo and in vitro. Many carbon nanomaterials, including single-walled carbon nanotubes (SWCNTs), graphene oxide (GO), fullerene, and carbon dots (CDs) have recently been used in the biomedical field due to their high surface area, mechanical strength, high electrical

40

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.12 Mechanism of aptamer-based FRET nanoflares for intracellular Kþ detection. Reprinted with permission from Ref. [79].

conductivity, and photoluminescence. These unique properties offer SWCNTs and graphene good opportunities for biosensing and bioimaging applications. For example, DNA aptamers can be adsorbed onto SWCNT/graphene and released through interaction with their specific intracellular target. By taking advantage of the high quenching efficiency of carbon structures, Cha et al. [81] were able to develop an intracellular insulin sensor with a wide detection range from 10 mM to 2 mM. Tang et al. [82] developed a novel photoresponsive drug delivery system based on GO-wrapped mesoporous silica nanoparticles (MSN@GO) for light-mediated drug release and aptamer-targeted cancer therapy (Fig. 2.13). In the beginning, Dox was loaded by MSN, and then negatively charged GO nanosheets were wrapped around the surface of the positively charged MSN through electrostatic interactions. Afterward, the Cy5.5-labeled AS1411 aptamer was attached to GO, determining the quenching of the dye. After the uptake of the complex into the target cell, the fluorescence of Cy5.5 was restored. In the absence of laser irradiation, GO acted as a gatekeeper to prevent the loaded Dox from leaking. After laser irradiation, the photoresponsive drug delivery system is activated, causing the release of fluorescent Dox. It is important to note that the MSN-Dox@GO-Apt platform used for killing cancer cells could introduce the synergism of chemotherapy and photothermal therapy, which is much more effective than monotherapies, providing a new approach to cancer treatment. Beltr
an-Gastélum et al. [83] proposed a strategy for amplified breast cancer 1 (AIB1) intracellular sensing by using GO nanocostructs. AIB1 is a member of the p160 steroid receptor coactivator family and it is frequently overexpressed in breast cancer. Gold nanowires (AuNWs) were initially coated with GO; then, the FAMAIB1 aptamer was physically adsorbed on the surface of the GO/AuNWs, leading

Aptamers in biosensing: biological characteristics and applications

41

Figure 2.13 Scheme of photoresponsive drug delivery system consisting in Cy5.5-labeled AS1411 aptamer-GO-wrapped Dox-loaded MSN. The release of drug in the target cell was controlled by NIR light. The MSN-Dox@GO-Apt with two “off-on” switches were controlled by aptamer targeting and light triggering, respectively. Reprinted with permission from Ref. [82].

to a quenching of the dye fluorescence due to the FRET between the fluorophore (FAM) and GO. The FAM-AIB1-apt functionalized GO/AuNWs were then powered under acoustic waves in order to accelerate their penetration through the membrane of cancer cells. Once inside the cells, the presence of the specific AIB1 target molecule resulted in the displacement of the quenched FAM-AIB1-apt from the nano-motor surface and a fast fluorescence recovery. MCF-7 cell line was chosen as the experimental model due to the high expression of the AIB1 protein. Wang et al. [84] proposed a (FAM)-aptamer/GO-nanosheet (GO-nS) complex for ATP intracellular detection (Fig. 2.14).

Figure 2.14 Scheme of ATP sensing in living cells by using aptamer/GO-nS nanocomplex. Reprinted with permission from Ref. [84].

42

Aptamers Engineered Nanocarriers for Cancer Therapy

As shown in Fig. 2.14, ATP aptamer labeled with FAM was incubated with GO-nS to form aptamer-FAM/GO-nS. After 5 min incubation, fluorescence quenching was observed on aptamer-FAM/GO-nS because of FRET between FAM and GO-nS. After the aptamer-FAM/GO-nS cellular uptake, in the presence of ATP, ATP aptamer linked to ATP forming the duplex configuration, which was released from the surface of GOnS due to the weak adsorption with an extraordinary fluorescence recovery. Another interesting strategy for ATP intracellular detection and imaging by fluorescent aptamer probes physisorbed on GO is proposed by Liu et al. [85]. The use of aptamers-GO-nS is also very important in real-time sensing and imaging of intracellular metabolites in living cells. In the study realized by Jin et al. [86], a strategy using these nanocomplexes has been developed to monitor a specific microalgal metabolite in living cells. As a proof-of-concept, b-carotene, an antioxidant pigment that accumulates in most microalgal species, was chosen as a target metabolite. The specific aptamer was labeled with FAM and adsorbed onto GO-nS, resulting in an aptamer/GO-nS complex that protected the aptamer from nucleic cleavages. In this configuration, GO-nS was able to act as a quencher, inhibiting the aptamer fluorescence. The aptamer/GO-nS complex was delivered into the cells via electroporation and, after internalization, in the presence of the target, the aptamer bound the target with the consequent recovery of the fluorescence. Among the strategies proposed for monitoring intracellular levels of ATP, Wang et al. [87] described ultrathin lanthanide-based metal-organic framework (MOF) nanosheets as a carrier of two dye-labeled aptamers for the intracellular two-color imaging of ATP. Hong et al. [88] also reported an intracellular ATP aptamer sensor based on a photocleavable linker and DQAsomes (liposome-like cationic vesicles) used to target mitochondria for spatiotemporally controlled monitoring of ATP in mitochondria of living cells. The ATP aptamer was extended on the 50 end by five bases and labeled with Cy3; then, it was hybridized with a partially complementary DNA strand containing the photocleavable linker and an Iowa Black FQ quencher in 30 position resulting in the quenching of the fluorescence. Upon 365 nm irradiation, the photocleavable linker undergoes photolysis, splitting the photocleavable-DNA strand into two DNA fragments. In the presence of ATP, the aptamer binds the ATP molecule resulting in the emission of Cy3 fluorescence (Fig. 2.15). To verify the photocleavable complex internalization inside cells, photocleavableApt/DQAsome complexes were incubated with HeLa cells and, in the presence of ATP, the red fluorescence of ATP-aptamer complex was observed by using a confocal laser scanning microscope. At the same time, commercially available MitoTracker Green, which is able to exhibit fluorescence in the green channel, was used to verify colocalization with mitochondria. Another strategy for ATP sensing using aptamers was proposed by Qiang et al. [89]. A nanocomplex was developed, based on the fluorophore-labeled aptamer and polydopamine nanospheres (PDANS). The aptamer was adsorbed onto the surface of PDANS forming the aptamer/PDANS nanocomplex, and, in this condition, the fluorescence was quenched by PDANS through FRET. In an in vitro assay, the presence of

Aptamers in biosensing: biological characteristics and applications

43

Figure 2.15 Schematic illustration of the photo-regulated ATP aptamer probe for the detection of mitochondrial ATP in living cells. Reprinted with permission from Ref. [88].

ATP led to the dissociation of the aptamer from the PDANS and the recovery of fluorescence. The retained fluorescence of the nanocomplex was found to be linear with the concentration of ATP in the range of 0.01e2 mM, and the nanocomplex was highly selective toward ATP. Then, the nanocomplex was incubated with HeLa and MCF7 cells and, in the presence of ATP, fluorescence was visualized (Fig. 2.16). Nielsen et al. [90] described a nanosensor consisting of an ATP-responsive DNA aptamer switch probe (ATP-DNA-ASP) with a fluorophore-quencher pair (Texas Red and BHQ2, respectively) embedded in polyacrylamide nanoparticles. In the absence of the target, the fluorescence is not visible due to the close proximity of fluorophore and quencher molecules. After electroporation of the nanosensor inside yeast cells, in the presence of ATP, the aptamer changes conformation binding ATP and making the Texas Red fluorescence visible. Zheng et al. [91] described an aptamer-AuNP hybrid with fluorescent reporters, termed as nanoflare, which can quantitatively detect ATP molecules inside living cells. The aptamer modified nanoflares are highly stable constructs, readily taken by cells, and the authors demonstrated that they are able to detect intracellular ATP concentrations in the range of 1e2 mM in HeLa cells. A strategy for the intracellular detection of cancer biomarkers was described by Wang et al. [92]. A polydiacetylene (PDA) liposome-based sensing system was used to develop a fluorescence turn-on aptasensor for MUC1 detection. This protein biomarker is overexpressed in lung, colon, pancreatic, breast and ovarian cancers. In this system, Cy3-labeled MUC1-specific aptamers were immobilized on the surface of PDA liposome micelles, and their fluorescence was directly quenched due to the close proximity between Cy3 and the conjugated polymer backbone. When MCF-7

44

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 2.16 Scheme of aptamer/PDANS nanocomplex for ATP sensing in living cells. Reprinted with permission from Ref. [89].

cells, overexpressing MUC1, were incubated with the sensing system, the highly specific interaction between MUC1 and aptamers released the Cy3 dyes from the interface of the PDA liposome micelle, resulting in fluorescence recovery. This simple PDA liposome-based sensing system can be considered a good and simple strategy for fluorescent turn-on detection and imaging of MUC1 in living cells.

2.5

Conclusions

This overview emphasizes the potential fascinating applications that aptamers, combined not only with optical or electrochemical systems but also with a variety of nanomaterials, can have for in vitro real-time biosensing and imaging, useful for early detection of severe diseases accompanied by possible therapeutic effects (theranostics) and more controlled outcomes during pharmaceutical treatments.

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Mechanisms of multidrug resistance in cancer

3

Fatemeh Zahedipour 1,2 , Prashant Kesharwani 3 and Amirhossein Sahebkar 4, 5, 6,7 1 Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; 2Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; 3Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India; 4Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 5Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 6School of Medicine, The University of Western Australia, Perth, WA, Australia; 7Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3.1

Introduction

Drug resistance is considered as the major reason of more than 90% of mortalities among cancer patients. Chemoresistance promotes disease recurrence and metastasis, complicates the improvement of clinical outcomes for cancer sufferers, and is the most significant barrier for the treatment of cancer. As a result, comprehending its underlying molecular mechanisms seems to be essential to investigate innovative methods to treat different types of cancer [1,2]. The development of inherent or acquired drug resistance against chemotherapeutics is a primary cause of treatment failure in cancer victims [3]. Acquired resistance may be detected after chemotherapy, whereas intrinsic resistance may already exist when the cancer is diagnosed, and tumor cells do not response to typical chemotherapy from the start [4]. The resistant phenotype is related to cancer cells developing cross-resistance to variety of agents with various cellular targets and architectures, a condition known as multidrug resistance (MDR) [5]. When MDR develops, the use of high dosage medicines to combat resistance becomes inefficient and toxic, resulting in less effective treatment [6]. It is also reported that in the same tumor cells, MDR might have at least two distinct resistance mechanisms to the same treatment [7]. Conclusively, the potential of cancer cells to acquire resistant to several medicines at the same time remains a substantial barrier to successful chemotherapy, despite the fact that chemotherapy is the most effective way for the treatment of majority of cancers. Overall, a key objective in cancer research is the precise explanation of mechanisms underlying MDR and numerous methods to combat this issue. Different molecular mechanisms of chemoresistance [8e10] are shown in Fig. 3.1 and are discussed further in this chapter. These molecular mechanisms are highly complicated and tightly associated to each other at different stages as are mentioned in the context. Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00002-6 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 3.1 Various cellular and molecular mechanisms involved in cancer drug resistance.

3.1.1

The role of drug transporters in cancer MDR

The most frequent pathways for the MDR development are the expression of ATPdependent efflux pumps. These pumps are transporters that belong to the ATPbinding cassette (ABC) transporter family and share sequence and structural similarity [11]. ABC proteins are in charge of transporting a variety of substrates including ions, lipids, carbohydrates, xenobiotics, amino acids, and peptides. Transferring drugs across cellular membranes, ABC efflux pumps, lower intracellular drug concentrations, and induce resistance via an ATPase transporter or a channel protein [12]. These efflux pumps are known to impact drug like paclitaxel, anthracyclines, actinomycin-D, and vinca alkaloids [13]. The most important types of ABC transporters involving in cancer MDR include P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and lung resistance-related protein or major vault protein (LRP/MVP).

3.1.1.1

P-glycoprotein transporter and MDR

With a molecular weight of 170 kDa, Pgp/ABCB1 is the most frequent transporter in the cellular membrane among the ABC transporters implicated in MDR [14]. Pgp can

Mechanisms of multidrug resistance in cancer

53

bind a wide range of hydrophobic agents, notably chemotherapeutics such as vinblastine, vincristine, doxorubicin, daunorubicin, and taxol, and numerous widely used drugs such as antihistamines and antiarrhythmics as well [15]. Pgp is a single polypeptide that consists of two homologous sections, each of which has a hydrophobic transmembrane domain (TMD) and a nucleotide-binding domain (NBD). An intracellular linker region connects these components. Six membrane-spanning helices make up each TMD. NBDs involve in ATP binding and hydrolysis, while TMDs are important for the substrate specificity by creating channels [16]. The most evident theory for drug transport via Pgp transporter expresses that the drug molecule attaches to a particular location of Pgp and activates one of the ATP-binding domains. Subsequently, ATP hydrolysis produces a significant change in the structure of Pgp, resulting in the drug delivery into the extracellular environment [17]. MDR via Pgp can arise as a result of changes in ABCB1 overexpression and amplification [8]. Other MDR mechanisms include stabilization of ABCB1 mRNA, regulation at the level of synthesis and changes in protein processing [18]. Many human malignancies such as liver cancer, large and small intestine cancers, pancreatic cancer, kidney cancer, ovary cancer, testicle cancer, neuroblastoma, fibrosarcoma, myeloma, lymphoma, and leukemia have witnessed the overexpression of Pgp transporter [19]. P-gp transpoter, which is abundantly expressed on the surface of endothelial cells, leads to a limited chemotherapeutic drug entry to specific locations, particularly in the case of brain tumor therapy, in which anticancer drugs are typically unable to pass across the bloodebrain barrier (BBB) [20]. To overcome Pgp-mediated MDR resistance, a variety of methods have been explored. Inhibition of Pgp with inhibitors such as verapamil, quinine, and cyclosporine A, as well as dexverapamil, is one way to reverse MDR in cancer [21]. Alternative methods for overcoming MDR, such as peptides and monoclonal antibodies, have also been explored [22,23]. Furthermore, it is known that cytotoxic drug therapy induces MDR gene expression in tumor cells. As a result, if MDR gene expression is suppressed via a variety of methods, MDR might be reversed. Antisense oligonucleotides, and short-interfering RNA (siRNA), and ribozymes are some of the most specialized methods that can be used to combat MDR [8].

3.1.1.2

BCRP transporter and MDR

BCRP (BCRP/ABCG2, MXR1 or ABCP) is another member of ABC superfamily [24]. Doyle et al. identified the BCRP in doxorubicin-resistant breast cancer cell lines for the first time. Many normal organs and solid tumors contain BCRPs including the adrenal gland, testis, stem cells, placenta, liver, BBB, and small intestine [25]. Overexpression of BCRP have been reported in normal tissues, namely, the placenta, intestine, liver, bloodetestis barrier, BBB, stem cells, cancer cells with MDR phenotype for the efflux of cytotoxic agents such as daunorubicin, doxorubicin, topotecan, mitoxantrone, etc. [2]. BCRP has just one NBD preceding one TMD with six membrane spanning helices, unlike Pgp and MRP1 [26]. In low concentrations, BCRP substrates are delivered out of the cell, although at larger concentrations, they can inhibit BCRP. At high doses, the tyrosine kinase (TK) inhibitor imatinib, for example, inhibits the function of BCRP [27].

54

3.1.1.3

Aptamers Engineered Nanocarriers for Cancer Therapy

MRP1 transporter and MDR

MDR associated protein 1 (MRP1/ABCC1) is expressed in several tissues including the brain, intestine, and peripheral blood mononuclear cells and is expressed similarly to P-gp; however, it has lower expression in comparison with Pgp. Furthermore, in contrast to ABCB1, this protein is found on the basolateral side of endothelial cells in cerebral capillaries [28]. Overexpression of MRP1 has been reported in many types of cancers including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), breast cancer, prostate cancer, pancreatic cancer, tongue cancer, brain cancer, and non-small cell lung carcinoma [29]. The 190-kDa ABCC1 protein has three TMDs, each with 17 membrane-spanning helices, two NBDs, and an additional N-terminal domain. MRP1 is involved in redox homeostasis, steroid and lipid metabolism, as well as the physiopathology of many diseases. This transporter is involved in the efflux of a range of xenobiotics and endogenous organic anions [30]. It also carries anticancer medications such as vincristine, etoposide, anthracyclines, and methotrexate. Other drugs used in nonmalignant conditions, including as opiates, antidepressants, statins, and antibiotics, have been found to be transported via MRP1 [31,32]. MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10, all members of the MRP family, have been shown to have an impact in MDR. However, according to the evidence, just ABCC1 has been widely assumed to be accountable for drug resistance in patients among members of the MRP family [33].

3.1.1.4

LRP/MVP transporter and MDR

The MVP, also termed as LRP, was initially found in doxorubicin-resistant lung cancer cells as a novel 110 kD efflux pump [34]. It also interacts with estrogen receptor alpha, PTEN, and Poly (ADP-ribose) polymerase (PARP) in nuclear pore complexes. As a result, MVP might have a role in chemoresistance by regulating nucleocytoplasmic transport of hormones, ribosomes, mRNA, and pharmaceuticals. Non-small cell lung cancer (NSCLC), gliomas, and B-cell lymphoma have all been shown to overexpress this transporter [2]. This is hypothesized that this transporter transfers DNAtargeting cytotoxic agents and mediates initial chemoresistance. In patients with acute leukemia or ovarian cancer, LRP might be used as an independent marker to determine MDR and clinical prognosis [35,36]. Anti-LRP monoclonal antibody and MVPspecific antisense oligonucleotides were shown to enhance cellular ciplastin levels and improve therapeutic efficacy in ovarian cancer cells [37].

3.1.2

The role of signaling pathways in cancer MDR

Several signaling pathways are involved in cancer MDR (Fig. 3.2). These pathways are connected with each other in a very complicated manner. These pathways are briefly discussed below.

3.1.2.1

ERK signaling pathway and MDR

Extracellular signal-regulated kinase (ERK)1/2 is a protein kinase regulating meiosis, mitosis, and postmitotic functions in response to cytokines, growth factors, viral

Mechanisms of multidrug resistance in cancer

55

Figure 3.2 Molecular signaling pathways of cancer multidrug resistance.

infection, and G protein-coupled receptor ligands. G protein Ras can activate Raf and subsequent MEK by phosphorylating threonine and serin after Ras is generally activated by growth factors via receptor TKs and GRB2/SOS. MEK is in charge of Erk1/2 phosphorylation and activation. Following that, Erks stimulate several transcription factors including ETS Transcription Factor ELK1 (ELK1) as well as protein kinases downstream of cell proliferation, apoptosis, and MDR pathways. The K-ras mutation has been linked to primary MDR in lung cancer to gefitinib, erlotinib, and sunitinib [38]. N-Ras can improve myeloma cell fibronectin adhesion and MDR [39]. Also, the cisplatin resistance in ovarian cancer cells is mediated by Raf-1/Erk signaling pathway [40]. According to investigations, caveolin-1 src-mediated phosphorylation was required for activation of EGFR or its association with b1 integrin increased Fyn-dependent Src homology and phosphorylation of Erk1/2, attributed to chemoresistance [41]. The phosphorylation of dynamin-related protein 1 (DRP1) via

56

Aptamers Engineered Nanocarriers for Cancer Therapy

Erk leads to mitochondrial fusion, low mitochondrial reactive oxygen species (ROS) levels, a strong proglycolytic shift, and MDR in T-cell ALL cells treated with mesenchymal stem cells [42]. Overexpression of the degradation in endoplasmic reticulum protein 1 (Derlin-1) caused bladder cancer chemoresistance via PI3K/Akt and Erk/ MMP signaling [43]. Following MDR, ABL1/2 kinase expression is increased in melanoma cell lines and patient samples, and ABL1/2 induce BRAF and BRAF/MEK inhibitor resistance via promoting reactivation of MEK/ERK/MYC signaling [44]. ABL1/2 silencing or inhibition prevents this pathway reactivation and reverse chemoresistance to inhibitors of BRAF/MEK [45]. In addition to AKT and ERK1/2 activation, fibroblast growth factor receptor 1 (FGFR1) overexpression has been shown to rendered breast cancer cells metformin resistance. FGFR1 induced insulin receptor substrate 1 (IRS1) and insulin-like growth factor 1 receptor (IGF1R) activation, key regulators connecting metabolism and cancer, that was associated with resistance to metformin. Furthermore, there is a feedback loop between MAPK/ERK and IRS1, in which IRS-1 acts as a major mediator of the crosstalk between FGFR1 and IGF1R pathways [46].

3.1.2.2

PI3K/Akt signaling pathway and MDR

The PI3K/AKT pathway is triggered by the formation of 30 -phosphorylated phosphoinositide that is a key signaling pathway for drug resistance in a number of malignancies including leukemia, ovarian cancer, lung cancer, melanoma, hepatocellular carcinoma (HCC), and breast cancer [47e51]. The MDR phenotype is frequently associated with induction of the PI3K/AKT pathway, providing a survival signal that allows cancer stem cells to survive cytotoxic anticancer treatments and improves CSC features. In many situations, however, PI3K/AKT activation pathway is insufficient to induce MDR and transduction with upstream and downstream targets in a synergistic manner is necessary. Furthermore, the PI3K/AKT pathway is a crucial connection that connects many of the targets involved in modulating apoptosis, cell proliferation, and cellular metabolism, all of which are linked to the MDR process. As a result, the major regulators of MDR are signals implicated in apoptosis resistance and enhanced survival [52]. PI3K interacts with PH domain of AKT, triggering conformational changes that lead to AKT phosphorylation and activation. This cascade stimulates downstream molecular proteins like NFк-B and mTOR, either directly or indirectly [53]. MDR can be caused by PI3K/AKT/NF-B activation, leading to the transformation of cyclin D1 and the expression of G1/S, as well as an accelerated cell cycle process [54]. To induce MDR, aberrant activation of PI3K/AKT/NF-B controls the expression of P-gp. In addition, MDR in tumor cells is possibly induced by P13K/ AKT/mTOR-mediated deregulation of microRNA (miRNA) that possess crucial role in cancer MDR [55]. Glycogen synthase kinase-3 (GSK3) is a kinase that controls the cell growth in response to a variety of stimuli and is found in a wide range of cancers and involves in various molecular pathways associated with resistant to chemotherapy, radiotherapy, and targeted therapy [56]. GSK-3b is phosphorylated by PI3K/AKT and changed to inactive form, leading to b-catenin activation and translocation to the

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nucleus. Subsequently, b-catenin functions as a transcription factor and increases the expression of MDR-associated genes [57]. The PI3K/AKT pathway is important in the control of aerobic glycolysis, improving the capacity of ABC transporters to expel drugs by increasing energy supply [58]. Overall, the PI3K/AKT pathway is extremely complicated and may be influenced by a wide range of different factors, all of which affect MDR processes [51]. Tumor suppressor, PTEN (phosphatase and tensin homolog), has phosphatase activity that prevents the PI3K/Akt signaling pathway [59]. PTEN is commonly found mutated in skin cancers, brain, endometrium, and prostate, contributing to the MDR development by increasing the activation of the PI3K/Akt signaling pathway [60]. Both targeting PI3K/Akt signaling and increasing PTEN intracellular levels could be used to overcome MDR-induced PTEN mutations. PTEN expression is increased by PS341, an inhibitor of proteasome, thus, triggers apoptosis in human breast cancer cell lines resistant to trastuzumab because of increased sensitivity to chemotherapy [61]. PTEN upregulation via eukaryotic expression plasmid (pEAK8) containing PTEN transfection protected human gastric cancer cells from etoposide and doxorubicin treatment. Furthermore, when a PI3K inhibitor, wortmannin, was used on human gastric cancer cell lines, the cells become more sensitive to etoposide and doxorubicin treatment [62]. Inhibition of PI3K/Akt/mTOR signaling pathway through transfection of PTEN to PTEN-deficient PC3 human prostate cancer cell lines, the cells became sensitive to doxorubicin and vinblastine treatment [63]. Moreover, recent research suggests that autophagy suppression may make PTEN-deficient PC3 human prostate cancer cells more susceptible to inhibitors of Akt such as AZD5363 [64]. Another experiment has investigated that microRNA19 a/b has a major function in the regulation of MDR by targeting the activity of PTEN in gastric cancer cells [65]. The expression of the tripartite motif protein 25 (TRIM25) is been changed in several cancers. TRIM25 expression had been found to be higher in tissues and cell lines of HCC. TRIM25 knockdown enhanced the sensitivity of HCC cells to epirubicin, as evidenced by decreased cell viability, higher cell apoptosis, and downregulation of P-gp and MRP1. In addition, TRIM25 knockdown enhanced epirubicin’s effects on PTEN and phosphorylated-AKT [66]. It is clear that PETEN has a significant role in cancer progression and MDR via involvement in different cellular signaling.

3.1.2.3

NF-кB signaling pathway and MDR

Ranjan Sen was the first to identify Nuclear factor kappa B (NF-kB), which interacts with an eleven-base pair region in the immunoglobulin light-chain enhancer in B cells [67]. IкBs sequester NF-кB dimers in the cytoplasm in inactive state in unstimulated cells. Phosphorylation, polyubiquitination, and proteasomal degradation of the IкB protein trigger NF-кB activation [68]. Tumor necrosis factor a (TNFa), ROS, interleukin (IL)-1, isoproterenol, lipopolysaccharide (LPS), cocaine, ionizing radiation, viruses, and chemotherapeutic drugs are all examples of NF-кB inducers. Following penetration to the nucleus, active NF-kB up-regulates the transcription of Akt, Bcl2, Bcl-xL, survivin, and XIAP [69].

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EMT is another mechanism that is activated by NF-kB activation. NF-B activation increased the aggressiveness, enhanced tumorigenesis, drug resistance, and EMT in cisplatin-resistant bladder cancer cells [70]. In colon cancer cells, chemoresistance to 5-FU have been found to be significantly linked to NF-кB activation [71]. In CD133 positive osteosarcoma cells, DNA-dependent protein kinase C (DNA-PKC) downregulation reduced Pgp expression and induced cisplatin chemosensitivity via decreasing the Akt/NF-B pathway [72]. Furthermore, it has been demonstrated that the AKT/NF-B signaling pathway is the primary mechanism by which CD133 controls MDR1/P-gp expression in colorectal cancer [73]. The MDR of nasopharyngeal cancer cells could be controlled by activating the PI3K/Akt/NF-B signal pathway, subsequently enhancing P-gp and LRP expression. Thus, by blocking the PI3K/Akt/NF-B signal pathway, the MDR of nasopharyngeal cancer cells can be reversed [74]. A gene expression profiling and pathway analysis study on patients with estrogen receptorepositive breast tumor demonstrated that the NF-кB pathway was activated and EMT/stemness characteristics were enriched before and after neoadjuvant tamoxifen therapy. In estrogen receptorepositive breast tumors, the NFк-B pathway increases tamoxifen resistance and disease recurrence [75].

3.1.2.4

mTOR signaling pathway and MDR

The mechanistic target of rapamycin kinase (mTOR) is a major factor of cancer MDR. The mTORC1 and mTORC2 are two distinct mTOR complexes, incorporating in a diverse set of environmental signals like growth factors and nutritional factors, to guide cellular metabolism and growth [76e78]. In a growth-promoting milieu, mTOR directs the metabolism of cell toward higher protein, lipid, and nucleotide synthesis while inhibiting catabolic pathways. mTOR, in particular, suppresses autophagy, a process in which intracellular components are enveloped in bilayer membraned vesicles called autophagosomes, eventually merging with lysosomes, where the contents are destroyed and recycled into the cytoplasm [79]. Autophagy is triggered in response to nutrient deprivation and metabolic stress, for example, by AMPK (AMP-activated protein kinase), phosphorylating ULK1(Unc-51 Like Autophagy Activating Kinase 1) in reaction to cellular energy reduction [80,81]. mTORC1 additionally phosphorylates ULK1, this phosphorylation is suppressive and inhibits ULK1 activation by AMPK and thus prevents autophagy in nutrient-replete conditions [81]. Despite the fact that only a small number of malignancies have mutations that activate mTOR gene constitutively or direct regulators, mTOR complexes are critical effectors of the most frequent oncogenes including Ras/MAPK and PI3K/AKT pathways [82,83]. As a result, persistent mTOR signaling leads to MDR as seen in melanoma, lung cancer, and breast cancer [76,84e86]. Furthermore, mTOR is linked to chemotherapy resistance, through activating the Fanconi anemia DNA repair pathway, which resolves toxic crosslinks between DNA strand caused by platinum drugs such as cisplatin [87,88] Accordingly, mTOR inhibitors are suggested as a beneficial complement to chemotherapy or targeted cancer therapy to avoid or delay the development of MDR owing to prolonged mTOR signaling [76,77].

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EGFR signaling pathway and MDR

Epidermal growth factor receptor (EGFR), also recognized as ErbB-1 or Her-1, the product of the proto-oncogene C-erbB-1 expression, is a 170-kDa transmembrane TK receptor involved in homeostatic control of normal cells and inducing epithelial malignancies [89]. EGFR can activate PI3K/Akt/mTOR, JAK/STAT3, SOS/Grb2/ Ras, and src/FAK/ROS SOS/Grb2/Ras pathways, thus involving in cell proliferation, differentiation, transformation, and survival. Overexpression of EGFR leads to NF-кB activation and STAT3, resulting in MDR and a poor prognosis. This should be mentioned that EGFR inhibitors including erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, and rociletinib are presently used for the treatment of various types of cancers [90]. The mutant form of EGFR has been reported to be still active and induces MDR in lung cancer and glioma. Gefitinib, on the other hand, targets mutant EGFR in lung cancer and inhibits the activation of EGF- and HER3-mediated Akt MDR phenotype in cells [91]. Cisplatin resistance has been linked to heme oxygenase (HO)-1 via the EGFR-mediated PI3K/Akt and NF-кB pathways in lung cancer cells, which may be reversed with an Akt or EGFR selective TK inhibitor [92]. In non-small-cell lung cancer (NSCLC) with wild-type EGFR, resistance to cisplatin, paclitaxel, gemcitabine, and pemetrexed is the result of continuous therapy with an EGFR TK inhibitor [93]. According to further research, the hippo coactivator YAP1 upregulated the EGFR and induced 5-FU and docetaxel resistance through an intact TEAD-binding site in the EGFR promoter [94]. It has been shown that CD133 overexpression or CBL suppression of the E3 ubiquitin ligase, mediated MDR via stabilizing EGFR-Akt signaling or EGFR activation, respectively [95,96]. Mutations within TK domain of EGFR gene that activate it are seen in 10%e30% of NSCLC patients and are directly linked to the disease’s onset and development [97]. The overexpression of mitotic regulator, never in mitosis gene A-related kinase 2 (NEK2), is highly associated with EGFR activation and poor survival of EGFRmutant patients but not the patients with wild-type gene. The EGFR mutation causes NEK2 expression through stimulating the ERK signaling pathway. NEK2 overexpression increases accelerated cell cycle progression and proliferation in EGFR-mutant NSCLC cells, as well as impairing the efficiency of TK inhibitor therapy by suppressing apoptosis, whereas NEK2 depletion inhibits cell growth and restores TK inhibitor sensitivity in NSCLC cells [98]. Desmoglein-2 (DSG2) belongs to the cadherin superfamily and its overexpression in lung adenocarcinoma (LUAD) cell lines and tissues results in poor prognosis in cancer patients. Overexpression of DSG2 enhanced cell proliferation and migration, as well as increases resistance to the osimertinib EGFR TK inhibitor, while DSG2 knockdown reverses these effects. Furthermore, direct contact between DSG2 and EGFR in the cell membrane increases EGFR signaling, leading to the cancer progression, while DSG2 depletion contributes EGFR cytoplasmic translocation. Additionally, DSG2 is also needed for EGFR binding to Src. As a result of the inhibition of the EGFR-Src-Rac1PAK1 signaling pathway, DSG2 silencing reduces tumor cell aggressiveness [99]. Accordingly, EGFR has an essential and complex role in MDR mechanisms of cancer.

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3.1.3

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The role of autophagy in cancer MDR

Autophagy is one of numerous ways whereby cells maintain homeostasis and is controlled by more than 30 genes. Cancer development and MDR are aided by autophagy. Beclin-1, HMGB1, MIF, PTEN, p53, NF-2, MEG3, p62, RAC3, SRC3, LAPTM4B, mTOR, c-MYC, and BRAF are only a few of the autophagy-associated genes, involving in cancer initiation, progression, and drug resistance. Cell growth, cellular microenvironment, and cell division are all affected by these genes [100]. Autophagy is activated when cells have been exposed to nutritional deprivation, hypoxia, LPSs, or chemotherapy to breakdown damaged organelles or particles within the cell and recycle fatty acids or amino acids by autophagosome generation. Three types of autophagy include microautophagy, chaperone-mediated, and macro autophagy. Microautophagy envelops cytoplasmic molecules directly via lysosomal membrane engulfment. Chaperone-mediated cytosolic protein transport to lysosomes via the lysosomal LAMP-2A receptor, leading to invagination and destruction. Macroautophagy is the process through which phagophores sequester cytoplasmic molecules, resulting in the development of autophagosomes. Subsequently, they fuse with lysosomes to generate “autolysosomes” and degrade their cargo [101]. Increased metabolic demand or cellular stress activate autophagy is that can promote cell survival while also causing cancer progression and MDR. Thereby, utilizing hydroxylchloroquine or its derivatives inhibit autophagy, restores chemosensitivity, and enhances cancer cell death [102]. Autophagy defects also contribute to MDR in cancers such as osteosarcoma, ovarian, and lung carcinomas after chemotherapy with doxorubicin, paclitaxel, cisplatin, gemcitabine, and etoposide drugs [100]. Well over 30 autophagy-related genes (ATG) are involved in the regulation of autophagy [103]. With changes in Bcl-2 family proteins and PMAIP1/NOXA mRNA overexpression, Atg7 suppression increased sensitivity to genotoxic agents in AML. Aquaporin 3 has a key role in resistance to cisplatin via transforming LC3 (Microtubule-associated protein light chain) -I-to LC3-II and overexpression of Beclin 1 and Atg5 in gastric cancer cells, and autophagosomal heparanase contributed to autophagy and MDR. Chloroquine has been found to reverse both of them [104,105]. Autophagy has been shown to induce chemosensitivity in HCC under hypoxic condition, which can be inhibited by 3-Methyladenine (3- MA) or Beclin 1 siRNA [106,107]. In colon cancer cells, CD44v6 upregulation induced acquired resistance to 5-FU and oxaliplatin chemotherapy by triggering autophagy and EMT, as well as activating the Ras/Erk and PI3K/Akt signal pathways [108]. In neuroblastoma, Beclin 1 was found to be overexpressed and a combination of hydroxychloroquine and vincristine remarkabely slowed the progression of the disease. Also, in bladder cancer and osteosarcoma cells treated with cisplatin treated with gossypol, resistance to chemotherapy was associated with increased autophagy and decreased apoptosis [109,110]. Furthermore, in bladder cancer cells, capsaicin caused autophagic cell survival and induced EMT and MDR in a Hedgehog-dependent manner [111]. It has been reported that cisplatin activated the NFKB1/NF-B pathway, causing GFRA1 (GDNF Family Receptor Alpha 1) expression and AMPK-dependent autophagy [110]. Furthermore, doxorubicin, cisplatin, and etoposide increased autophagy in

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neuroblastoma cells by inducing HMGB1 (High mobility group box protein 1) expression and cytosolic translocation. increased levels of HMGB1 also facilitated MDR in neuroblastoma cells by inducing autophagy mediated by Beclin-1 [112]. Through the PI3K/ Akt/mTORC1 pathway, HMGB1 increased starvation-dependent autophagy in leukemia cells, while 3-MA decreased autophagy and MDR in leukemia cells [113]. The results of an experiment by Gremke et al. link mTOR-induced MDR to autophagy defects as a result of a metabolic liability. Tumor cells with acquired MDR are more sensitive to mechanistically distinct cancer metabolism inhibitors. They demonstrated that autophagy is required for tumor cells to deal with therapeutic metabolic perturbation and that inhibition of mTORC1-mediated autophagy is needed and adequate for inducing a metabolic vulnerability resulting in energy crisis and apoptosis [114].

3.1.4

The role of EMT in cancer MDR

The transition of tumor cells from nonmetastatic form to metastatic form is induced by a reversible biological mechanism that causes cancer metastasis. Epithelial cells are changed into mesenchymal cells, elevating invasive properties that is because the loss of intercellular adhesion and thus increased motility. EMT, formerly known as epithelial to mesenchymal transformation, is the name given to this process [115,116]. Apical to basal polarity is a feature of epithelial cells that allows them to tightly position themselves on a basement membrane by intercellular junctions [117]. Cancer cells lose cellular adhesion and achieve higher motility and invasiveness as a consequence of EMT. Activation of transcription factors, expression of particular cell surface proteins and cytoskeleton proteins, generation of enzymes, degrading extracellular matrix, and alteration in the expression of specific types of microRNAs are all required for EMT to begin [118]. Furthermore, cells that have undergone EMT are frequently resistant to senescence and apoptosis. The pattern of gene expression in EMT is regulated by EMT-transcription factors (EMT-TFs) such as Twist1, Snail, and the zinc-finger E homeobox-binding 1(ZEB1) either directly or indirectly [119]. EMT induction by EMT-TFs is linked to cancer stem cell phenotype, invasion, propagation, and metastasis [120]. Moreover, EMT-TFs are associated with MDR, radiation, and targeted therapy resistance [121]. Twist1, Snail, and ZEB1 are three primary EMT-TFs linked to MDR as well [122]. According to experiments, Snail is involved in resistance to 5-Fu, cisplatin, and doxorubicin [123e125]. Twist is involved in resistance to cisplatin, doxorubicin, epirubicin, erlotinib, and paclitaxel [126e128]. ZEB1 is also associated with resistance to 5-Fu, cisplatin, doxorubicin, butyrate, EGFR TK inhibitor, oxaliplatin, and epirubicin [122]. Multiple intracellular signaling pathways regulate EMT-TFs. receptor TK, TGF- receptor, Frizzled, and Notch are receptors for growth factors, transforming growth factor beta (TGF-b), Wnt, and the Jagged family bind to respectively, thus initiating intracellular signal transduction [129]. By binding receptors to their ligands, receptors transmit intracellular signals through pathways that regulate the expression and stability of EMT-TFs such as PI3K/Akt, NF-кB, b-catenin, Smad or the mitogen-activated protein kinase (MAPK) signaling pathway [119]. EMT-TFs inhibit epithelial markers like E-cadherin, cytokeratins, and tight junction proteins while inducing mesenchymal

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markers like matrix metalloproteinases (MMPs), N-cadherin, vimentin, fibronectin, lethal giant larvae protein homolog 1/2 , and a-smooth muscle actin [115,129]. Ecadherin deficiency is the most reliable indicator of cancer stem cell phenotype and MDR [130,131]. This coordination of intracellular pathways and gene expressions in relation to EMT-TFs is critical for the EMT process and MDR. GINS2 (Go-Ichi-Ni-San 2) is a recently discovered oncogene that is upregulated in a variety of cancers such as pancreatic cancer. GINS2 overexpression induced EMT by promoting cellular morphology of EMT, increasing cell motility, and inducing the EMT and ERK/MAPK signal pathways, all of which make a contribution to the advanced clinical stage of pancreatic cancer patients and promote MDR [132].

3.1.5

The role of cell cycle events in cancer MDR

Unrestrained cell proliferation is a key feature of cancer cells arising from defects in the cell cycle progression at the G1, G2, S, and mitotic phases. Checkpoints of cell cycle, which include a network of protein kinase signaling pathways, protect cells from chemotherapeutic-induced DNA damage and give them enough time to fix the damage [133]. Thereby, cell cycle checkpoints defects could lead to cancer initiation and MDR. The G2 phase cell cycle arrest of allows cells to protect their viability following drug treatment. This arrest necessitates the activation of DNA damage checkpoint elements like the major checkpoint kinase, Chk1 [134]. It was discovered that activating the Chk1 kinase pathway could postpone entrance to mitosis while enhance the cells’ ability to survive and that a lack of cellular Chk1 could make cells sensitive to drugs hypersensitivity that cause DNA damage [135]. MDR-associated cell cycle events are most frequent in combination therapies, where the first drug may influence the cell cycle, making the next drug less effective [136]. The checkpoint pathways are important for chemotherapy as well as radiotherapy sensitization because they cause a delay in the cell cycle at the G1, S, and G2 phases, potentially permitting for the repair of damaged DNA. The ataxia-telangiectasia mutated (ATM) is a checkpoint protein that is activated when the DNA strand breaks. Next, CHEK2 phosphorylate and activate the kinase activity of ATM, resulting in phosphorylating both p53 and MDM2. Likewise, ATM and Rad3 related (ATR) activates CHECK1 signaling in response to single-strand DNA breaks [137]. Cell cycle arrest in response to DNA damage can be disrupted by p53 mutations, which play an important role in the ATM/CHEK2/p53 pathways. Because of their critical roles in the regulation of cell division cycle protein-2 (Cdc-2), CHECK1 and CHECK2 have potent antitumor characteristics. Thus, suppression of these two proteins can improve cytotoxicity and DNA damage sensitivity in malignant cancer cells [137]. As a result, preventing DNA repair by targeting the cell cycle checkpoint could be a potential cancer treatment and reversal of MDR.

3.1.6

The role of apoptosis in cancer MDR

Apoptosis is a type of programmed cell death that is important for the cell development, tissue homeostasis, and defense against unwanted and possibly hazardous cells

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Figure 3.3 Extrinsic and intrinsic pathways of apoptosis and the way that these pathways could be inhibited due to cancer multidrug resistance.

[138]. MDR is linked to apoptosis resistance (Fig. 3.3) and consequently, poor clinical prognosis [139,140]. Extrinsic or death receptors pathway and intrinsic or mitochondrial signaling pathway signals can both trigger apoptosis by causing a synchronized activation of a family of cysteine proteases known as “caspases” [141]. Fas ligand (FasL) and TNF interact with their respective apoptotic receptors namely FAS receptor (CD95) and TNF receptor (TNFR), respectively, to initiate the extrinsic apoptotic pathway [142,143]. Various apoptotic signals, including oxidative damage, DNA damage, withdrawal of growth factor, loss of cell adhesion, and steroid hormones, activate the mitochondrial pathway. This pathway is the primary mechanism for removal of cells during normal development of organisms [144]. Cancer cells can avoid apoptosis by turning off pro-apoptotic genes like P53 and BAX, or by inhibiting their function [145,146] and by overexpression of antiapoptotic proteins like Bcl-2 [147]. Elevated levels of antiapoptotic proteins that are essential for cell survival might be potentially addictive for cells [148]. Antiapoptotic proteins overexpression is caused by several mechanisms include 1) genetic changes at the DNA level (mutations, translocation), 2) RNA alterations (alternative splicing, miRNAs), and 3) protein regulation via adjusting the stability of antiapoptotic protein. Therefore,

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understanding these mechanisms can help guide therapeutic approaches that target proteins involved in apoptosis to overcome MDR [149]. P53, and Bcl-2 family are pro- and antiapoptotic proteins involve in MDR that their role is discussed below.

3.1.6.1

p53 and MDR

TP53 tumor suppressor gene regulates the transcription of target genes and induces cell cycle arrest or apoptosis in response to DNA damage. Over half of all human cancers have TP53 mutations that cause loss of function. The emergence of MDR and the rejection of cancer chemotherapy are linked to loss of function mutations in p53 gene [150]. Cisplatin is one of the most often utilized anticancer medicines in clinic cancer therapy [151,152]. Multiple factors that are involved in the development of cisplatin drug resistance are regulated by p53. The Nrf2, a key transcription factor, makes a significant contribution to anticancer drug resistance. In this regard, Nrf2 is significantly suppressed by wild-type p53 and overexpressed by mutated p53. Mutant p53 has recently been discovered to tune the Nrf2-dependent antioxidant response, while also promoting cancer cell survival. The gain of function mutant p53-Nrf2 axis was also discovered to overexpress the proteasome and immunoproteasome machinery in cancer, conferring resistance to inhibitors of proteasome [153]. By regulating Nrf2, p53 regulates the expression of the apoptosis regulator BCL2, BCL2-like 1 (Bcl-XL), and hemeoxygenase 1 (HO-1), leading to Nrf2-mediated cisplatin resistance. A research indicates that when p53 was present in high concentrations in the cytoplasm, it had a strong suppressive activity on cleaved caspase-9 [154]. The mutations R248Q and R273C in the p53 gene have been linked to the development of MDR to doxorubicin and paclitaxel [155]. Lung carcinoma resistance to paclitaxel that is developed as a result of upregulation of the p38 MAPK-EGFR signaling pathway could be overcome by suppression of p38 MAPK or EGFR activity through induced MDM2 degradation and p53 stabilization. It indicates that the p38-MAPK-p53-EGFR pathway has an important effect on resistance to paclitaxel in NSCLC [156]. Resistance to Paclitaxel, 5-fluorouracil, Temozolomide, Etoposide, Idasanutlin, Carfilzomib, Capsaicin, and Apigenin has also been investigated to be linked to the P53 loss of function mutations [157].

3.1.6.2

Bcl-2 family and MDR

The Bcl-2 family comprise of both proapoptotic proteins like Bax, Bak, and Bid and antiapoptotic proteins like Bcl-2, Bcl-xl, and Mcl-1 [147,158]. Bcl-2 overexpression has been found to be increased in leukemias, lymphomas, breast carcinomas, prostate carcinomas, and glioblastomas [149,159]. Furthermore, in patients with diffuse large B cell lymphoma and bladder cancer, upregulation of Bcl-2 is a poor prognostic indicator for chemotherapy and radiation therapy [139]. Enhanced Bcl-2 levels can be attributed to both increased transcription and increased protein stability. Genetic changes, miRNA expression, or reduced activity of E3-ligases that enhance proteasomal degradation of Bcl-2 are among the underlying mechanisms. Bcl-2 upregulation was first

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identified in follicular lymphoma by cause of a [8,14] chromosomal translocation [160]. Bcl-2 inhibits apoptosis associated with c-myc while dose not inhibit the progression of cell cycle, resulting in increased cancer progression [161]. A method for increasing Bcl-2 stability is to eliminate the proapoptotic ARTS protein. The Sept4 gene encodes ARTS, acting as a scaffold to bring Bcl-2 into near vicinity with XIAP (X-Linked Inhibitor of Apoptosis). With its E3-ligase activity, XIAP ubiquitylates Bcl-2 and facilitates its proteasomal degradation [162]. Bcl-xl activation is linked to tumor progression, reduced survival rates, and MDR in many cancers. Colorectal cancer, liver cancer, non-Hodgkin lymphoma, and chondrosarcoma all have high levels of Bcl-xl expression [149,163,164]. A further member of Bcl-2 antiapoptotic family protein is Mcl-1 that is overexpressed in several cancers, including prostate carcinoma, leukemia, HCC, pancreatic cancer, and oral cancer and lead to MDR [149]. As a result, antiapoptotic Bcl-2 proteins could be used as potential therapeutic to restore apoptosis in cancers with MDR phenotype.

3.1.7

The role of DNA repair mechanisms in cancer MDR

Following a strict cell cycle regulation, DNA damages are repaired in G1/S, intra S, and G2/M phase checkpoints in normal cells, thus fixing DNA damages or undergo apoptosis, whereas cancer cells circumvent the strict control system and have mutations in DNA repair system [59]. Some anticancer agents, such as platinum, directly induce DNA damage, whereas others such as irinotecan, doxorubicin damage the DNA indirectly through suppression of topoisomerase enzyme. The effectiveness of chemotherapy agents is affected by the capability of some cancer cells to repair DNA damage [60]. A mechanism by which the MDR in cancer cells is reversed directly or indirectly is called DNA damage response (DDR) and the amount of chemotherapeutic sensitivity is determined by the strength of this mechanism in the cells. In many malignancies, cancer cells witnessed control problems or the degradation of certain genes in DDR pathways due to mutation or epigenetic silencing [61]. Various DDR mechanisms can be activated to correct the faulty route, and enhance the DNA repair activity and subsequently, increase MDR [61]. One of the nucleotide excision repair (NER) members that is responsible for repairing DNA damage induced by platinum drugs, is excision repair cross-complementing 1 (ERCC1). It is overexpressed in several cancers such as colorectal, breast cancer, esophageal squamous cell carcinoma, and others [62]. The regulatory effect of ERCC1 on 5-FU resistance in gastric cancer has been investigated and it has been proposed that ERK 1/2 and p38 signaling controlled ERCC1 expression through stimulation of the transcription factor c-jun/activator protein (AP)1 [63]. Besides the resistance to cisplatin mediated by ERCC1 in lung cancer, focal adhesion signaling and PDGF/PDGFR may also play a role [64]. Mismatch repair (MMR) systems are another repair system necessary for preserving genomic integrity, and mutations in the MMR gene can induce microsatellite instability. In this regard, resistance to certain platinum medications is caused by MMR deficiency and mutations in MMR genes [61].

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The role of microRNAs (miRNAs) in cancer MDR

MicroRNAs (miRNAs) are noncoding RNAs, some of which are implicated in MDR mechanisms. MiRNAs are short noncoding RNAs comprise of 18e22 nucleotides that bind to the 30 -untranslated region (30 -UTR) of their target mRNAs and mediate posttranslational degradation and proteins down-regulation, fine-tuning various cell processes to preserve homeostasis [65,66,165]. MiRNAs have been examined as a probable explanation for MDR mechanisms offered by evidence of their participation in almost all cancer hallmarks due to their essential function in cellular processes [166]. The miRNAs-mediated MDR mechanisms have been linked to various biological processes such as apoptosis, cell cycle modification, EMT, cancer stem cells, changes in drug targets, and drug efflux transporter modulation (Fig. 3.4) [167e172]. Numerous areas of research are examining the application of miRNA antagonists (e.g., locked nucleic acids and anti-miRs) for both silencing miRNAs with oncogenic potential and for re-expression of miRNAs that undergo reduced expression because of genomic, epigenetic, and transcriptional mechanisms and resulted in MDR [173,174].

Figure 3.4 The impact of miRNAs on biological processes involved in cancer multidrug resistance including apoptosis, cell cycle modification, epithelial to mesenchymal transition (EMT), cancer stem cells (CSCs), changes in drug targets, and drug efflux transporter modulation.

3.1.9

The role of inflammation and growth factors in cancer MDR

Acute inflammation promotes tumor elimination; however, a prolonged immune response promotes tumor development and invasion, according to the results of cumulative experimental and clinical evidence. In MDR cancer cells, autocrine synthesis of

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growth factors such as IL-1, IL-4, IL-6, and IL-8 are increased compared to drugsensitive cancer cells [175e177]. By elevating the expression of ABCB1 gene and activation of the CCAAT enhancer-binding protein family, IL-6 has been shown to regulate a variety of biological processes including cell proliferation, differentiation, metabolism, and death [175]. Moreover, compelling evidence shows a link between the activity of IL-6 in cancer-related fibroblasts occurring in MDR and the tumor stroma in gastric cancer cells [176]. According to the findings, IL-6 is a chromatin assembly factor-1 (CAF)specific secretory protein that confers MDR in gastric cancer cell via paracrine signaling. Furthermore, using tocilizumab, a monoclonal antibody against IL-6 receptor, restored the apoptosis inhibition induced by CAF in both in vitro and in vivo experimental settings. Therefore, IL-6 inhibitors have potential therapeutic application in reversal of MDR of gastric cancer cells. Wang et al. discovered a strong link between IL-8 overexpression in ovarian cancer tumor tissue, serum, cyst fluid, and ascites and low sensitivity to a range of anticancer drugs utilized during cancer treatment in patients. As investigations highlighted, MDR in ovarian cancer cells mediated by increased IL-8 expression, activation of Ras/MEK/ERK and PI3K/Akt signaling and overexpression of ABCB1 efflux pump, overexpression of apoptosis inhibitors such as XIAP, Bcl-xL, and Bcl-2 proteins, while decreasing the proteolytic activation of caspase-3. This is why modulating the IL-8 expression might be a possible MDR ovarian cancer therapeutic approach [175]. MDR in cancer can be boosted by both intracellular factors and elevation in extracellular fibroblast growth factors (FGFs) levels presented in solid and metastatic tumor medium. Drugs with various modes of action such as 5-FU, doxorubicin, and paclitaxel, were found to be ineffective against tumors with high amounts of these extracellular factors. Song et al. used suramin, a known inhibitor of these factors, to demonstrate the importance of FGFs in the development of cancer MDR. Suramin effectively reversed the ten-fold increased resistance witnessed in a combination of intracellular and extracellular factors [178]. In an in vitro analysis on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells substantial correlations between the FGFs function (for instance, FG2, FGF9, and FGF10), and proliferation, apoptosis, and drug sensitivity have been reported in a cell-specific manner [179].

3.1.10 The role of cancer stem cells in cancer MDR Cancer stem cells, also defined as tumor-initiating cells (TICs), are a subset of cancer cells having self-renewal feature and develop in the way as normal stem cells do. CSCs can contribute to cancer progression and relapse [180,181]. Furthermore, increased expression of drug efflux pumps and other genes leads to inherent resistance of CSCs to chemotherapy [182,183]. Because CSCs have a slower cell-cycle kinetics than rapidly dividing cells, chemotherapy medicines are less effective against them [184]. Besides to the well-known ABC efflux pumps, several other MDR mechanisms have been discovered that highlighted the role of CSCs in MDR, including aldehyde dehydrogenases (ALDHs), epigenetic changes, EMT, variables impacting the tumor microenvironment, like hypoxia, and signaling pathways [185e189].

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ALDH is a stem cell biomarker because it helps differentiate stem cells by changing retinol to retinoic acid and detoxifies them by aldehyde to carboxylic acid conversion [185]. ALDH-1 has been linked to CSCs in a number of studies. Breast, lung, and other malignancies are managed by gefitinib, an EGFR inhibitor. ALDH1A1-expressing CSCs, on the other hand, have been shown to be more resistant to gefitinib than ALDH1A1-negative ones [102]. ALDH-1 production is controlled by high levels of Snail, a transcription factor that controls metastasis and, thereby, promotes CSCs to acquire resistance to chemotherapy [190]. One of the most prevalent posttranslational changes, histone acetylation, is linked to MDR in CSC. The acetylation of lysine residues is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). As a result, inhibiting histone acetyltransferases, inhibits cell growth, and causes apoptosis Furthermore, hypermethylation of promoter regions of DNA silences tumor suppressor genes, rendering chemotherapy ineffective against many malignancies [191]. Other important signaling pathways involved in MDR of CSC are Notch, Hedgehog (Hh), and Wnt/-catenin signaling pathways [192]. Therefore, this is obvious that by identification of exact role of each of these pathways in cancer MDR, suitable therapeutic approaches can be designed.

3.1.11

The role of exosomes in cancer MDR

Exosomes are formed when the membrane of an endosome buddings inward, resulting in the development of multivesicular bodies, which then undergo fusion with the plasma membrane to release exosomes [193,194]. MDR can be regulated by exosomes interacting directly with drugs, altering the transcriptome of cancer cells, and affecting the immune response. Exosomes can affect various molecular mechanisms involved in cancer drug resistance that are mentioned in this chapter. The contents of exosomes, including nucleic acids, proteins, lipids, and metabolites, determine their involvement in MDR [195]. A rising number of researches on the function of miRNAs in MDR have been published. Exosomal miRNAs cause treatment resistance in the majority of instances. Exosomal miRNAs generated from drug-resistant tumor cells can give susceptible cells the resistant phenotype. Exosomal miR-100-5p and miR-222-3p from NSCLC cells, for instance, target the mammalian target of rapamycin and the suppressor of cytokine signaling, respectively, promoting MDR in recipient cells [196,197]. Exosomal mRNA and DNA are areas with few investigators, yet they play important roles in MDR induction. Exosomal O-6-methylguanine-DNA methyltransferase (MGMT) mRNA has a crucial function in Temozolomide (TMZ) resistance in CNS malignancies. In vitro and in vivo, normal human astrocytes can protect glioma cells from TMZ-induced death by releasing exosomal MGMT mRNA [198]. MDR is influenced significantly by protein absorbed into exosomes. Exosomal proteins are classified as enzymes, transcription factors, membrane proteins or receptors, and secretory proteins like cytokines based on their methods of action in generating MDR. For example, heparanase expression was recently revealed to be increased in multiple myeloma cells that had withstood treatment. It was found that exosomes

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containing heparanase enhanced the breakdown of heparan sulfate, culminated in the activation of ERK signaling and shedding of syndecan-1 [199].

3.1.12 Drug inactivation and cancer MDR The major drug-metabolizing enzymes include cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), dihydropyrimidine dehydrogenases. Based on the tissue in which they are expressed, these enzymes play a remarkable role in xenobiotic detoxification as well as drug metabolism. They induce resistance by metabolizing chemical compounds and making them inert when found in tumor tissues [200]. Found in the endoplasmic reticulum membrane, CYPs are a diverse class of enzymes. They are terminal oxidases from the heme-thiolate multigene family. CYPs require molecular oxygen and a connection to NADPH reductase. They participate in oxidative processes that are important in the metabolism of many commercialized therapeutics [201]. Cytochrome P450s have developed to defend organisms from toxic chemicals [202]. CYPs are involved in the xenobiotics detoxification and a variety of endogenous substances such as steroids, bile acids, leukotrienes, prostaglandins, and unsaturated lipids [203]. Many studies have found that the CYP3A and ABCB1 transporters have the same substrate specificity [204]. By lowering the concentration of active drugs in both systemic circulation and target cells, this combinational mechanisms may develop to MDR [205]. GSTs are members of the detoxifying enzyme family that preserves cellular macromolecules from reactive electrophile assault [204]. GST expression in cancer cell lines renders them resistant to treatments, contributing to the concept of using GSTs as a tumor progression marker. GSTP1 expression is strongly linked to the development of drug resistance in tumor cell lines. The substrates of GST are the alkylating compounds that function as anticancer agents. In addition to their detoxifying function, GSTs regulate apoptosis by inhibiting JNK1, a kinase implicated in apoptosis, stress response, and cell proliferation [204]. The activity of the Nrf2 raises the amount of GST. Nrf2 is responsible for the transcription of antioxidative genes like GST in the nucleus. The sulfur atom of GSH on the electrophilic group of numerous xenobiotics is attacked by nucleophilic assault of GST, leading to decrease their activity. As a result, GSTs’ catalytic activity may be important in the detoxification of chemotherapeutic drugs including cyclophosphamide, chlorambucil, ifosfamide, melphalan, cisplatin, carmustine, busulfan, and mitoxantrone, thiotepa [201]. DPD (dihydrouracil dehydrogenase, dihydrothymine dehydrogenase, or uracil reductase) is a catalytic enzyme that catabolizes nitrogen-containing compounds such as pyrimidines uracil and thymine. DPD is also essential in 5-FU metabolism. It is the most important enzyme in the catabolism of 5-FU, metabolizing around 85% of the dose into the molecule 5-fluoro-5,6-dihydrouracil (5-FUH2). Anabolism of 5-FU and consequently total metabolism are reduced as a result of its inactivation. Thus, increasing DPD levels in malignancies result in drug resistance based on nitrogen heterocycles [206].

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Aptamers Engineered Nanocarriers for Cancer Therapy

Alterations in drug targets and decreased drug uptake and cancer MDR

The biological target of chemotherapeutics might be changed or even lowered or raised to the point where it loses therapeutic value. Patients develop resistant to endocrine therapy during antiestrogen (e.g., tamoxifen) therapy for breast cancer because of evident loss of estrogen receptors in resistant tumor cells that were thought to be no longer dependent on estrogen for their proliferation [207]. Another method to reduce drug accumulation in cancer cells is to reduce drug uptake. Drugs enter cells by a variety of ways, including diffusion through the plasma membrane, drug loading on particular receptors, and nonspecific or receptormediated endocytosis [3]. Mutations that block or alter receptors may lead to the development of MDR in some cancer cells. Endocytosis, specifically receptor-mediated endocytosis, is also recognized to play a crucial role in the transport of certain therapeutics into cells, and a dysfunctional endocytic pathway leads to drug resistance [3,8].

3.3

Conclusion

Studies looking into the molecular mechanisms of MDR clearly indicate that anticancer drug resistance is caused by a variety of factors. In this chapter, we have covered the most important mechanisms. The development of novel highthroughput and multiplex screening technologies has facilitated the identification of intrinsic, and extrinsic cellular pathways, contributing to MDR in cancer. Using knockdown techniques or chemical agents to target key genes encoding membrane transporters or other proteins that contribute to the development of the MDR phenotype has a great potentiality in the cancer treatment. These strategies would allow for the reversal of MDR, and cancer patients would be treated entirely. Clinical methods, on the other hand, require more in vivo research and larger clinical trials. More significantly, potentially innovative therapeutic targeting and delivery methods with minimum side effects must be developed. These strategies help to target and eradicate cancer cells with MDR phenotype rather than normal cells. However, the FDA has not yet authorized any new effective pharmaceuticals for reversing chemoresistance. Despite this, the complex and redundant nature of tumor chemoresistance continues to be a major impediment to the development of safe and effective approaches to reverse MDR. It seems unlikely that we will be able to conquer chemoresistance in the near future until we have completely explained all of the factors that regulate and govern it.

Competing interests None of the authors has a direct conflict of interests with respect to the subject of this review. Funding None.

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[200] G. Kaur, S.K. Gupta, P. Singh, V. Ali, V. Kumar, M. Verma, Drug-metabolizing enzymes: role in drug resistance in cancer, Clinical and Translational Oncology 22 (10) (2020) 1667e1680. [201] N. Penner, C. Woodward, C. Prakash, Drug metabolizing enzymes and biotransformation reactions, in: D. Zhang, S. Surapaneni (Eds.), ADME-Enabling Technologies in Drug Design and Development, John Wiley & Sons, Inc, Hoboken, NJ, 2012. [202] F.P. Guengerich, M.R. Waterman, M. Egli, Recent structural insights into cytochrome P450 function, Trends in Pharmacological Sciences 37 (8) (2016) 625e640. [203] S. Rendic, F.P. Guengerich, Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of xenobiotic and natural chemicals, Chemical Research in Toxicology 28 (1) (2015) 38e42. [204] D.M. Townsend, K.D. Tew, The role of glutathione-S-transferase in anti-cancer drug resistance, Oncogene 22 (47) (2003) 7369e7375. [205] L. Huang, S.A. Wring, J.L. Woolley, K.R. Brouwer, C. Serabjit-Singh, J.W. Polli, Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors, Drug Metabolism and Disposition 29 (5) (2001) 754e760. [206] S. Pathania, R. Bhatia, A. Baldi, R. Singh, R.K. Rawal, Drug metabolizing enzymes and their inhibitors’ role in cancer resistance, Biomedicine & Pharmacotherapy ¼ Biomedecine & Pharmacotherapie 105 (2018) 53e65. [207] Y.A. Luqmani, Mechanisms of drug resistance in cancer chemotherapy, Medical Principles and Practice: International Journal of the Kuwait University, Health Science Centre 14 (Suppl. 1) (2005) 35e48.

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Payam Bayat 1 , Aida Gholoobi 2, 7 , Khalil Abnous 3 , Seyed Mohammad Taghdisi 4 , Peyman Asadi 5 and Rezvan Yazdian-Robati 6 1 Faculty of Medicine, Department of Immunology and Allergy, Immunology Research Center, BuAli Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 2 Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 3Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 4Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 5Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 6Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 7Metabolic Syndrome Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

4.1

Introduction

Aptamers (chemical antibodies) as a new class of molecular targeting agents are, single-stranded DNAs or RNAs with short lengths (15e100 nt) screened through a repetitive approach called SELEX (systematic evolution of ligands by exponential enrichment) technology [1,2]. In This cell-free process the aptamers are isolated from a library of nucleic acids against different targets and molecules such as purified proteins, antibiotics, and whole cells, basically in three steps including (1) Binding (2) Elution and (3) Amplification. In this process, nonbinding oligonucleotides (DNAs or RNAs) are removed by several washing steps (Fig. 4.1) [3]. Aptamers through hybridization of complementary sequences can be easily folded into tertiary or quadruplex structures and further form three-dimensional spatial structures. Aptamers are known for their highly selective binding ability to the predefined target, mirroring interaction antigen/antibody, through different forces, such as hydrogens bonds, electrostatic attraction as well as van der Waals, [4]. Importantly, aptamers can cross the bloode brain barrier (BBB) as the main barrier for transferring the drug into the brain [5]. Compared with antibodies, aptamers have the following positive properties: stronger affinity, greater specificity, simple preparation and modification, lower toxicity, small size, good stability and reproducibility lower molecular weight as well as easy to store [6]. The ability of aptamers to specially bind to broadly targets such as living cells or tissues, applicable to them either as a therapeutic agent or as a targeting ligand for Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00017-8 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Figure 4.1 Schematic illustration of SELEX process. Adapted from the published work [7].

diagnosis, imaging analysis or targeted therapy. However, the therapeutic efficiency of aptamers for the treatment of diseases can vary, because they were introduced by different researchers via SELEX process. Treatment of various cancer using aptamers is the most interesting field for the majority of Scientifics. So in the following section, some famous aptamers in cancer therapy are introduced.

4.1.1

As1411 aptamer (AGRO001)

AS1411(50 -GGTGGTGGTGGTTGTGGTGGTGGTGG-30 ), as the most famous example of a guanine-rich quadruplex aptamer, is a nonSELEX aptamer that was discovered incidentally by Bates et al. [8]. This aptamer with 26 base-pair has good nuclease resistance and thermal stability properties. It binds specifically to nucleolin (C23). nucleolin is a protein that is aberrantly expressed on the cell membrane of cancer cells, and consider a tumor-selective target. Nucleolin has a variety of biological functions including implications in the synthesis of ribosomes and regulating multiple vital processes such as proliferation and differentiation of cells, chromosome replication, division and even metastasis of tumor cells, which brings out the important status of nucleolin in cancer biology [9]. AS1411 aptamer, not only has been employed as a transporter of therapeutic drugs for targeted tumor therapy but also can kill tumor cells directly without any drug [10]. This aptamer is internalized and shuttled efficiently even at a very low concentration [11]. However, growthinhibitory functions of AS1411were proved against a broad range of cancer cells, such as metastatic renal cell carcinoma, colon and breast cancer and acute myeloid

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leukemia, but, the precise mechanism of cancer-targeting by this aptamer is not completely understood [12,13]. A selective and effectual therapeutic strategy was designed for the treatment of colorectal cancer by Lohlamoh et al. through intercalating doxorubicin (DOX) into duplex sites of AS1411 aptamers [14]. Dox-loaded AS1411 aptamers were internalized into the Human colorectal adenocarcinoma cell line (SW480) via a specific interaction between AS1411 aptamer and nucleolin. Intercalated DOX was released inside the cell by lysosomes, resulting in a significant decrease in procaspase-3 and cell apoptosis [14]. Very recently our team employed DNA nanotechnology to produce a codelivery system for the delivery of both drugs (Epirubicin) and gene (antimiR-10b) into breast cancer cells. A bivalent aptamer consisting of AS1411 and FOXM1 Aptamer was prepared to target breast cancer cells with a Linear structure with bifunctional molecules. This highly stable system, not only provides the efficiently targeted ability to deliver Epirubicin and antimiR-10b to the tumor cells but also inhibits tumor growth meaningfully much more than free Epi [15]. An up-conversion nanoparticle (UCNP)-based nanocage system loading with antiVEGF siRNA has been fabricated using the thermal decomposition method for targeting overexpressed nucleolin on the surface of NCIeH889 lungadenocarcinoma cell line cells. The inhalation-mediated local delivery approach was exploited to maximize the targeted UCNP nanocage distribution in the lungs. By comparison of the region of interest (ROI) analyses, the accumulation of UCNP- antiVEGF siRNA-AS1411 nanocages in the lung was significantly higher than that in other organs, confirming that the system not only possesses excellent tumor targeting ability but also has supporting gating characteristics in transferring the siRNA to the tumor cells due to the presence of AS1411 aptamer [16]. A cationic liposome was prepared by thin-film hydration and sonication method for the loading of miR-29b by the Jiang group in 2020 [17]. The thiol-maleimide reaction was used to conjugate AS1411 aptamer onto the liposome through the formation of thioether linkage [18]. AS1411-Conjugated liposome was then diffused into A2780 ovarian cancer cells, where the targeted liposome bound to nucleolin through AS1411 and entered the nucleus. The result of the fluorescence microscopy nuclear morphology assay, revealed the brightest fluorescence intensity in the miR-29b treated cells, confirming enhanced nuclear degradation and cell apoptosis in the cancer cells via nucleolin-mediated endocytosis [17]. More importantly, the AS1411 aptamer is also able to inhibit Glioblastoma multiforme (GBM) cell migration and invasion by p53up-regulation and Bcl-2 and Akt1 down-regulation [19]. To guide the delivery of 5-fluorouracil (5FU) and Dox to GBM, a dual-stimuli responsive nanosystem comprise bio-synthesized Au NPs (gold nanoparticles) and chitosan (CS) introduced by Sathiyaseelan group in 2021 [20]. This nanosystem can enter LN229 cells through the mediation of AS1411 aptamer and effectively prompted apoptosis of LN229 cells by arresting the cell cycle at G0/G1, G2/M and S phases. Here, chitosan could improve drug loading and drug encapsulation efficiency, which is a critical parameter in drug delivery nanosystems [21]. However, although AS1411 aptamer has been withdrawn from clinical trial studies, the improvement of the targeted nanoplatforms with AS1411aptamer as a targeted agent can make important contributions to the treatment of cancer and is it still a hot research issue for scientists [14,22e27].

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4.1.2

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Sgc8-c aptamer

For the first time using the cell-based aptamer selection approach, Shuangguan group (2006) introduced a group of aptamers, named sgc aptamers including (sgc3, sgc6, sgd3, sgc4, sgc4a, sgc5, sgc7, sgc8, sga16, Sgd2) for the specific recognition of T cell acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM [28]. In this process, an ssDNA library containing random sequence regions was incubated with CCRFCEM cells. In order to minimize nonspecific aptamers, B cell lymphoma cell line (nontarget cells) was considered as a counter selection. Among identified sequences, sgc8 aptamer (88-mer) recognized the CCRF-CEM with the highest affinity (Kd ¼ 0.80  0.09 nM). So, this aptamer can be easily and efficiently exploited for cancer cell detection when is tagged with fluorescent probes and even used for MRD determination [29]. Sgc8-c, a truncated form of sgc8 aptamer, is a DNA bifunctional aptamer with 41 nucleotides long that selectively recognizes (protein tyrosine kinase) PTK7-receptor overexpressed in several types of tumors [30]. Both sgc8 and Sgc8-c aptamers are internalized specifically by the lymphoblastic leukemia cells via receptor-mediated endocytosis (RME) [7,31]. Sgc8-c can be employed as a molecular imaging probe for in vivo diagnosis of metastatic and nonmetastatic melanoma [32]. With respect to this aptamer, a variety of drug delivery systems using gold nanoparticles and carbon nanotubes along with different drugs have been developed by our group [33e35]. In order to reduce the side effects of cytarabine, glutathioneresponsive nanoassemblies based on Sgc8 aptamer and cytarabine (Ara-C) were reported by Fang et al. in 2019 [36]. The Sgc8 aptamers targeted nano-formulation were tested in vitro and in CCRF-CEM tumor-bearing nude mice model. Enhanced tumor growth inhibition, improved survival rate and decreased side effects were observed in mice treated with aptamer-cytarabine compared to cytarabine treated mice indicating that Sgc8 aptamer has therapeutic potential for lymphoblastic leukemia [36]. Currently, the only clinical trial (early phase I) (NCT03385148) is registered for the treatment of colorectal cancers.

4.1.3

NOX-A12 (Olaptesed pegol)

NOX-A12, a PEGylated L-stereoisomer RNA Spiegelmer aptamer (L-RNA) consists of 45 ribonucleotides, specifically binds to CXCL12 (SDF-1) at a Kd of 0.2 nM and disrupts binding of CXCL12 to CXCR4 (Fig. 4.2) [37]. CXCL12 secreted by tumor stromal fibroblastsis responsible for tumor metastasis. The inhibition of CXCL12CXCR4 signaling pathway results in an increase in CLL cell migration from the bone marrow (BM) to peripheral blood, exposing CLL cells to chemotherapeutic drugs to increase tumor cell apoptosis [38]. Data from phase IIa clinical trial (NOX-A12 plus Bendamustine/Rituximab, ClinicalTrials.gov: NCT01486797) showed high ORR (The Overall Response Rate) (86%) and median PFS (progression-free survival) of 15.4 months in ITT (intent-to-treat ) population in comparison to the single agents alone [39]. These promising results, prove the valuable role of this aptamer in the treatment of CXCL12-derived tumors.

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Figure 4.2 Schematic representation of inhibition of the CXCL12/CXCR4 axis by NOX-A12 aptamer. Adapted from the published work [40].

4.1.4

NAS-24

Vimentin belongs to the type III intermediate filament protein and forms microtubules, maintain cytoplasm integrity and intracellular structure stability as well as regulation of cell migration [41]. It was to be found, that vimentin’s overexpression in various epithelial cancers and associated with augmented tumor growth and enhanced metastatic capacity [42]. For the first time, Zamay et al. isolated a DNA aptamer called NAS24 (80-nucleotide), which specifically binds to vimentin [43]. They injected targeted complex (NAS-24/arabinogalactan) into the peritoneum of mice with adenocarcinoma and observed efficient inhibition of adenocarcinoma growth [43]. Using this aptamer, our team developed; a targeted codelivery system based on PEI-PEG selenium nanoparticle (SeNPs) for the optimization of epirubicin delivery. In this way, SeNPs decorated by two aptamers 5TR1 aptamer as the targeting moiety to improve the antitumor efficacy of epirubicin and NAS-24 aptamer to target vimentin in MCF-7 and C26 cells and induce apoptosis. This targeted nanosystem diminished tumor growth in cancerbearing mice as compared to the control [44]. Following this line, our group introduced a theranostic system comprising two nanocomplex. First, one that induced apoptosis in breast cancer cell lines using graphene oxide-MUC1/NAS-24 aptamers due to the binding of NAS-24 aptamer to vimentin and the second nanocomplex (Graphene oxide-MUC1/Cytochrome C aptamer) was used to check the function of the first nano-complex [45].

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4.1.5

Aptamers Engineered Nanocarriers for Cancer Therapy

CD44 aptamer

CD44, a transmembrane glycoprotein belonging to the adhesion receptors family, is instrumental in cellular pathways such as adhesion, proliferation, differentiation, motility and migration [46]. In addition, the molecule recognizes and binds to hyaluronic acid and thereby is involved in many normal and pathological conditions [47]. There are more than 20 isoforms of CD44 generated via alternative splicing. Cell distribution of CD44 isoforms differs between cell types, both normal and tumor cells. For example, CD44H is a hematopoietic isoform expressed in all cell types of blood, whereas CD44v6 is overexpressed in breast and colorectal cancers [48]. Overexpression of CD44 in a variety of tumor cells including breast cancer, hepatocellular carcinoma (HCC), colon cancer, ovarian cancer, nonsmall-cell lung cancer and glioma on the one hand and its expression in cancer stem cells (CSCs) that is thought to be responsible for metastasis and resistance of tumor cells to common therapeutic strategies [49e51]. On the other hand, has attracted the attention of researchers to target CD44 as a tumor marker. In this regard, CD44 aptamer-doxorubicin conjugates in different aptamer-drug molar ratios were applied to induce cytotoxicity in human breast cancer cells. The obtained results indicated that proper internalization and accumulation of aptamer-drug conjugates at a 1:2 aptamer-drug molar ratio resulted in proliferation inhibition of CD44-overexpressing breast cancer cells compared to control cells [52]. A single aptamer called CD44-Apt1 was demonstrated to recognize and bind to both CD44E and CD44s isoforms overexpressed in HCC cells. The high affinity of CD44-Apt1 for both CD44E and CD44s isoforms (Kd values of 1.22 and 2.09 nM, respectively) proposed CD44-Apt1 as a novel carrier for efficient delivery of 5FU to HCC cells. In vitro assay showed that complexes of CD44-Apt1-5FU were able to enhance drug toxicity up to more than 6000 times. Moreover, a significant reduction in size and weight of tumor tissue was observed following treatment of mice bearing CD44E or CD44s-positive HCC xenograft with CD44-Apt1-5FU compared to control mice. Tumor growth inhibitory effects of CD44-Apt1-5FU were more efficient than those of 5FU alone, as well. The study indicated that aptamer-mediated specific delivery of 5FU protects major organs including the heart, brain, liver, kidney, lung, intestine, stomach and spleen from detrimental side effects of the therapeutic agent [50]. It has been shown that aptamer against CD44 on its own is capable of inducing cell death in CD44þ ovarian cancer cell lines including SKOV3, OVCAR8 and ES2 in a dose-dependent manner. Interestingly, bispecific CD44-EpCAM (epithelial cell adhesion molecule) aptamer exhibited much greater potency in decreasing the cell viability of mentioned cell lines. EpCAM is a glycosylated membrane protein overexpressed in more than 70% of ovarian cancers. In addition to much-improved cytotoxicity of bispecific CD44-EpCAM aptamers, resulting nanostructures showed better half-life in blood circulation in contrast to either single CD44 or EpCAM aptamers. Apoptosis-inducing effects of bispecific CD44-EpCAM aptamer were also observed in mice injected with OVCAR8-Luc cells. EpCAM aptamer alone showed no significant decrease in tumor growth. Although CD44 aptamer was able to significantly reduce tumor burden, the results pertained to bispecific aptamer displayed higher tumor growth retardation compared to each aptamer alone and even a combination of

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nonconjugated CD44 and EpCAM aptamers [53]. In a similar strategy, nanomicelles loaded with gefitinib were conjugated with CD133 and CD44 aptamers to enhance the delivery of gefitinib to lung cancer-initiating cells. Both CD44 and CD133 are specific markers for lung cancer-initiating cells endowed with stem cell properties such as tumorigenic spheres generation. CD44/CD133 aptamer-conjugated nanomicelles loaded with gefitinib (CD133/CD44-NM-Gef) significantly reduced 50% inhibitory concentration (IC50) of gefitinib for CD44/CD133 positive lung cancer cells. Besides, tumorspheres formation ability was also suppressed by more than 60% in H446 and A549 lung cancer cells treated with CD133/CD44-NM-Gef in comparison with untreated cells. The study demonstrated that using CD44 and CD133 aptamers is promising to tackle lung cancer-initiating cells resistant to gefitinib [54].

4.1.6

EpCAM aptamer

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein associated with cell proliferation, migration and invasion. Overexpression of EpCAM has been evidenced in many kinds of cancer cells including breast, bladder, prostate, pancreas, ovarian and colon cancers and in some cancer stem cells (CSCs) such as breast, colon, pancreas and prostate [55,56]. Higher expression of EpCAM in tumor cells has turned this molecule into an attractive target for designing new therapeutic strategies against a wide variety of tumor cells. For instance, EpCAM-positive HT29 (colorectal cancer cell line) internalized EpCAM Apt-DOX much more efficient than EpCAM-negative HEK293T cell line followed by a higher accumulation of DOX in the nucleus of target cells. Retention of the drug inside the cells is of great importance for better effectiveness of the drug, the goal that was achieved by conjugation of DOX with EpCAM Apt. Improved accumulation and retention of EpCAM Apt-DOX led to a 16.7-fold reduction of HT29 cancer stem cells frequency compared to that of free DOX. Antitumorigenicity properties of EpCAM Apt-mediated targeted delivery of DOX were not limited to HT29 as Apt-DOX could effectively disrupt the tumorigenicity of CSCs in ovarian (SKOV3) and breast (T47D) cell lines. Antitumor efficacy of EpCAM Apt-DOX was further confirmed via the treatment of HT29 colorectal tumor-bearing mice with Apt-DOX where tumor volume decreased by 3-fold compared to free DOX treatment [57]. In a more complicated method, colorectal cancer cells were targeted by one of the maytansine derivatives called DM1 (a microtubules-depolymerizing agent) delivered to tumor cells by mesoporous silica nanoparticles (MSNs) decorated with hydrochloride dopamine (PDA), polyethylene glycol (PEG) and EpCAM aptamer (APt). In this system, PDA served as a pHsensitive moiety for better release of the drug in an acidic tumor milieu and EpCAM aptamer enabled more efficient delivery of DM1 to cancer cells. Aptamer-mediated smart delivery of an encapsulated cytotoxic drug (DM1) in MSNs resulted in much stronger depolymerizing effects in treated SW480 cells compared to the drug alone. The downstream effects of microtubule assembly disruption were reflected in the form of apoptosis induction in target cells so that the percentage of dead cells increased from 4% in control cells to 73% in cells treated with MSN-DM-PDA-PEG-Aptamer. These results were in keeping within vivo experiments in which a significant tumor

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growth inhibition was reported in nude mice bearing SW480 tumor cells treated with MSN-DM-PDA-PEG-Aptamer [58]. In addition to drug-based cancer therapy, cancer immunotherapy has been in the spotlight in the past few decades. Nonetheless, tumor cells adopt different mechanisms to escape from the immune response and resist getting killed by cytotoxic lymphocytes. To overcome tumor cell evasion of the immune response, increasing cancer immunogenicity is needed to improve immunotherapy. A novel strategy used was to knock down genes in mice bearing breast cancer to induce tumor neoantigens. To do so, EpCAM aptamer-linked small interfering RNA (SiRNA) chimeras (AsiC) were used to knockdown Upf2, Parp1, CD47 and Mcl1 in EpCAMþ mouse breast cancer cell line (4T1). The expression of target genes was silenced in 4T1 cells from 50% to 90% upon treatment with AsiC whereas the EpCAM-negative cell line (L929) was not affected. Downstream consequences of gene expression inhibition mediated by AsiC were tumor growth inhibition and enhancement of antitumor T cell immunity. Each of the four EpCAM-AsiC on its own reduced tumor size in orthotopic 4T1 tumor-bearing mice but the EpCAM-AsiC mixture showed more efficient antitumor effects. Mice treatment with EpCAM-Asic was followed by increased expression of genes associated with T cell activation (Fos, Zap70, Junb and CD69 and proliferation Ifng, Tnf and Il2). In addition, the genes involved in CD8þ T cell cytotoxicity including Gzmb, Gzmk and Prf1 were upregulated in EpCAM-AsiC treated mice, implying enhanced anti-tumor activity of T cells. On the whole, the results indicated that EpCAM-AsiC had the potential to restore the functionality of the exhausted tumor-infiltrating lymphocyte to kill tumor cells [58].

4.1.7

Anti-PDeL1 aptamer

PD-1 (Programmed Death 1, CD279) as a coinhibitory molecule, belongs to the CD28 family and is expressed on activated T cells, B cells, dendritic cells (DCs), macrophages, regulatory T cells (Tregs), natural killer (NK) cells and, of note, CD8 tumor-infiltrating lymphocytes. The interaction of PD-1 with PD-L1 (programmed death ligand 1) on tumor cells results in inhibition of the T cell immune response through different signaling pathways [59]. Based on these perspectives, blocking the PD-1/PD-L1 pathway via targeting either PD-1 or PD-L1 immune checkpoint can be considered an important alternative to the current antibody basedmainstream, by restoring the proliferation of T cells blocked by the PD-1/PD-L1 axis, to induce a long-lasting clinical response. In this regard, different aptamers as inhibitors, targeting the PD-1/PD-L1 axis, were isolated by various research teams. For instance, Lai et al. introduced an aptamer against PD-L1 that inhibited the interaction between PD-1 and PD-L1, and mediated tumor growth arresting by increasing lymphocyte proliferation [60]. In this line, our team in 2017, isolated successfully an aptamer against PDeL1 (APT5) with high specificity and affinity using protein SELEX. This aptamer could bind to PD-L1 protein with Kd of 64.77 nM. Moreover, the PD-L1 aptamer could also recognize, bind as well as internalized PDL1-positive ovarian carcinoma cells (A2780) [61]. The internalization ability of APT5 aptamer is very important in targeted drug delivery strategies [30]. In another study, Gao et al. selected PD-L1 aptamers using cell-SELEXs with an affinity for PD-L1 with a

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Kd of 95.73 nM. This selected aptamer was able to block the interaction of PD-1/PDL1 and suppress the growth of the CT26 colon carcinoma [62]. A special tetravalent DNA nanostructure (Holliday Junction) designed four PD-L1 aptamers with an average size of 13.22 nm preventing leaking out by renal clearance and nuclease degradation. This cross-shaped structure, had a stronger cellular binding Capacity to CT26 murine colon cancer in comparison to free PD-L1 aptamer [63]. In an attempt to increase the biological stability of PD-L1 aptamer, two threoses nucleic acid (TNA) aptamer (S42 and N5), which have a unique structure different from native DNA and RNA reported. Here, TNA aptamer N5 (Kd around 400 nM) blocked the binding of PD-L1 to PD-1 and significantly arrested colon carcinoma growth in vivo, indicating the precise and specific targeting ability of the TNA aptamer N5 [64]. To our knowledge, in 2020 and 2021, five anti-PD-L1 aptamers were introduced that highlight the importance of this signaling pathway in cancer therapy [65].

4.1.8

MUC-1 aptamer

Mucin 1 (MUC1), an O-linked glycosylated protein, is one of the members of the membrane-bound mucin subfamily, which resides on the apical face of most normal secretory epithelial cells. Whereas the MUC1 is overexpressed by most human epithelial cancer cells, its glycosylation differs from naturally expressed ones. Abnormal hypo-glycosylated MUC1 has been found in cancers such as lung, breast, colon, pancreas, ovarian, uterus, prostate, and to a lesser degree in hematopoietic cells as well as platinum-resistant tumors [66]. In cancer cells, due to the underglycosylation of MUC1, the immunogenic epitopes of the N-terminal region will expose to the immune system so that new antigens will develop including Tn antigen, sialyl-Tn antigen, and TF antigen. Moreover, MUC1 is not apically overexpressed in cancer cells, this glycoprotein spreads across the entire cell membrane of cancerous cells, even in the cytoplasm, nuclei, and mitochondria [66]. MUC1 has been shown to act as an oncogene leading to tumorigenesis and cancer formation. It is responsible for tumor metastasis, angiogenesis, and drug resistance. Hence, inhibiting MUC1 as a targeting ligand would be a strategic approach in cancer treatment [67]. In this regard, several aptamers were selected against different epitopes of MUC1, based on their exposed peptide sequences, in 2006. The selected aptamers included S1.1, S2.2, 5TR1, 5TRG2, MA3, and GalNAc3. One of these, the S2.2 aptamer, which binds to a variable-number tandem repeat of MUC1 peptides have shown higher affinity and specificity than the monoclonal antibody C595 [68]. The MA3, S2.2, and 5TR1 are the most frequently used aptamers in experimental studies with the Kd values of 38.3, 13.6, and 21 nM, respectively. In a study in 2018, 5TR1 aptamer was applied as a targeting molecule to conjugate to PEGylated liposomal doxorubicin. The delivery system was injected into the BALB/c mice-bearing C-26 colon carcinoma, which showed a high drug accumulation in tumor tissue. 5TR1 aptamer improved the therapeutic efficacy of PEGylated liposomal doxorubicin in the C26 animal model [69]. Another study in 2019 used a 5TR1 aptamer to deliver epirubicin and antimir-21 loaded on PLGA and PbAE, respectively, as a nano complex platform. 5TR1 aptamer-modified nano complex could remarkably prevent tumor growth in the C-

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26 colon carcinoma tumor-bearing C-26 colon carcinoma mice model [70]. A DNA sensor-capped doxorubicin-loaded mesoporous silica nanoparticle was designed to target the MUC-1 motif to trigger the release of doxorubicin through a conformational switch induced by MUC-1. A hairpin structure of MUC1 aptamer was designed as a gatekeeper of drug-loaded nanoparticles. In this way, a conformational change would trigger the release of the drug through a switch in position. This platform improved tumor accumulation and retention of doxorubicin in the tumor-bearing mice model [71]. It has been shown that GalNAc aptamer with an amphiphile structure conjugated to nanoparticles was capable of promoting the doxorubicin accumulation in target cells and tissue in the mice model [72]. A MUC1-targeting aptamer was applied for targeted codelivery of doxorubicin and KLA (a proapoptotic peptide) using a micellar DNA nanoparticle. In vitro assay showed that the construct was specifically delivered to MCF7 cells. Moreover, the tumor growth remarkably regressed following the in vivo experiment [73]. In another strategy, aptamer-guided DNA tetrahedrons were synthesized for targeted delivery of TMPyP4 as a photosensitizer. MUC1 aptamer was linked to the tetrahedral nanostructures, a carrier for TMPyP4, and selectively delivered the photosensitizer to MUC1 positive cells [74]. A DNA aptamer selected against MUC1/Y, an isoform of MUC1, has been shown to recognize a wide range of human cancer cells and reduce the growth of MUC1/Y-expressing cell lines. Besides, MUC1/Y aptamer inhibited the tumor growth of breast cancer cells in vivo studies [75].

4.1.9

Forkhead Box M1 (FOXM1)

For the first time, FOXM1 aptamer was introduced in 2017 that binds to FOXM1 protein [76]. Upregulation of FOXM1 protein is correlated with tumorigenesis, metastasis, angiogenesis as well as drug resistance of many cancers [77]. Also, in a study conducted by Shahriari et al. FOXM1 aptamers were selected to conjugate to the surface of polymersomes loaded with doxorubicin and camptothecin for maximum prevention of proliferation in cancer cells resulting in a robust combinatorial delivery platform against nonsmall cell lung cancer [78]. In another study, liposomal targeted delivery of doxorubicin by FOXM1 aptamers was introduced as a new strategy to overcome Dox resistance. In addition, a significant increase in dox cytotoxicity and Dox-induced apoptosis in cancer cells was reported [79].

4.1.10

PSMA aptamer

Prostate-specific membrane antigen (PSMA) is a prominent extracellular domain, which can bind to the ligands and internalize them into the cells. PMSA is expressed much higher in prostate cancerous tissue than noncancerous one. Therefore, PMSA is considered a prostatic biomarker for diagnostic and therapeutic purposes [80,81]. Research shows that this membrane glycoprotein is certified as a feasible target to detect metastatic prostate cancer tumors using an anti-PSMA antibody [82]. Nucleic acid aptamers are another targeting moiety that can be applied as a ligand for PSMA. In 2002, two aptamers were selected against PSMA recombinant protein

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named xPSM-A9 and xPSM-A10, each of which binds native protein and then inhibits its glutamate carboxypeptidase enzymatic activity [83]. To obtain the minimal functional unit of these target molecules, the aptamers were truncated, which caused xPSM-A9 aptamer to lose its enzymatic ability, whereas xPSM-A10-3 aptamer (a refined form of xPSM-A10) maintained its property [83]. Rocky et al. in 2011 made a rational truncation of aptamer A9 bioinformatically, named Ag9 with Kd of 130 nM [84]. Later on, another group made an isoform of A10-3, termed A10-3.2, whose Kd value was increased up to 2.9 nM. The selected and/or truncated aptamers can be conjugated with therapeutic drugs or SiRNA for targeted treatment of prostate cancer. Farokhzad et al. for the first time explained the conjugation of Aptamer 10 to PLA-PEG-COOH nanoparticles for controlled release of rhodamine-labeled dextran, which showed successful delivery of nanoparticles to prostate cancer cells [85]. Afterward, they synthesized PLGAebePEG nanoparticles attached to the A10 aptamer that enabled much more efficient drug delivery to the prostate tumor compared to nontargeted nanoparticles [86]. In 2013, Boyacioglu et al. selected a DNA aptamer against PSMA and synthesized a dimeric complex of the aptamer and a CpG sequence to bind to doxorubicin. The dimeric complex showed high selectivity for PSMApositive cells similar to that of the A10 RNA aptamer. They also showed that the complex had prolonged retention in plasma. Besides, it displayed enhanced permeability and retention (EPR) effects and augmented specificity for PSMA-positive cells [87]. A9g aptamer inhibited the enzymatic activity of PSMA. In vitro study showed repressed cell migration and invasion. Furthermore, in the mice model, pharmacokinetics and biodistribution studies demonstrated target specificity and reduced off-target effects [88]. Wu et al. used A10e3.2 aptamer as a targeting molecule to functionalize gas-filled nanobubbles made of PLGA encapsulated paclitaxel for targeted delivery to prostate cancer using ultrasound. The targeted compound enhanced cell apoptosis and paclitaxel release in vitro. In vivo study demonstrated an enhanced tumor inhibition rate with no apparent systemic toxicity [89].

4.1.11 HPV E6/E7 aptamers High-risk human papillomavirus (HPV) E6 and E7 proteins are the main elements in transforming to the malignancy of HPV-positive tumor cells [90]. The E6 and E7 viral proteins are small proteins that can interact with the p53 and pRb (retinoblastoma) tumor suppressor pathways, respectively, leading to inactivating them. E6/E7 inhibition prompts senescence in HPV-positive tumor cells. As a result, these two viral oncogenes are noticeable therapeutic targets in HPV-positive neoplastic lesions. Nearly 12 HPV types are categorized as high-risk HPVs strains with carcinogenic potential, for example, HPV16 and -18 are responsible for more than 70% of all cervical cancer cases. Cancers related to high-risk HPVs are mainly cervical cancer, head and neck cancer, and anogenital malignancies [90,91]. Therefore, using specific aptamers to E6/E7 oncoproteins might pave the way for target-specific therapy. In 2011, an isolated RNA aptamer inhibited the interaction between E7 and pRb [92]. The RNA aptamer transfected into the HPV-positive tumor cells led to E7 loss and apoptosis [93]. Gourronc et al. isolated RNA aptamers against HPV16 transformed tonsillar

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cells. internalization ability of their selected aptamers proposed them as tools for targeted drug delivery [94]. In 2014, an RNA aptamer to E6 was derived from HPV16transformed cervical carcinoma. This aptamer was capable of inhibiting the interaction between E6 and PDZ-binding domain, which might have potential as a therapeutic molecule [95]. Moreover, we selected two different DNA aptamers against HPV16positive tumor cells (CaSki), which bind to cell surface proteins with high affinity and specificity as diagnostic or targeting agents [96,97].

4.2

Conclusion

In this chapter, the newest research advancement of aptamer and their applications in the treatment of cancers are reviewed. With a surge of advances in the treatment of different cancers, the development in the selection of aptamers, as new targeting agents, is essential to assist the science in the cancer field. Aptamers can bind to different tumor markers with high affinity and specificity, and provide more effective treatment based biological characteristics of cancers. However, some major problems such as nuclease degradation and fast renal clearance in vivo and critical issues, limited application of aptamers for targeted therapy and essential to be resolved by implantation of new strategies.

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Aptamers as smart ligands for the development of cancer-targeting nanocarriers

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Yadollah Omidi 1 , Jaleh Barar 2, 3 , Somayeh Vandghanooni 2 , Morteza Eskandani 2 and Hossein Omidian 1 1 Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL, United States; 2Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran; 3Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran

5.1

Introduction

To date, as an effective approach, various targeted therapy modalities have been developed using targeting agents such as antibodies (Abs), aptamers (Aps), peptide ligands, and small molecules (e.g., folic acid). Such modalities can be used to modulate different biological entities (e.g., genes, peptides, and proteins) and even cells/tissues. These types of treatment modalities are developed using organic/inorganic nanocarriers decorated with targeting agents, which can interact with the cognate biological entity with high affinity and specificity. In the case of cancer, the prime goal of targeted therapy modalities is to specifically induce maximal therapeutic impacts on the diseased cells/tissue with no/trivial effects on the healthy normal cells [1e3]. Several homing agents (e.g., Abs, intrabodies, nanobodies, Ab fragments, nonantibodyderived scaffolds like affibodies, macrocyclic peptides, and Aps [4e16]) are used for specific targeting of cancerous cells that can be used to develop multimodal nanosystems (NSs) against cancer. Accordingly, Abs or Aps have been exploited to arm various advanced nanobiomaterials such as gold nanoparticles (AuNPs) [17], magnetic NPs (MNPs) [18e22], quantum dots (QDs) [23e25], carbon nanotubes (CNTs) [26,27], and polymeric/lipidic NPs [19,28e36]. Aps (either as DNA or RNA), which are small single-strand oligonucleotides with three-dimensional (3D) nanostructure, display high binding affinity toward a large variety of small molecules, biomacromolecules, and even cells. The 3D structure of Aps favors their interaction with small molecules via the formation of specific binding pockets with different forces [37]. Given that Aps are considered as Abs’ alternatives, much attention has been paid to their applications as targeting agents. Unlike Abs, Aps show high stability. Like Abs, Aps display a high binding affinity with marked specificity toward designated antigen (Ag). Abs are bulky and complex; however, Aps with their smaller sizes, are better candidates in the development of targeted therapies as they can easily

Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00001-4 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Aptamers Engineered Nanocarriers for Cancer Therapy

access the epitopes of target proteins/Ags [38]. Since Aps can interact with the cell surface receptors with the possibility of internalization, they can potentially serve as homing devices for targeting intracellular compartments and cell sorting. Once tagged with an imaging agent such as QDs, the cell-penetrating Aps can also be used for cell imaging in vitro and in vivo [24,39e41]. While the production of Abs is a laborious and complex process that often fails to provide Abs with desired high-affinity binding, the selection process of Aps appears to be relatively simple. Aptamers are technically produced through an in vitro chemical combinatorial technique, the so-called systematic evolution of ligands by exponential enrichment (SELEX). Collectively, SELEX can be used to select Aps specific to a wide range of targets from small molecules and ions to biomacromolecules and even whole cells [42e45]. Unlike Abs, Aps may simply undergo chemical modification without losing their function to improve their stability and bloodstream half-life [37]. Nontoxicity and the absence of immunogenicity of Aps make them the targeting agent of choice for many applications [46]. In addition, due to their fast clearance rates, Aps have been used in the development of nanoscale multifunctional radiopharmaceuticals that can be exploited as theranostics for simultaneous diagnosis and treatment of cancer [47e49]. Besides their fine-tuning and modifications potential, Aps can be used as antidotes to control drug toxicity [50e52]. Further, Ap-drug conjugates and Ap-based bispecific chimeric NSs have advanced the field [42,53e55]. Such functions of Aps can largely expand the clinical usesda field so-called aptamedicine [56e58]. For instance, Aps have been selected and used against anticoagulant drugs such as bivalirudin [59]. Aps are considered a new class of antidotes with a high binding affinity, which can control the possible toxicity of drug overdoses. Further, such capacity can be used to markedly enhance the seminal therapeutic impacts of drugs through Ap-tagged entities [38,60]. It is noteworthy that the production of mAbs might be expensive and complex; therefore, developing and building clinically sound immunotherapeutics, bispecific Abbased scaffolds, and Ab-drug conjugates can be extremely costly. Such pitfalls might limit the applications of Ab-based therapeutics [61]. Altogether, because of the unique characteristics of Aps, they have been used in the development of drug delivery systems (DDSs), biosensors, and multifunctional theranostics for the targeting, imaging, and treatment of cancer [62e64]. In the current chapter, we provide a deep insight into the Ap-based NSs and Ap-drug conjugates. We further elaborate on their production and applications in targeted therapy of human malignancies.

5.2

Selection of Aps

Because of the 3D structure of DNA/RNA aptamers, they are used in the detection of a wide variety of biomolecules and cells. They can simply be produced through in vitro cell-free amplification utilizing polymerase chain reaction (PCR), and as such they are considered remarkable biomolecules from the synthetic chemistry standpoint. The production of Aps can be performed using random sequence oligonucleotide (ONT)

Aptamer-armed cancer-targeting nanocarriers

105

libraries employing the SELEX technique [65], in which the larger the diversity of library is, the better the aptamer selection will be [66]. For instance, libraries consisting of about 1015 different ONTs can be made via chemical synthesis. Such a library with a large diversity can then be used to select Aps with high binding affinity. The Ap selection process can be integrated with the automated microfluidic system to maximize the speed and accuracy of the process [67e69]. Furthermore, a protocol has also been reported for the non-SELEX selection of Aps, in which repetitive steps of partitioning with no amplification in between are involved in the selection process [70]. Arraybased evolution of DNA aptamers using microarray technology has further advanced the aptamer selection process [71,72]. In 1990, Tuerk and Gold capitalized on the application of a combinatorial ONT library to identify RNA aptamers specific to bacteriophage T4 DNA polymerase (gp43) [65]. Capitalizing on an RNA pool randomized at specific positions, they selected gp43 binding sequences, which were termed SELEX [65]. In 1992, using a library of random sequences of DNAs specific to Cibacron blue and reactive green 19, Ellington and Szostak selected a few DNA aptamers [73]. In this line, the HTPSELEX database has also been developed [74]. Technically, the conventional SELEX methodology can be defined as a method with repetition of several successive steps, comprising (i) the selection step that includes binding, partitioning, and eluting; (ii) the amplification step that can be run by PCR process; and (iii) the conditioning step. Notably, the success of the selection step largely depends on the quality and diversity of the library used. In this line, a library with a large number of ONTs (RNA/ DNA) is routinely used (Fig. 5.1). Such a library consists of randomly generated sequences with a central random region of 20e80 ONTs flanked by specific sequences of 18e21 ONTs as primer binding sites required for the PCR. In selecting DNA aptamers, the library can be used without pretreatments, while for the RNA aptamers, the library has to be transformed into the RNA library. As shown in Fig. 5.1a, the special sense primers with an extension at the 50 end contain the T7 promoter sequence, in which the antisense primers are required for the conversion of the single-stranded (ssDNA) library into a double-stranded (dsDNA) library. The dsDNA is then transcribed by the T7 RNA polymerase using randomized RNA library. In practice, the randomized RNA/DNA library is first introduced to the target molecules. Second, the bound Ap-target complexes are partitioned from the unbound and/ or weakly bound ones. Third, the Ap-target complexes are eluted to obtain the desired Aps. Forth, the obtained Aps are amplified by the cell-free PCR methodology. The RTPCR technique is used in the case of RNA libraries to transcribe them to the cDNA before amplification by PCR. Various methods are then used in the conditioning step to obtain the relevant ssDNA or transcribed through T7 RNA polymerase in the case of RNA libraries. After several rounds of selection, amplification, and conditioning followed by the final amplification step (Fig. 5.1b), the selection process is considered complete [76]. Notably, the initial diversity of the library might be decreased by the repeating steps, which is a determinant factor in attaining the desired Aps specific to designated sequences with high binding affinity. The final PCR amplified products are further subjected to sequencing steps and in-silico analyses [77]. Often, post-SELEX modifications are required to enhance the stability of the selected Aps, or to further functionalize them (e.g., the substitution of the 2ʹ-OH group of ribose to 2ʹ-F, 2ʹ-NH2 or 2ʹ-O-methyl) [78].

106

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 5.1 The schematic representation of systematic evolution of ligands by exponential enrichment (SELEX) for the selection of aptamers (Aps). (a) The DNA/RNA SELEX process. A chemically synthesized DNA oligonucleotide (ONT) library is used. Each ONT possesses an internal randomized sequence of 20e80 nt and two fixed sequences. In the case of RNA SELEX, dsDNA is produced by PCR using two special primers, in which the sense primer has an extension sequence containing the T7 promoter sequence, and T7 RNA polymerase is used to transcript the dsDNA to RNA entities. (b) The schematic illustration for the standard SELEX process. Repeated rounds of selection, amplification, and conditioning are performed, in which the selected ONTs are used for the next round. In practice, 8e16 cycles are needed to select desired Aps. Adapted with permission from Ref. [75].

5.3

Recent advances in aptamer selection technology

To date, several advancements have been made to optimize not only the aptamer libraries but also the separation schemes and the amplification of the selected libraries. Furthermore, the identification of aptamer sequences from enriched libraries has also been improved [79,80]. The SELEX technology can be applied both in vitro (i.e., toward immobilized peptide/protein, or cell-based models) and in vivo using various animal models. The microfluidic-based SELEX (M-SELEX), the so-called on-chip aptamer selection technology, together with high-throughput sequencing (HTS) and “particle display” (PD) screening system for transforming solution-phase Aps into “Ap particles” have further advanced the selection of Aps with high affinity [81]. For instance, capitalized on the on-chip SELEX with clinical cancer tissue samples, Liu et al. [68] selected Aps with high specificity to ovarian cancer (with a dissociation constant of 129 nM).

Aptamer-armed cancer-targeting nanocarriers

5.4

107

Diagnostic applications of Aps

Due to such unique features and the low cost of production of aptamers, Ap-based optical and electrochemical diagnostic modalities are increasingly becoming one of the preferred diagnosis tools (Table 5.1). Such an approach has widely been applied in the early detection of cancer [104,105]. Aps have been exploited to devise electrochemical or optical sensors/biosensors for the detection of a wide range of biological targets [106,107]. Notably, their conformational changes in association with the cognate targets make Aps seminal detection agents used in the development of optical/electrochemical biosensors. A light-responsive probe can be conjugated onto Ap, upon which a signalon/off reaction can be granted based on the interaction of Ap with the target entities [106]. Aps serve as flexible seminal sensing probes to create a new class of biosensors, so-called aptasensors, which can be used to detect cancerous cells. An ideally featured electrochemical aptasensor is theoretically composed of an electrode decorated with Ap molecules specific to a designated molecular target. Once exposed to the cognate biological target, the Ap molecules change their structural conformation and bind with the analyte entities. Such association can alter the current flow that results in electrochemical signals different from the unassociated ones [108]. The interaction of Aps with their related targets is the foundation of the development of various electrochemical and optical aptasensors used to detect cancers [109e111].

5.5

Therapeutic applications of aptamers

Several nanosized targeted therapies have so far been developed using various advanced biocompatible bioactive nanomaterials. The nanosized carriers offer a large surface areada feature that favors the high loading of drugs. They can also be functionalized with other moieties such as targeting and imaging agents. Such unique characteristics make them preferred vehicles for the development of delivery systems for active targeting of cancer. To actively target the cancer cells, these nanoscale DDSs can further be functionalized with homing agents such as antibodies, aptamers, peptide ligands, and even small molecules. To maximize the therapeutic impacts with minimal side effects, the targeted therapy of the diseased cells seems to be the best strategy [112,113], which can be accomplished via passive and/or active targeting mechanisms. As a result, several advanced multifunctional nanomedicines have been developed for targeted therapy of different types of malignancies using homing devices such as Abs, peptides, small molecules, and Ap. Among targeting agents, Aps offer notable features, including inexpensive production and ease of functionalization among others. As a result, many Ap-based targeted therapies have been developed (Table 5.2).

5.5.1

Ap-conjugated NPs

Ap-conjugated organic/inorganic NPs have been developed to serve as advanced DDSs in the active targeting of cancer cells. Upon further functionalization of these

108

Table 5.1 Aptamer-based biosensors. Sensor type

Carrier

Diagnostic use

Target

Refs.

Optical biosensor

Quantum dot/Au nanoparticle Au-capped nanoparticles

Ion detection Antigen-antibody reactions

[82] [83]

PAMAM-Dendrimers/AuNPs Label-free sensor surface Split DNAzyme halves

Ovarian cancer Circulating tumor cells Homogeneous allosteric sensor for adenosine Protein’s detection

Mercury (II) ions Fc portion of the human IgG1 subclass CA125 Mammaglobin-A Adenosine

Polymer complexes Au electrode Au electrode AuNps electrodeposited apta-cyto biosensor Au electrode Screen-printed electrode Au electrode Dialdehyde cellulose/carbon nanotube/ionic liquid nanocomposite Polyacrylonitrile/polypyrrole nanofibers modified pencil graphite electrodes Bimetallic PdeAu nanoparticles Au electrode CD63-modified electrodes in a microfluidic chamber Au nanoneedles

Cocaine sensors Detection of nanomolar Kanamycin A Detection of circulating human MDAMB-231 breast cancer cells Detection of cancer biomarkers

[87] [88] [89] [90] [91]

Breast cancer detection Antibiotics Detection of proteins

Prostate-specific antigen (PSA) HER2 protein Aminoglycoside Thrombin

[92] [93] [94]

Non-small cell lung cancer detection

A549 cells

[95]

Detection of proteins Ion detection in human serum samples Exosome detection

Thrombin Potassium ion CD63 exosomes

[96] [97] [98]

Detection of human a-thrombin in complex real sample

Thrombin

[99]

Aptamers Engineered Nanocarriers for Cancer Therapy

Electrochemical biosensor

Ions or human athrombin Cocaine Kanamycin A MDA-MB-231cells

[84] [85] [86]

Aptamer-armed cancer-targeting nanocarriers

Boron-doped diamond (BDD) electrode decorated with Au nanoparticles Silane self-assembled monolayer (SAM) onto ITO surface Screen-printed carbon electrode modified by gold nanoparticles and aptamer

Proton detection

Proton

[100]

Detection of human lung adenocarcinoma Detection of human prostate cancer

A549 cells

[101]

Prostate-specific antigen

[102]

For details, see Ref. [103].

109

Aptamer

Apt-DOX conjugates

CD44 specific Ap AS1411 Ap AntiEGFR RNA Ap AS1411 Ap Sgs8c DNA Ap A10 RNA Ap MUC1 Ap

AgNPs coated albumin AuNP-Ap

SPION-Ap

SWNT-Ap

Sgs8c DNA Ap

Molecular target

Drug or treatment

Cancer cells/model

Refs.

CD44

DOX

CD44-expressing cancer cells

[114]

Nucleolin

Radiosensitizing effect

Glioma

[115]

EGFR

His-BIM protein

HeLa cells

[116]

Nucleolin

Radiosensitizing effect

[117]

PTK7

DOX

MCf-7, MDA-MB-231, and mammosphere cells CCRF-CEM

[118]

PSMA

DOX

LNCaP

[119]

MUC1

MR imaging and photothermal therapy DOX

HT-29, CHO and L929 cells Molt-4

[120]

PTK7

[121]

Aptamers Engineered Nanocarriers for Cancer Therapy

Delivery system

110

Table 5.2 Aptamer-armed drug delivery systems for active targeting of cancer.

MSN-DOX@GO-Ap

QD-Ap

PLA-PEG NPs Ap-Albumin NPs

Ap-MSNPs PEG-PCL-Ap

AS1411 DNA Ap Sgs8c DNA Ap AS1411 DNA Ap AS1411 DNA Ap MUC1 DNA Ap A10 RNA Aps GBI-DNA Ap A10 RNA Ap AntiHER2 Ap EpCAM Ap GMT8 DNA Ap

Nucleolin

DOX

NIH3T3

[122]

PTK7

DOX and FITC

Hella

[123]

Nucleolin

DOX and curcumin

MCF-7 and HEK-293

[124]

Nucleolin

DOX

MCF0 7

[125]

Mucin-1

DOX

A2780/AD

[126]

PSMA

Cancer imaging

LNCaP

[127]

Tenascin-C

Cancer imaging

U251

[128]

PSMA

Dtxl

LNCaP

[129]

HER2

Curcumin

HER2 positive cancer cells

[130]

EpCAM

DM1

[131]

Glioblastoma

Dtxl

EpCAM-positive colorectal cancer U87

Aptamer-armed cancer-targeting nanocarriers

MSN-Ap

[122]

111

Continued

112

Table 5.2 Aptamer-armed drug delivery systems for active targeting of cancer.dcont’d

Aptamer

PLGA- lecithin-PEG-Ap

AS1411 DNA Ap AntiCD30 RNA PSMA RNA Ap 5TR1 DNA Ap CD44 Ap

PEI-citrate-Ap Ap-liposomes

PEG-PLA-Ap micelles (APP)

PEGePLGA-Ap

AS1411 DNA Ap Sgs8c DNA Ap FB4 RNA Aps A10 RNA Ap A10 RNA Ap

Molecular target

Drug or treatment

Cancer cells/model

Refs.

Nucleolin

Paclitaxel

MCF-7GI1 cells

[132]

CD30

ALK siRNA

ALCL

[133]

PSMA

DOX

LNCaP

[134]

Mucin-1

DOX

C26 tumor-bearing mice

CD44

Gene silencing by siRNA

Nucleolin

DOX and ammonium bicarbonate (ABC, a bubble-generating agent)

CD44 positive murine triple-negative breast cancer MCF7 cells

[136]

PTK7

Dextran-FITC

CEM

[137]

TfR

Flurbiprofen

bEND5 cells

[138]

PSMA

DOX

CWR22Rv1

[138]

PSMA

Docetaxel

LNCaP

[139]

[135]

Aptamers Engineered Nanocarriers for Cancer Therapy

Delivery system

Ap-MNPs

Multistage stimuli-responsive nanostructured lipid carrier PLL-alkyl-PEI-Ap

EGFR RNA Ap MUC1 DNA Ap HER2 Ap (HB5) AS1411 DNA Ap

EGFR

Cisplatin

HeLa cells

[140]

MUC1

Targeted hyperthermia

MCF-7, HepG2 cells

[141]

HER2

Epigallocatechin gallate and protamine sulfate Bcl-XL shRNA

SK-BR-3 cells

[142]

Whole-cell detection (A549 cells)

[143]

Nucleolin

Ap, aptamer; AuNPs, gold nanoparticles; AgNPs, silver nanoparticles; BIM, apoptosis-inducing protein; DOX, doxorubicin; Dtxl, Docetaxel; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; MNPs, magnetic nanoparticles; MSN-Dox@GO-Ap, graphene oxide wrapped DOX-loaded mesoporous silica nanoparticles; MSN, mesoporous silica nanoparticle; MSNPs, mesoporous silica nanoparticles; PEG-PCL, poly(ethylene glycol)-poly(ε-caprolactone); PTK7, protein tyrosine kinase 7; PLA-PEG-COOH, poly(lactic acid)-blockpoly(ethylene glycol) (PEG) copolymer; PSMA, prostate-specific membrane antigen; PLGA-lecithin-PEG, poly (lactide-co-glycolide) (PLGA)-lecithin-poly(ethylene glycol) (PEG); PEI, polyethyleneimine; PLL, poly(L-lysine); SPION, superparamagnetic iron oxide nanoparticle; SWNT, single-wall carbon nanotube; TfR, transferrin receptor. For details, see Refs. [56,75,144].

Aptamer-armed cancer-targeting nanocarriers

Albumin-cisplatin-Ap

113

114

Aptamers Engineered Nanocarriers for Cancer Therapy

NSs, they can be used for simultaneous imaging and targeted therapy of malignant cells as Ap-based theranostics. As presented in Table 5.2, to actively target cancer cells/tissue, nanoscale DDSs can further be decorated with Aps. In this line, various organic and/or inorganic nanocarriers can be used. These include AuNPs, superparamagnetic iron oxide nanoparticles (SPIONs), hybrid MNPs, functionalized CNTs, QDs, lipid-based nanovesicles (NVs), polymer-based NVs (polymersomes), micellar NVs, dendrimers, exosomes/ethosomes, DNA structures, and even virus-based NPs [48,145e149]. For instance, based on the notion that DNA can be used as 3D polyhedra nanofabricates, the DNA-based NSs have been exploited for intracellular delivery of drugs. Capitalized on DNA NSs using well-used primer sequences, Chang et al. fabricated a distinct Ap-conjugated six-point-star motif to construct DNA icosahedra to serve as NSs for the delivery of doxorubicin (DOX) [150]. Based on their findings, they proposed the engineered Ap-conjugated DOX-intercalated DNA icosahedra as an effective NSs that can specifically internalize and deliver cytotoxic drugs to eradicate the diseased cancer cells. Further, the CG-rich duplex containing prostate-specific membrane antigen (PSMA) aptamer-armed thermally cross-linked SPIONs (TCL-SPIONs) have been used as targeted nanocarriers with potential prostate tumor detection in vivo through magnetic resonance imaging (MRI) as nanotheranostics. Similarly, PSMA-specific Ap armed TCL-SPIONs loaded with DOX displayed superior binding affinity targeting PSMA-positive prostate-cancer LNCaP cells in vitro, which was detectable by T(2)-weighted MRI in vivo and showed selective targeting in the LNCaP xenograft mouse model with high efficacy [151]. In a study, the impacts of EpCAMfluoropyrimidine specific Ap-functionalized PLGA-b-PEG loaded with DOX-loaded were examined in vitro (SK-MES-1 and A549 cells) and in SK-MES-1 non-smallcell lung-cancer nude mice xenografts. The results revealed enhanced cytotoxicity in vitro and profound tumor inhibition in nude mice models [152]. In another study, to specifically target the cancer cells, Powell Gray et al. [153] developed conjugates of aptamers with highly toxic chemotherapeutics. They showed that the developed Ap-drug conjugates using truncated E3 Ap can specifically be taken up by prostate cancer PC-3 cells but not the healthy normal prostate cells (Fig. 5.2). The conjugation of Ap E3 to the cytotoxic agents, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), was found to impose extremely high toxicity, leading to profound eradication of cancerous cells both in vitro and in vivo without damaging the normal prostate epithelial cells (Fig. 5.3). Lv et al. recently showed that the Ap-drug conjugates (sgc8-5FU) can be internalized through caveolin-mediated endocytosis as a cytoskeleton-dependent transport in association with microfilaments and microtubules, which are then trafficked into the acidic endosomes near the cell nucleus in the cytoplasm of breast cancer MCF-7 cells [154]. In a similar study, mertansine (DM1) was conjugated with HER2-specific RNA aptamers. The Ap-DM1 conjugates were examined in terms of cell-binding affinity, cytotoxicity, and efficacy both in vitro and in vivo in BT-474 breast tumors bearing mice. The nanoconjugates resulted in specific and efficient binding to the HERpositive BT-474 cells but not to the HER2-negative MDA-MB-231 cells, inducing specific toxicity. Based on the in vivo experiments in the mice xenografts of

Aptamer-armed cancer-targeting nanocarriers

115

Figure 5.2 The identification of the E3 aptamer with 36 nt by the cell-SELEX in prostate cancer cells. (a) Schematic representation of the selection of 20 F pyrimidine-modified RNA aptamers. (b) The flow cytometry result of the PC-3 prostate cancer cells’ internalization of different selection rounds (i.e., RNA pools from rounds 0, 7, and 9). (c) The mfold software modeled the secondary structure of the minimized 36-nt version of the E3 aptamer. Adapted with permission from Ref. [153].

BT-747 cells, the Ap-drug conjugates showed marked inhibitory effects [155]. Further, to tackle the anaplastic thyroid cancer (ATC), which is known as an undifferentiated extremely aggressive type of thyroid cancer that highly expresses the CD133 stem cell marker, the CD13-specific aptamers (AP-1 and truncated AP-1-M) were conjugated with DOX. The Ap-DOX conjugates were shown to specifically target the ATC both in vitro and in vivo [156].

116

Aptamers Engineered Nanocarriers for Cancer Therapy

Figure 5.3 The E3 aptamer conjugated monomethyl auristatin E (MMAE-E3) and monomethyl auristatin F (MMAF-E3). (a) The molecular structures of E3 aptamer conjugated to maleimide-caproyl-valine-citrulline-p-aminobenzylcarbamate-MMAE or maleimidocaproylMMAF. (b) The targeted region of the E3 aptamer (purple line). (c) The PC-3 cells flow cytometry results upon incubation with 250 nM of either DL650-C36 or DL650-E3  10-fold excess antidote 5. (d) The viability of 22Rv1 cells treated with 100 nM of MMAF-E3 or control conjugate MMAF-C36  2-fold excess of antidote five or with (e) 100 nM of MMAEE3 or control conjugate MMAE-C36  2-fold excess of antidote 5. Viability was determined 144 h postaptamer addition. (f) The NIR fluorescence images of the tumor-bearing mice injected with 2 nmol of either AF750-E3 or AF750-C36 (24 h postinjection). (g) Tumor growth in challenged mice. (h) KaplaneMeier survival curves in mice treated with MMAF-E3. DL650: far-red fluorescence dye, C36: nonspecific control aptamer, Antidote 5: an antidote oligonucleotide to control aptamer activity. Adapted with permission from Ref. [153].

5.5.2

Hybrid Ap-based structures

The hybrid Ap-based systems (the so-called “chimeric Ap”) can be attained through the conjugation of a designated Ap with another non-Ap molecule (i.e., small molecules, peptides/proteins) or a new Ap. The conjugation of an Ap with another Ap results in the production of the bispecific Ap-Ap system [55]. Based on the final application, various non-Ap molecules can be grafted to an Ap, including drugs, dyes, therapeutic oligonucleotides like antisense/siRNA, toxins, peptides/proteins, enzymes, and Abs/Ab fragments. In addition to the conjugation chemistry, the physical

Aptamer-armed cancer-targeting nanocarriers

117

interactions can be utilized in the preparation of the Ap-based hybrid systems [157]. While one molecule detects the target cells, the other applies its diagnostic/therapeutic effect. Table 5.3 represents several applications of different Ap chimera. It should be pointed out that Aps with binding ability to specific cell surface receptors can be isolated against any given CMM [180], including nucleolin (NCL) [181], PSMA [182], mucin 1 (MUC1) [183], and protein tyrosine kinase-7 (PTK7) [184]. The nonspecific impacts of the traditional cytotoxic agents on both the cancerous and healthy cells/tissue can induce intrinsic side effects failing chemotherapy treatment modality [185e187]. Various anticancer drugs (e.g., paclitaxel (PTX), DOX, cisplatin, methotrexate, mitoxantrone, and tamoxifen) can be conjugated to an oncomarker targeting Ap. In addition, Aps have been used to arm a nanoscale DDS to develop Ap-mediated targeted therapies [24,41,48,56,75,103,120,144,188e193]. Multifunctional Ap-miRNA scaffold was engineered by grafting an Axl receptor (GL21.T RNA Ap) specific RNA Ap to the human let-7g miRNA [194]. To have a sound NS, the passenger strand of let-7g miRNA was grafted to the 30 end of the Ap and the double-strand of miRNA was formed. It was shown that the GL21.T could specifically bind with the Axl receptor and enter the cells, significantly inhibiting the Axl tyrosine kinase signaling pathway and hence suppressing the proliferation of the targeted cells. Such impacts were further validated in the xenograft model of adenocarcinoma. Aptamers have been used for the modulation/regulation of the membrane proteins whose overexpression might be linked with the initiation and/or progression of various diseases such as malignancy. Miao et al. [176] reported on a bispecific Ap chimeras platform for the targeted degradation of membrane-associated proteins. They developed bispecific Ap chimeras, which were able to bind with both the cell-surface lysosome-shuttling receptor and the therapeutically relevant membrane-bound proteins such as mesenchymal-epithelial transition (MET) receptor, and Protein Tyrosine Kinase 7 (PTK-7). They demonstrated that the bispecific Ap chimeras could effectively transport the membrane proteins Met and PTK-7 to the lysosomal compartments where the shuttled proteins were degraded by enzymatic degradation machinery (Fig. 5.4).

5.5.3

Bispecific Aps with antitumor immunity function

Innate and adaptive immune defense systems not only control the infections but also play key roles in the suppression of cancer. Upon the initiation of a solid tumor, the cancer cells progress via forming a unique setting, the so-called tumor microenvironment (TME), with aberrant features such as the formation of leaky irregular microvasculature and remodeled immune system. In the TME, the immune system cells are deceived by cancer cells and reprogrammed. As a result, they coadapt with the cancer cells in the TME. Such a cooperation results in the disfunction of immunosurveillance, leading to further proliferation, progression, and invasion of cancer cells. In solid tumors, activated CD8þ and CD4þ T cells are the most tumor-infiltrating lymphocytes (TILs). However, in most cases, these cells are not as responsive as they should be and

Type of chimera ApssiRNA/ ShRNA

Ap-chimera

Target/effect

Cell line/in vivo

Refs.

PSMA Ap-survivin siRNA EpCAM Ap-siRNA PSMA Ap- DNA-PK siRNA Ap-siRNA chimera

Survivin mRNA

LNCaP cells

[158]

Upf2, Parp1, Cd47, and Mcl1 DNA-dependent protein kinase

Transgenic mice LNCaP/Systemic administration in human tumor xenografts Gastric cancer cell line MKN45 and nude mice model CD4þ T/humanized mice Breast cancer MCF-7 and MDA-MB-231 cells LNCaP cells BT474, SKBR3, MDA-MB-231, MCF7, and Hs578T cells; BT474 cells xenografted mice LNCaP cells

[159] [160]

CD4 Ap- CCR5 Ap-siRNA chimera PSMA Ap- EEF2 siRNA Bivalent HER2 aptamerEGFR siRNA chimeras PSMA Ap- DNAPK shRNAs EpCAM ApeEp CAM siRNA (EpAptsiEp) CD38 Ap-doxorubicin Sgc8c Ap-combretastatin A4 CD117 Ap-MTX PSMA Ap-DOX AP-1-M Ap-DOX ABCG2 Ap-DOX Sgc8c Ap-DOX

Drug-resistant gene CeC chemokine receptor type 5 EEF2, PLK1, GRK4, SKIP5 Eukaryotic elongation factor 2 mRNA EGFR DNA-dependent protein kinase, catalytic subunit EpCAM

Induction of apoptosis Inhibiting tubulin polymerization and eliciting apoptosis Eliciting apoptosis Inducing apoptosis CD133 oncomarker ABCG2 oncomarker Inducing apoptosis

[161] [162] [163] [164] [165] [166]

MCF7 cells

[167]

MM1S cells Pancreatic cancer cells

[168] [169]

HEL cells LNCaP cells CD133 positive FRO cells Drug-resistant MCF7/MX breast cancer cells CCRF-CEM

[170] [171] [156] [172] [173]

Aptamers Engineered Nanocarriers for Cancer Therapy

Ap-drug (ApDC)

118

Table 5.3 Selected hybrid Ap chimeras used for targeted therapy.

4-1BB–PSMA bivalent Ap Multivalent 4-1BB Ap IGFIIR Ap- membranebound proteins Aps CD16a -c-Met bivalent Ap MRP1-CD28 bivalent Ap TE02-LD201t1 multi valent, bispe cific Ap

Costimulation at the tumor site

CT26 colon carcinoma cells bearing mice

[174]

Costimulation of activated T cells Different membrane-bound proteins (Met and PTK-7) Mimic ADCC by recruitment of natural killer cells to tumor cells Deliver the CD28 costimulatory signal to tumor-infiltrating lymphocytes Targeted B cell and T cell interactions

CD8þ T cells Modulation of membrane proteins

[175] [176]

PBMC, NK, GTL-16 cells

[177]

B16-MRP1,CD8þT, Melanoma-bearing mice

[178]

Jurkat, Ramos cells

[179]

ADCC, antibody-dependent cell-mediated cytotoxicity; Ap, aptamer; DOX, doxorubicin; EpCAM, epithelial cellular adhesion molecule; EEF2, eukaryotic elongation factor 2; GRK4, G proteincoupled receptor kinase 4; IGFIIR, a cell-surface lysosome-shuttling receptor; LD201t1, Ap for Jurket (T cell line); MET, mesenchymal epithelial transition receptor; MTX, methotrexate; PLK1, polo-like kinase 1; PSMA, prostate-specific membrane antigen; PTK-7, protein Tyrosine Kinase 7; SKIP5, sphingosine kinase interacting protein; ; Sgc8c, Ap for CEM cells; sgd5a, Ap for Toledo cells; TE02, Ap for Ramos (B cell line). For details, see Refs. [56,75,144].

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Ap-Ap (bispe cific Ap)

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Figure 5.4 Degradation of targeted protein of cell membranes by bispecific aptamer chimeras. (a) The A1-L-A2 construct is composed of an IGFIIR aptamer (A1), a linker (L), and an aptamer (A2) specific to cell membrane proteins. (b) Three different types of A1-L-A2 (D1, D2, and D3). (c) The schematic mechanism of degradation of the targeted membrane protein by A1-L-A2 via IGFIIR-mediated lysosomal protein degradation machinery to lead the target membrane proteins to lysosomal compartments. Adapted with permission from Ref. [176].

fail to control the progression of the tumor. As a result, the immune cells in the TME become either nonresponsive or less responsive to tumor-associated antigens (TAAs). Such disability is presumably associated with marked dysfunction macrophages and the incompetency of tumor-associated dendritic cells (DCs) in presenting TAAs to the immune cells in various solid tumors [195,196]. For the active targeting of tumor cells, a few strategies have been developed to engage the immune system cells. These include innate and adaptive immune system cells such as natural killer cells (NK), DCs, and effector T lymphocytes. Given the importance of the antibody-dependent cellular cytotoxicity (ADCC), several bispecific Abs (biAbs) have so far been developed and used for the active targeting of different types of malignancies via activating the immune system [197e203]. Notably, compelling evidence corroborated the clinical impacts of blinatumomab as the trailblazing

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T-cell engaging biAb (T-biAb), in addition to other types of Ab scaffolds and also chimeric antigen receptor T cells (CAR-Ts). Among T-biAbs, the NK-cell engaging biAbs (NK-biAbs) are also emerging as alternative immunotherapy agents in addition to other types of biAbs developed to engage the molecular components and cellular entities of both innate and adaptive immune systems against cancer [202e206]. Aligned with the biAbs, some bispecific Aps (biAps) have also been developed to specifically target the chemotherapy-resistant tumors expressing various oncomarkers. Given the importance of the ADCC in Ab-based tumor therapies through NK cells via binding of the Fcg receptor III (CD16), Boltz et al. produced chimeric DNA aptamers using SELEX technology. The biAps were able to simultaneously bind with CD16a and overexpressed oncomarker c-Met [177]. The CD16a specific DNA Aps with high specificity and affinity were selected and two optimized CD16a specific Aps were coupled to c-Met specific Aps using different linkers. The engineered BiAps showed appropriate binding potential to both molecular markers and elicited marked cellular toxicity, in which the displacement of the biAps from CD16a validated the proposed mode of action of biAps-mediated cellular cytotoxicity. Likewise, multidrug resistance-associated protein 1 (MRP1) and the T cell expressed protein CD28 involved in co-stimulatory signals required for T cell activation and survival were targeted using MRP1-CD28 specific biAps [178]. This can potentially activate the TILs against the chemotherapy-resistant melanoma-bearing mice inducing substantial antitumor immune responses. The Ap-based T cells activators and modulators have also been developed. Remarkably, T cells need Ag-specific and costimulatory signals to be activated. The first signals are engaged in the interaction of the T cell receptors with MHC molecules. The costimulatory signals interact with the relative receptors on T cells such as CD28 via expressed molecules like B7.1/B7.2 on APC or other cells like B cells/macrophage. Based on this concept, McNamara II et al. developed an Ap that binds with the 4-1BB receptor expressed on the surface of activated T cells [207]. They showed that proper bivalent/multivalent Ap-based constructs can induce the costimulation of T cells against cancer cells. Capitalized on a Y-shaped DNA Ap scaffold, Zheng et al. developed biAps to redirect NK cells for enhanced adoptive immunotherapy against hepatocellular carcinoma [208]. They demonstrated that the proposed Y-type biAp could show potent avidity toward NK cells and tumor cells, inducing higher secretion of cytokine with substantial antitumor effects.

5.5.4

Ap-based multimodal NSs

Recently, much attention has been paid to the design and development of Ap-based multimodal NSs, which are composed of targeting, imaging, and active therapeutics agents. Such multifunctional NSs are referred to as theranostics/diapeutics [57,209e212]. Ap-based NSs have been designed to specifically target designated cells and impose desired multimodal impacts such as imaging. Subjects related to the imaging device

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and surface modification and bioconjugation paradigms have already been well reviewed [210,213]. Various imaging modalities have been clinically used for the diagnosis of cancer, including some optical imaging techniques, computed tomography (CT), ultrasound (US), MRI, and positron emission tomography (PET). Despite their unique advantages, the use of such imaging and detection technologies might associate with some shortcomings such as (i) inadequate sensitivity, (ii) deficiency in spatial resolution, and (iii) lack of specificity. Such issues might impose difficulties in obtaining images with high accuracy and precision. As a result, the use of advanced NSs for precision imaging can be greatly beneficial. Markedly, the multimodal NSs with imaging potential can be advantageous based on their combined use (e.g., PET/CT or PET/MRI) [214,215]. Such approaches can be much more powerful and sound detection techniques upon the integration with theranostics. To date, many nanoscale formulations such as magnetic NPs and gold NPs have been engineered to serve as nanoprobes in multimodal imaging, including optical/MR, optical/CT, optical/PET, PET/CT [215]. Fig. 5.5 represents various organic and inorganic Ap-armed PEGylated radionuclide-armed drug-loaded NSs. To date, some works have been carried out in terms of the advancements of nanoscale multimodal imaging probes. These multimodal nanostructures possess at least two imaging agents. Such nanostructures provide much greater detailed information about anatomy and biology of the designated target disease. Notably, such NPs might possess intrinsic imaging potentials. For instance, MNPs and AuNPs have been used in optical and MR imaging. Various multimodal imaging nanoprobes could be established through conjugation or coencapsulation processes, which can be designed as polymer-/lipid-based nanoformulations with conjugated targeting and imaging agents [216]. Zhu et al. produced Ap-tethered DNA nanotrains (aptNTrs), via selfassembly of two short DNAs, to serve as vehicles in targeted drug delivery of cancer [217]. The aptNTrs were shown to induce potent antitumor efficacy with minimal side effects. Their further integration with fluorophores allowed designated imaging as cancer theranostics. To improve the photoresponsive AuNPs properties (e.g., thermal conductivity), Ye et al. produced a novel irregularly-shaped CueAu alloy nanostructure via a one-pot procedure [218]. Such nanostructures demonstrate advantages including, (i) broader intense near-infrared (NIR) absorption (from 400 to 1100 nm), (ii) greater thermal stability and performance upon laser irradiation at various wavelengths, and (iii) higher photostability. To engineer multimodal Ap-based NS for imaging and targeted therapy of leukemia, CueAu alloy nanostructures were coated with Cy5-labeled Sgc8c Ap to attain selective fluorescence imaging that could serve as the NIR photothermal therapy agent. The engineered NS showed high target recognition with improved signal stability for molecular imaging, which also rigorously eradicated the target cancer cells in mice after 5 minutes of irradiation at 980 nm. Altogether, distinct types of Ap-based multifunctional NSs have been developed using different nanomaterials and biomaterials. Such advanced systems need to be fully evaluated in terms of pharmacokinetic and pharmacodynamic properties as well as toxicity at cellular and molecular levels.

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Figure 5.5 Various organic and inorganic Ap-based theranostics. Ap-armed PEGylated radionuclide-linked drug-loaded NSs can be formulated using organic and inorganic materials such as (a) lipids, (b) polymers, (c) MNPs, and (d) AuNPs. Ap, aptamer; AuNP, gold nanoparticle; MNP, magnetic nanoparticle; PEG, poly(ethylene glycol).

5.6

Concluding remarks

Aptamers, as oligonucleotide (i.e., DNA/RNA) nanostructures, can specifically interact with their cognate targets ranging from small to large molecules and even cells. They can be selected through the SELEX process. Aptamers offer unique characteristics, including specific binding affinity, great stability in biological fluids (especially

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for DNA Aps), small size, and simplicity in terms of the selection process. All these features make Aps attractive macromolecules not only to serve as a targeting agent for the diagnosis and development of targeted therapies but also as the therapeutic agents. These macromolecules can simply be functionalized and modified using various functional groups. As a result, they can be conjugated onto nanoscale DDSs and nanomaterials and implanted for distinct pharmaceutical and/or biomedical applications. Given their remarkable pharmacokinetic and pharmacodynamic characteristics, various Ap-based multifunctional NSs (aptamedicines, biAps, Ap-armed DDSs, and aptasensors) can be developed. Such nanoscale multimodal systems are envisioned to be developed in a tunable quality-by-design fashion and applied to control/treat various ominous malignancies.

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Aptamer-functionalized liposomes for targeted cancer therapy

6

Seyedeh Alia Moosavian 1 , Prashant Kesharwani 2 , Vanshikha Singh 2 and Amirhossein Sahebkar 3, 4, 5,6,7 1 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran; 2Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India; 3Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran; 4 Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran; 5Department of Biotechnology and Nanotechnology, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran; 6School of Medicine, The University of Western Australia, Perth, WA, Australia; 7Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan Province, Iran

6.1

Introduction

Despite many advances in cancer therapy, Current chemotherapy still faces challenges such as cytotoxicity, drug resistance, and lack of specificity. The therapeutic index of most cancer drugs is narrow, and they are associated with wide off-target toxicity [1]. The use of nanoparticles as delivery systems has been extensively studied during the last decade [2,3,4,5]. Using nanoparticles to deliver anticancer drugs offers great promise for cancer therapy. Despite significant advances, nanoparticle-based delivery systems still suffer the lack of tumor specificity. Hence, finding the best therapeutic delivery system is still an ongoing process. Since the discovery of liposomes by Bangham and colleagues in the 1960s, the potential of liposomes as drug delivery carriers in cancer therapy has been extensively investigated [6]. The presence of aqueous and lipid phases in the liposome structure allows them to entrap both hydrophilic and hydrophobic agents [7]. In addition, owing promising characteristics such as high biocompatibility, low toxicity, and lack of immunogenicity, high stability, and high drug loading efficiency makes them a promising carrier to deliver chemotherapeutics [8,9]. PEGylated liposomal doxorubicin, Doxil is the first FDA-approved liposomal platform, which is indicated for the treatment of solid tumors. Encapsulation of doxorubicin in PEGylated liposomes significantly modified pharmacokinetic profile of doxorubicin [10]. The circulation

Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00014-2 Copyright © 2023 Elsevier Ltd. All rights reserved.

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half-life (T1/2 ) increased and the clearance rate reduced. Moreover, doxorubicin associated cardiotoxicity is significantly lower with liposomal formulation [11]. Other liposomal products approved for cancer treatments include cisplatin, daunorubicin, cytarabine, and vincristine [12]. The access of all these liposomal formulations to the tumor site is through “enhanced permeability and retention” (EPR) effect mechanism which involves defective tumor blood vessels and blood flow. The discovery of EPR in solid tumors has led to the development of nano-particulate delivery systems for cancer treatments [13]. The EPR effect-based nanoparticle delivery has been discussed in details extensively [14e17]. Liposomes are of prime importance for receiving the benefits of the EPR effect. Tumor accumulation of liposomal formulations depends on two parameters: (1) tumor microenvironment and (2) physicochemical characteristics of liposomes. The natural response of body to physicochemical characteristics of the delivery system determines passive targeting [1]. In addition to improving the pharmacokinetic profile of chemotherapy drugs, passive targeting has also brought new challenges to the treatment process. Taking Doxil as an example, although tumor accumulation of doxorubicin is improved, only a modest increase in treatment efficacy can be observed [18e23]. Due to a high in vivo stability and EPR effect Doxil is delivered to tumor tissues, but it has a low rate of drug release from the liposomes, which is a crucial issue [24]. This challenge also applies to liposomal formulations of other chemotherapeutic agents such as cisplatin [25]. Drug resistance may also occur due to the slow release of drugs from stealth liposomes [18]. It has been reported that the effectiveness of stealth liposomes are limited in many cancers like nonesmall-cell lung cancer, sarcoma, or hepatoma [26]. Despite improving pharmacokinetic profile and ameliorating cardiotoxicity of doxorubicin, the slow drug release in Doxil can lead to newfangled side effects like hand-foot syndrome, myelosuppression and mucositis, which could be consequences of the slow release of the encapsulated drug [27,28]. In addition, in hypovascular tissues such as prostate, pancreas and liver tumors, we cannot rely on EPR effect, hence employing stealth liposomes is not effective [29e31]. Meta-analysis demonstrated that even in tumors with high EPR only less than 1% of NPs reach tumor sites [32]. Finally, strong evidence suggests that passive targeting is primarily challenged by slow release of encapsulated agents from sterically stabilized liposomes in tumor interstitium and cellular uptake of released agents [1,14]. By improving the targeting efficiency of nanoparticles (NPs) and increasing retention at the target site, active targeting has been developed as a complementary strategy to EPR-based drug delivery systems to improve tumor localization of NPs (Fig. 6.1). Incorporating targeting ligands on the surface of NPs increases their cellular internalization in target sites without affecting their overall tumor localization [33,34]. A variety of ligands including antibodies, antibody fragments, peptides, nucleic acids and small molecules have been employed for this purpose [35]. Over the past 40 years, most the studies have focused on monoclonal antibodies as a potent modulators of target protein functions. The most significant problem with commercial antibody reagents is reproducibility [36]. Thus, there is an emerging consensus that researchers should switch to antibodies made from traditional sources. Among these ligands, aptamers are considered as a promising candidate with unique features for targeted delivery [37e39].

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Figure 6.1 Aptamer-targeted liposomes are promising delivery platform for chemotherapy drugs to tumors by passive tissue targeting and active cellular targeting. Passive tissue targeting uses the increase permeability of tumor vasculature and the poor lymphatic drainage of tumors (EPR effect), allowing the release of chemotherapeutic agents in the vicinity of the tumor. Active cellular targeting is achieved by functionalization of the surface of liposomes, containing chemotherapy drugs, with aptamers that recognize cell surface receptor with high affinity and efficiency, then aptamer-receptor mediated cell internalization of liposomes via endocytosis.

Aptamers are short, synthetic and single-stranded DNA or RNA molecules that are capable of binding their target with a high affinity and specificity [40]. Systematic evolution of ligands by exponential enrichment (SELEX) is a process which identifies aptamers from a random nucleic acid library [40]. Aptamers have noteworthy features including small size, high affinity and selectivity to their target, simple synthesis process, low immunogenicity, and stability across a wide range of physicochemical conditions that make them a promising agent as targeting ligands. Furthermore, aptamers can be synthesized in large amount out of the biological systems with minimum risk of viral or bacterial contaminations [37]. Therefore, owing privileged characteristic over antibodies, they are potential alternatives to antibodies (Table 6.1). Aptamers do not have the Fc region which stimulates the immune system in systemic administration. In comparison with antibodies, aptamers have higher tumor penetration, retention and their distribution are more evenly that attributed to their smaller size [45e47]. Due to the smaller size, decorating the surface of nanoparticles with aptamers causes less steric hindrance than antibodies. Aptamers attach more readily and more

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Table 6.1 Properties of aptamers versus monoclonal antibodies [41e44]. Monoclonal antibodies

Aptamers

Binding affinity Development process

Nanomolar to picomolar range Requires an immune response in animal model

Manufacturing process Cost of production Reproducibility

In vivo production; cell culture

Nanomolar to picomolar range SELEX (systematic evolution of ligands by exponential enrichment) process In vitro, chemical synthesis

Reasonably high and timeconsuming Little batch-to-batch variation

Modification

Antibodies are typically conjugated with one type of signaling or binding molecule

Stability

Susceptible to high temperatures and pH changes; irreversible denaturation Significant immunogenicity Higher chemical diversity as the building blocks of antibodies are 20 different amino acids

Immunogenicity Structural diversity

Size Target size

w150e170 kDa
600 Da

Inexpensive and take a few weeks Activity of antibodies varies from batch to batch Wide variety of chemical modifications are introduced to diversify properties and functions Fairly stable at wide range of temperature; reversible denaturation No evidence of immunogenicity Lower chemical diversity, as aptamers have only four building blocks (nucleotides). However, their diversity can be increased by chemical modification w12e30 kDa
60 Da

reproducibly to nanoparticle surfaces than antibodies [41,48]. Unlike antibodies, they can be supplied in dried form at room temperature and reversibly thermally denatured by changing the surrounding conditions [49]. Hence, replacement of antibodies by aptamers is a potential strategy to improve tumor penetration of nanoparticles. Owing to these features, aptamers are considered as promising ligands for active targeting purposes. Surveying literatures show there is growing interest in employing aptamers that target cell surface receptors for use in the targeted delivery of therapeutics [50,51]. Despite the broad application of actively targeted nanoparticles in research, only a handful of them have advanced to clinical trial stages of development [52]. Apparently, incorporation of ligand poses significant challenges toward translation of NPs from bench to clinic [2,53]. As witnessed by many studies, liposomes are by far the most successful type of nanoparticles used in drug delivery. They are well-investigated nanocarriers owing to their unique ability to entrap a diverse range of hydrophilic and hydrophobic drugs. Liposomes are also biocompatible delivery systems with the capacity of selfassembly. The characteristics of liposomes can be modified to achieve optimal biological properties

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without losing their stability or function [54]. Moreover, aptamers are promising targeting ligands with optimal specificity and selectivity. Herein, we provide an overview of studies dealing with the application of aptamers as targeting ligands on liposomes. The methods used for the preparation of aptamer-liposome conjugates are also elaborated.

6.2

Conjugation strategies in aptamer-targeted liposomes

Aptamers are often chemically conjugated or physically adhered to the surfaces of liposomes. Adherance can be achieved by electrostatic interactions between negatively charged aptamers and cationic liposomes. This association rarely used in targeting studies because interferes with aptamers second structure and deactivate it as targeting molecule. Noncovalent adherence usually has been used in gene delivery studies that nucleic acid-based molecules delivered to cells by liposomal carriers and depends on factors such as size, surface charge, functional groups, and hydrophobicity of the liposomes and aptamers, as well as the interaction medium [55,56]. For ligand-targeting purposes covalent attachment is used even in cationic liposomes because it provides a stable platform in the biological environment and it is less affected by pH, temperature or presence of other biomolecules [55,57,58]. For covalent linkage, conjugation usually accomplished through functional group that added at the 30 and/or 50 terminals of aptamers and lipid chains or free terminus of polyethylene glycol (PEG) chains (Fig. 6.2). The widely used method in studies is conjugation of aptamers via PEG chain, but in some studies the linking were performed through the lipid part. Common functional groups that have been studied in aptamer-liposome conjugation are the conjugation of maleimide with thiols, and the conjugation of carboxyls with amines mediated by N-hydroxysuccinimidyl (NHS) along with carbodiimide (EDC). The following sections summarize the methods that are widely used for functionalization of liposomes with aptamers in studies, i.e., membrane anchor method and post-insertion method.

6.2.1

Membrane anchor method (pre-conjugation strategy)

In this method, aptamers are integrated into the liposome structure during the preparation process of liposomes. The bio-conjugation is accomplished in a single step which facilitates the purification of prepared liposomes [14]. In preconjugation method, aptamers are exposed to organic solvents that could renders the folded structure of aptamers and thereby the targeting ability of aptamers could be affected [14]. Moreover, during bioconjugation a portion of the aptamer molecules is located in the inner surface of liposomes. This could reduce targeting efficiency of platform, occupy the internal space of liposomes and interact with payload, and make liposomes susceptible to hydrolytic degradation [59]. Willis et al. decorated liposomes by NX213 aptamer. They conjugated aptamer to di(otadecyl)glycerol (DAG) and then liposomes prepared by DAG-conjugated-NX213. They used this platform to delivery of NX213 as vascular endothelial growth factor (VEGF) inhibitor. They reported about one-third

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Figure 6.2 Illustration of a typical aptamer-targeted liposome (a), aptamers conjugated directly on the phospholipid headgroups of PEGylated liposomes (b); andbaptamers conjugated on the free terminus of PEGylated chains (c).

of the aptamers were located in interior space of liposomes. They used Ribonuclease T1 digestion method to determine the amount of aptamers was located in the internal space of liposomes(Fig. 6.3a) [60]. Tan and coworkers conjugated 30 -thiol-modified sgc8 aptamer to maleimide-terminated PEG-DSPE (MalPEG) and prepared liposomes (HSPC/cholesterol/mPEG-DSPE/MalPEG-DSPE) by extrusion method. Aptamer conjugation significantly improved cellular uptake of liposomes [61]. In two separate studies, membrane anchor method was used to functionalization of doxorubicin and cisplatin liposomal formulations with AS1411 aptamer. Aptamers incorporated into liposomes via cholesterol [62,63]. As Xing et al. reported, cholesterol-modified AS1411 were immobilized onto the surface of liposome by intercalating the 30 cholesterol modification into the lipid bilayer. As their results shows a small fraction of aptamers are located in interior space of liposomes [62].

6.2.2

Postinsertion method

Postinsertion is a simple, flexible method which is compatible with variety of ligands for prepare ligand-targeted liposomes. In this method, ligands of interest like aptamers are firstly covalently linked to polyethylene glycol (mPEG)-lipid micelles, and

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147

Figure 6.3 Schematic representation of liposome-anchored aptamers. It is clearly representing a portion of aptamers are located in the inner surface of liposomes (a); Schematic showing the incorporation procedure of DSPE-PEG-aptamer micelles in liposomes structure by postinsertion method (b).

afterward prepared micelles are incubated with preformed drug-loaded liposomes, during incubation with gentle mixing, the micelles transfer into the liposomal bilayer Fig. 6.3b [64,65].

6.3

Factors affecting the efficiency of aptamerfunctionalized liposomes

The process and efficiency of aptamer-liposomes conjugation is dependent on factors such as environmental situation, aptamer, and liposome characteristics. Environmental related factors include pH, temperature and biological enzymes. The following sections will discuss the parameters that must be considered in aptamer-targeted liposome formulations.

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6.3.1

Aptamers Engineered Nanocarriers for Cancer Therapy

Conjugation chemistry of aptamers and liposomes

Carbodiimides and Thiol maleimide coupling chemistry are frequently used bioconjugation method for aptamer-lipid attachment. Thiol-maleimide reaction is widely used for aptamer bioconjugation [58]. In this process, maleimide polyethyleneglycol (PEG-Mal) is conjugated to the thiol-modified aptamer. Carbodiimide coupling is commonly used to conjugation of aptamer to the surface of polymeric nanoparticles and liposomes [58,66]. We have exploited this method in several experiment to functionalize liposomes with different aptamers [67e70]. In this process, carboxyl group of PEGylated1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE-COOH) is activated by ethyl(dimethylaminopropyl) carbodiimide (EDC) and NHS reagents. Afterward, the activated carboxylate is incubated with amine-modified aptamer [70]. In some studies, carboxylate-modified aptamer has been used to bind to PEG-DSPE-NH2 micelles. Carbodiimide bioconjugation approaches efficient and stable conjugate. While SH-modified aptamers have potential of oxidation in thiol site and disulfide bond may form between aptamers [58]. The addition of reducing agents such as tris (2-carboxyethyl) phosphine to prevent oxidation during the reaction process is necessary. In Carbodiimide bioconjugation, unreacted carboxylate may affect zeta potential of liposomes. However, in cationic liposomes this could prevent nonspecific reactions between negatively charged aptamers and liposome surface [58]. In another study, Li et al. conjugated carboxylmodified aptamers to the amine group of chitosan using EDC/NHS reagents; then, incorporated the aptamer-chitosan bio conjugate into the liposome structure [71].

6.3.2

The spacer structure

Aptamer-liposome attachment is typically facilitated by spacer molecules located between the surfaces of aptamers and liposomes in order to minimize liposome interference that could compromise their ability to recognize ligands. Exploiting spacer is highly recommended for truncated aptamers because direct attachment to the surface of liposomes could interfere with folding of aptamers and inactive it. Different spacers have been used in including PEGs, hydrocarbons or nucleotide chains with different size and hydrophobicity. The length of spacer must be long enough to expose aptamer on the surface of liposomes and prevent liposome interference with aptamer binding. PEG chain is commonly used as spacer in aptamer-liposome conjugation. Another common spacer is obtained by elongation of aptamer for example by addition of several thymine bases to the 3’ or 50 terminal of aptamer. The orientation of aptamers on the surface of liposomes and addition of spacer from 30 versus 50 terminal should be optimized [72]. Spivak et al. showed that correct orientation facilitate high binding efficiency because it influence on correct folding of aptamer [73]. The length of spacer could affect the performance of attached aptamer. Kawano et al. found that increasing the length of the PEG chain improves cellular uptake of folate-targeted liposomes, without any changes in cytotoxicity [74]. In the same study, Thakur and colleagues showed folate-targeted dendrimers had higher tumor-targeting efficiency when the length of PEG chain was longer [75]. The hydrophilicity of spacers is another important factor that could influence on membrane diffusion of nanoparticles and their

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stability in blood circulation [76]. Kokkoli and coworkers investigated the effect of spacer characteristics on the binding affinity of aptamers to the amphiphile moiety [77]. Their results showed aptamers bind to their targets most effectively when nucleotide spacers are used. PEG spacer was in the second place. Hydrocarbon chains and direct conjugation of aptamer to lipid without using spacer had the lowest cytotoxicity. In 2016, Xing et al. studied the effect of employing different spacer with various length and type on the targeting efficiency of aptamer-decorated liposomal doxorubicin. Their results revealed the length of oligoT (thymine) spacer affect on the interaction between AS1411 aptamer and PEG chain on the surface of liposomes [78]. They demonstrated lengthening thymine chain could evade the hindrance effect of PEG. Because to reach the maximum targeting efficiency the fully exposure of aptamer on the surface of liposomes is crucial [78]. Dai et al. also revealed that to overcome hampering effect of PEG, the length of spacer should be longer than backfilled PEG [69,70,79,80]. Employing aptamer-tethered DNA is another way to increase the exposure of aptamer from the surface of liposomes. Baek and coworkers attached RNA aptamer to Peg chain via a tethering DNA chain and to obtain a proper exposure [81]. Spacers enable aptamers to be fully exposed on the surface of liposomes and facilitate aptamer-target interaction. The length of spacers, rather than their structure, seems to be more important for achieving the best targeting effect.

6.3.3

Aptamer characteristics

The secondary structure of aptamers is critical factor on aptamer-target interaction. Any alteration in secondary structure and folding of aptamer may lead to loss of functionality [82]. Hydrophilic interactions like hydrogen bonding and, polar and electrostatic interactions mediate aptamer-target interaction. Modifying aptamers to provide additional hydrophobic interactions between aptamers and their targets have been investigated in different studies [83e85] the presence of guanine-rich sequence in the structure of aptamers provides a unique structure that binds to the target with high affinity and specificity. guanine-quadruplex (G-quadruplex) containing aptamers are highly resistant against serum nucleases thermal changes and are more efficient to recognize their target [86,87]. Methods of enhancing aptamers’ affinity have been comprehensively reviewed by Hasegawa et al. [88]. Chemical modification of aptamers is a common way to enhance stability and efficacy of aptamers. Aptamers are sensitive against nuclease degradation in serum environment. Modification of natural nucleotides (i.e., 20 -OCH3, 20 -NH2 or 20 flouropyrimidine), substituting natural nucleotides with hydrocarbon linkers or capping terminal ends of aptamers are some chemical techniques to overcome this challenge [58,89]. The length of aptamers is also important issue that should be considered. The folded structure of aptamers is crucial to recognizing the target receptor. The Long chain allows aptamers to fold in more complex form and enhance binding efficiency. Truncation optimization after SELEX process is a way to shorten length of aptamer and improve specificity and robustness of aptamer.

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Keeping the loop sequence is crucial to save aptamer’s binding ability [90,91]. Elongation allows aptamers to fold properly after biconjugation, while short aptamers need spacer to avoid inactivation after immobilization on the surface. In truncated aptamers the addition of spacer is highly recommended to evade steric hindrance [72]. One of the important advantages of aptamers with smaller size is the less interference with size and surface charge of liposomes which in turns reduces pharmacokinetic profile alteration after functionalization [14,92]. Unlike antibodies that can changes the diffusion and penetration of nanoparticles into the tumor tissue [93], We have previously reported that small size aptamers have a minimum effect on the pharmacokinetic properties of liposomes in animal models [69,70]. According to studies, the advantages of minimizing the length of aptamers outweigh the disadvantages [94,95]. A direct benefit of truncation is reducing production costs and increasing production yields of aptamers. Some studies have found that shorten aptamers chain can have a beneficial effect on the binding characteristics and reduce the risk of immunological reactions of aptamers [49,95e99].

6.3.4

Surface density of aptamers

The surface density of aptamers affects the surface charge of liposomes and enhances its recognition by immune cells. Gu et al. showed how the optimization of surface density of aptamers improves the tumor penetration of nanoparticles [100]. An excessive amount of aptamers reduces the surface charge and inhibits efficient cellular uptake [92]. Employing multivalent aptamers instead of monovalent aptamers reduces the surface density and enhances their binding efficiency [38].

6.4

Aptamer-mediated targeted delivery of liposomes

Active targeting of liposomes by attaching aptamers allows the selective accumulation in tumor cells, (b) increase cytotoxicity in tumor cells, and decrease off-target toxicity. Generally, aptamer-targeted liposomes work perfectly in vitro (Fig. 6.4aec). Aptamers improve cellular uptake of liposomes via receptor-mediated endocytosis. The mechanism of cellular uptake has been discussed comprehensively in previous studies [101,102]. In brief, interaction of aptamer with surface receptor of target cells and liposomes internalize via engulfment. In cytoplasm liposomes fuse with early endosomes and Early endosomes differentiate into late endosomes. In endolysosomes, liposomal payload releases by hydrolytic enzymes and pH change [66,103]. Even though aptamer-targeted liposomes hold great promise, it faces several challenges. Surveying literatures reveals, aptamer-targeted liposomes are more effective platform in vitro; however, the results in animal models are controversial. For example, Rudnick et al. showed in antibody functionalization, higher affinity to cell surface receptor does not necessarily improve tumor penetration and targeted nanoparticles are not able to penetrate to central part of tumor [93]. It is possible to generalize

Figure 6.4 The uptake of various aptamer functionalized liposomes by their target cells. Histogram of the alteration in cellular uptake of liposome at presence and absence of aptamers. Target cells were treated with different free drug, encapsulated drug in nontargeted liposomes and encapsulated drug in aptamer-targeted liposomes for 1, 3 and 6 h of incubation at either 37 C or 4 C. Then, the cells were lysed and percent of drug associated with cells was measured (a) [70] Flow cytometry analysis indicate the uptake of the anti-EpCAM RNA aptamertargeted liposomal doxorubicin and Caelyx in C26 cells at and 37 C (b), and The results of cell internalization of doxorubicin on C26 cell lines visualized by fluorescent microscopy. Cells stained with DAPI. Both free doxorubicin and anti-EpCAM RNA aptamer-targeted liposomal doxorubicin have higher level of doxorubicin internalization compared to the Caelyx. Cells inspected under 200 magnification (c) [67]. Reproduced with permission (License code: 5210071267774, 5210060191555).

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this finding to other targeting ligands like aptamers. Although aptamer have small size, but the animal studies show they can alter pharmacokinetic of liposomes [46]. In 2011, Laurent and coworkers suggested that targeted nanoparticles are covered by dynamic cove of biomolecules in serum environment which is called protein corona. This phenomenon can reduce targeting capability of ligand-targeted nanoparticles which leads the inconsistency of the in vitro and in vivo results [104e107]. Varnamkhasti et al. showed the targeting efficacy of MUC1 aptamer-functionalized SN-38 is affected by the addition of bovine serum albumin [108]. Ding et al. explored how the presence of human blood serum affects targeting efficacy and release profile of aptamer-targeted gold nanoparticles. They showed surface adsorption of proteins could induce immune response, which in turn increases clearance rate of nanoparticles. They found the effect of protein aggregation on the surface of nanoparticles is more critical in smaller nanoparticles [109]. In our studies, the doxorubicin concentration of liver and spleen in aptamer-targeted liposome received mice was higher than nontargeted liposomes received groups, while the circulation half-life had no significant difference [69,70]. Whereas, some studies reported an increase in the circulation time of liposomes after aptamer conjugation [110]. It is supposed that the negative charge of aptamer-liposome conjugates prevents the interaction of liposomes with negatively charged immune cells. In addition, the smaller aptamers have less effects on zeta potential and pharmacokinetic properties of liposomes [69,70]. Perschbacher et al. showed guanosine-rich aptamers improve tumor penetration and pharmacokinetic profile of NPs [111] our findings show, despite the decrease half-life of aptamer-functionalized liposomes, tumor accumulation and survival time increases (Fig. 6.5) [69,70]. Studies on in vivo efficiency of aptamer-targeted liposomes reveal aptamer functionalization improve tumor accumulation of liposomes irrespective of any changes in pharmacokinetic parameters [55,62,69,70,81,110,113]. The quest to explain the fate of aptamer-targeted liposomes is still a big challenge and more studies are demanded. This apparent paradox could be attributed to the mechanism of delivery of liposomes at the tissue and cellular level. At the tissue level, liposomes accumulate in the tumor by the EPR effect and passive targeting. After tissue accumulation, targeted liposomes are able to enter the cells via ligand-mediated internalization which is facilitated by targeted aptamers. In other words, enhanced localization leads to the release of the drug near to its site of action and increases antitumor efficacy of the drug [33,114]. A major obstacle in aptamer-liposome delivery in solid tumor model is heterogeneous distribution in tumor tissue owing to limited penetration in tumor tissue. The poor tumor penetration hampers therapeutic efficiency of drug-loaded aptamer-targeted-liposomes. High tumor density, high interstitial fluid pressure and dense tumor matrix significantly reduce homogenous tumor penetration [1]. Significant improving in therapeutic efficacy in tumor bearing animal models could be attributed to rapid growth of solid tumors, high abnormal blood vessels, and high ratio of tumor weight to total body weight in mice models. Therefore, it is unlikely translate animal data to humans [1,115]. Therefore, it is necessary to find a proper preclinical model to evaluate the effectiveness of targeted-delivery systems. Given the findings

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Figure 6.5 In vivo antitumor efficacy of subcutaneous xenograft model and safety evaluation. Photographs of tumors from each group after treatment with Saline, Liposomes (NPs), miR139-5p loaded liposomes (MNPs) and Anti-EpCAM RNA aptamer targeted MNPs (MANPs) (a) and Tumor weight of the different groups after treatment (b) [112], Therapeutic efficacy of various liposomal preparations in female BALB/c mice after i.v. administration of a single dose of 15 mg/kg RNA aptamer-targeted and nontargeted liposomal doxorubicin or dextrose5% on day 8 after tumor inoculation Tumor growth rate (c) and survival curve (d) (Data represented as mean  SE, n ¼ 5; P-value < .05) [70]. (a and b). Reprinted with permission from Zhao et al. [112]. Copyright {2019} American Chemical Society. (c and d). Reproduced with permission (License code: 5210060191555).

by various groups discussed above, we can conclude despite alteration of the pharmacokinetic profile of liposomes and poor tumor accumulation or penetration, aptamertargeted liposomes are efficient in reduce tumor growth compared with nontargeted liposomes. As Storm et al. described, active targeting of liposomes may increase their retention time in tumors and prevent them from rapid reentry to the systemic circulation [1]. By this way, aptamer-targeted liposomes increases residence time in tumor tissue and enhance antitumor efficacy compared with nontargeted liposomes. In the last few decades, many aptamers have been developed to recognize cancerspecific biomarker (Table 6.2). In the following sections, we review aptamerfunctionalized liposomal systems with an emphasis on the receptors targeted on the surface of cancer cells or related tissues.

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Table 6.2 Aptamers targeting cancer biomarkers. Aptamer/ type

Nucleotide number

Target

AS1411/ DNA 5TR1/DNA S2.1/DNA

26

Nucleolin

25 25

Mucin-1

EpCAM/ RNA

19

EpCAM

xPSMA9/ RNA A10/RNA

40 40

TA1/DNA Apt1/RNA

30 90

PSMA (prostate specific membrane antigen) CD44

Anti-EGFR/ DNA

76

EGFR (epidermal growth factor receptor)

HB5/DNA

86

A30/RNA

49

HER2 (human epidermal growth factor 2) HER3 (human epidermal growth factor receptor 3)

6.4.1

Therapeutic application Induce bcl-2 mRNA instability Prevent cancer cell invasion through beta catenin Regulate gene expression of cmyc, e-fabp, cyclin, and modulate EMT Prevent hydrolysis of N-acetylaspartylglutamate for overproliferation Inhibit cell proliferation, differentiation, migration, and angiogenesis Inhibit cell proliferation, invasion and metastasis Inhibition of tumorigenic signaling Reduction in drug resistance

References [116] [117]

[118]

[119]

[120,121]

[122]

[123]

[124]

Protein tyrosine kinase 7 (PTK7)

Protein tyrosine kinase 7 (PTK7) is a transmembrane receptor highly expressed on human acute lymphoblastic leukemia cells (CEM). For the first time, Tan et al. developed scg8 aptamer targeted liposomes loaded with fluorescein-dextran as a drug mimic. 30 -thiol-modified sgc8 conjugated to lipid chain via thiol-maleimide conjugation. they showed scg8 aptamer significantly improves the uptake of liposomes into CEM cells [61]. Vincristine (VCR) is a cytotoxic compound widely used in hemato-oncology, especially ALL. But due to dose limiting neurotoxicity, its therapeutic potential has been limited. The development of VCR-loaded and Sgc8 aptamer-conjugated liposomal drug delivery system (Sgc8/VCR-Lipo) significantly increase the therapeutic effects of VCR against leukemia and off-target toxicity was significantly reduced due to specific drug accumulation in tumor tissue [125].

Aptamer-liposome conjugates for cancer therapy

6.4.2

155

E-selectin

E-selectin is a protein expressed by inflamed endothelial cells and plays an important role in the adhesion of tumor cells to the vascular endothelium [126]. Mann et al. functionalized fluorescein-encapsulated liposomes by employing E-selectin-specific thiolated aptamer (ESTA). In vivo studies showed ESTA-liposomes accumulated in tumor tissue more than nontargeted liposomes. Pharmacokinetic analysis showed ESTA-functionalization slightly increased circulation half-time and decreased clearance rate of liposomes, while no significant change was observed in the volume of distribution (Vd) [110].

6.4.3

CD44 protein

CD44 is a receptor protein that highly expresses in many tumors and is one of the wellstudied cancer stem cell surface markers [120,127]. Alshaer et al. developed CD44 RNA aptamer (apt1) and studied it on active targeting of liposomes. They showed apt1-targeted-liposomes had higher selectivity to CD44þ cell line compared with nontargeted liposomes. Thiol-maleimide reaction was used to apt1-liposome conjugation [127,128]. In a further work by Alshaer et al., a noncationic PEGylated liposomal system loaded with siRNA: protamine was developed and then anti-CD44 aptamer was postinserted into liposomes to actively target CD44 expressing TNBC cells. The construct was able to inhibit the expression of the reporter gene of luciferase (luc2) in vitro and a prolonged inhibition in vivo on an orthotopic MDA-MB-231 breast cancer model [129].

6.4.4

Prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a well-characterized membrane protein that is overexpressed on the surface of prostate cancer cells [38]. PSMA is a promising target for the treatment of prostate cancer (PC) and various other solid tumors. A9 and A10 RNA aptamers discovered by Lupold et al. using in vitro SELEX against PSMA [119]. Baek et al. employed xPSM-A9 RNA aptamer for targeted delivery of liposomes containing rhodamine or doxorubicin. Their results revealed that xPSM-A9 significantly increased cellular uptake of liposomes in a PSMA-positive cell line. The animal experiments demonstrated tumor growth rate was decelerated in targeted-liposome received groups compared with nontargeted liposomes received and control groups [81]. PSMA-specific aptamer-liposome systems have also been employed in antivascular radiotherapy with radioactive actinium (225Ac) [130]. Stuart et al. reported decoration of zinc chelator-laden liposomes by anti-PSMA SZTI01 aptamer reduces tumor growth in mice with human prostate cancer xenograft. Zinc chelator N,N,N0 ,N0 -tetrakis(2pyridylmethyl)-ethylenediamine (TPEN) induces cell death owing to unbalancing oxygen species [113].

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6.4.5

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Nucleolin protein

Nucleolin is a well-studied multifunctional protein which its abnormal expression is related to tumor growth and metastasis [131]. AS1411 is an antinucleolin 26-mer Guanine-rich DNA aptamer which inhibits cell proliferation in a wide range of cancers that discovered by Bates and coworkers [116,132]. AS1411 is the first anticancer aptamer that had reached clinical stage evaluation [116]. AS1411 has been employed as therapeutic or targeting agents in many studies. In 2009, Lu and coworkers used AS1411 for targeted delivery of liposomal cisplatin. They functionalized liposomes by incorporation of AS1411-cholesterol into liposome structure. They showed cytotoxicity of liposomal cisplatin increases by AS1411 functionalization. They Authors also employs AS1411 to functionalization of liposomal calcein as a fluorescent dye and showed MCF7 cells treated with AS1411-liposomes showed a strong fluorescent signal compared to nontargeted liposomes or nonexpressing AS1411 cells [63]. They further explored the impact of AS1411 aptamer in target delivery of liposomal doxorubicin. Their findings showed that AS1411 significantly increased antitumor efficiency of liposomal doxorubicin in vitro and in vivo [62]. Sung et al. studied the effect of AS1411 functionalization on improvement of drug release from thermosensitive liposomal doxorubicin. Their results revealed cytotoxicity was increased after treatment of cells with AS1411-coupled liposomes followed by hyperthermia. Moreover, a better result was achieved in mice at 42 C compared to 37 C [133]. Du et al. also developed an AS1411etargeted thermosensitive liposome platform to deliver docetaxel. They used dual targeting strategy by employing AS1411 and S2.2 aptamers against nucleolin and MUC1 receptors. Aptamers attached to liposomes via gold nanoshells. The prepared liposomes were highly biocompatible and showed higher tumor docetaxel accumulation with less off-target tissue concentration [134]. AS1411coupled thermosensitive liposomes were also exploited as diagnosis agents for magnetic resonance imaging [135]. As siRNA delivery system, Yung and coworkers prepared AS1411 targeted-liposome for delivery of anti-BRAF siRNA (siBraf) [55]. They reported strong silencing of BRAF gene and inhibition of melanoma growth in xenografted mice. Recently, Jiang et al. formulated nanosized cationic liposomes loaded with microRNA29b (miR-29b) and decorated with AS1411 aptamer [57]. The miR-29b, are endogenous, small, noncoding RNAs, is involved in DNA methylation by targeting methyltransferase and/or regulates DNA demethylation pathway members [136] and plays an important role in cell proliferation, differentiation and apoptosis [137]. flow cytometry and confocal laser scanning microscopy experiments demonstrated cellular uptake and cytotoxicity of miR29b loaded AS1411-liposome was increased and in ovarian cancer cells A278 [57]. Yu et al. engineered a cationic liposome delivery system for codelivery of paclitaxel and PLK-1 targeting siRNA in breast cancer [138]. Polo-like kinase one is a highly conserved serine/threonine protein kinase and plays a critical role in cell division. Codelivery of chemotherapeutic agents and PLK-1 targeting RNA interference (siRNA) is a promising way to increase therapeutic efficacy and reduce drug resistance [139].

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The liposome composed of DOPE, sphingomyelin, cholesterol, DSPE-PEG2000 and didodecyldimethylammonium bromide functionalized with AS1411 aptamer to further enhance tumor targeting capability. They reported about 79% knockdown in the expression level of PLK1 mRNA after treatment of MCF-7 with such liposomes. AS1411 functionalization significantly reduced tumor growth rate and increased survival time in mice model. The codelivery of paclitaxel and PLK1 siRNA increased the number of apoptotic cells and reduced angiogenesis which demonstrated the synergistic antitumor activity [138]. In a similar study, 5-fluorouracil (5-FU) as an anticancer drug, was loaded in a PEGylated liposome conjugated to AS1411 aptamer for improving of the therapeutic effect against basal cell carcinoma. In vitro antitumoral experiments indicated the superiority of aptamer functionalized liposomes loaded with 5-FU in killing basal cell carcinoma TE354. T cell line compared to the nonfunctionalized ones [140]. Ding et al. developed new liposomal platforms using Liposomegold nanorod hybrids and functionalized it by exploiting AS1411 aptamers for delivery of Morin [141]. Morin is a kind of natural medicine with anticancer, antioxidant, and antiinflammatory effects [142]. The release percentage of Morin from pH sensitive liposome was correlated to the pH of the environment which was about 54% at pH: 5. In vitro study showed effective suppression of proliferation of SGC-7901 cells. Administration of Apt-.Au-MSL could inhibit tumor growth in SGC-7901 tumor xenograft mouse model with the promotion of tumor apoptosis [141]. Recently Dai et al. developed thermo-sensitive liposomes for multimodal phototheranostics of triple-negative breast cancer. The liposomes fabricated by integrating the semiconducting polymer, azo compound and targeted with AS1411 aptamer. They showed this platform is a promising candidate for spatiotemporally specific imaging and treatment of triple-negative breast cancer [143].

6.4.6

Transferrin receptor

The transferrin receptor is another well-studied cell surface receptor that is widely studied in targeted delivery systems in cancer treatment [144]. Wilner and coworkers designed RNA aptamers against transferrin and then employed it as targeting ligand in liposomal formulation. They prepared cationic liposomes with positively charged 1,2dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) lipid. Thiolated-Aptamer was linked to DSPE-PEG-maleimide and incorporated into liposomes. The results showed cell association and gene knockdown efficiency were improved by aptamer targeting [145].

6.4.7

Epidermal growth factor receptor (EGFR)

Epidermal growth factor receptor (EGFR) is a growth factor receptor that overexpresses in many solid cancers and its activation is associated with cancer development including: cell proliferation, invasion, metastasis, angiogenesis, and decreased apoptosis in malignant cancers [122]. Li and coworkers functionalized liposomal erlotinib using anti-EGFR aptamer via aptamer-chitosan anchoring. Aptamer-targeted liposomes enhanced cytotoxicity of erlotinib compared with nontargeted counterparts

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in a lung cancer cell line [71]. Dong et al. developed a gen drug delivery platform by liposome-polycation-DNA complex loaded with SATB1 siRNA and decorated with anti-EGFR aptamers for treatment of choriocarcinoma [146]. Choriocarcinoma is an aggressive and vascular cancer that is characterized by increasing the expression of SATB1 by 19-fold more compared with the normal chorionic cell lines. So the inhibition of SATB1 could improve the treatment of choriocarcinoma [147]. The multilayer liposomes composed of DOTAP and cholesterol were extruded to prepare single layer liposome. Liposome-polycation-DNA complex (LPD) was prepared by blending protamine solution, calf thymus DNA, siRNA and DSPE-PEG-Mal. EGFR aptamers were then added to the DSPE-PEG-Mal modified LPD to form EGFR-LPDS. The results of in vitro experiments showed significant inhibition of SATB1 expression, growth suppression activity and apoptosis in EGFR overexpressing choriocarcinoma cells. In addition, the expression of SATB1 was decreased in vivo studies with a consequent decrease in tumor size with the rate of 81.4% in mice bearing JEG-3 choriocarcinoma [146]. Dou and coworkers, screened the aptamer library targeting human epidermal growth factor receptor 3 (HER3) to find aptamer with a high affinity against HER3. They developed a HER3 extracellular domain aptamer functionalized liposome encapsulation Dox (Apt-lip-Dox) and evaluated its efficacy in vitro and in vivo. It was observed that the Apt-lip-Dox system could effectively deliver the Dox into MCF-7 cell line in a lower concentration of Dox compared to controls groups which reduced the cardiac toxicity and prolonged survival time in tumor-bearing mice [148].

6.4.8

Epithelial cell adhesion molecule (EpCAM)

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that highly expressed in most of the solid tumors. It has been suggested that it is a potential cancer stem cell marker [149]. Anti- EpCAM aptamers have been developed and investigated in different types of studies [118,121,149e154]. Zhao et al. developed a cationic liposome delivery system loaded with miR-139-5p and conjugated with anti-EpCAM aptamer for targeted therapy of colorectal cancer [112]. miRNAs play a vital role in proliferation, differentiation, survival, and migration [155]. Dysregulation of miR -139-5b is reported in various types of cancer. MiR-139-5p is overexpressed as a tumor suppressor in colorectal cancer which assisted in the inhibition of cancer progression. It inhibits the invasion and migration of colorectal cancer by targeting Notch 1 [156]. Lipids including HSPC/DOTAP/Chol/DSPE-PEG2000COOH were employed for the construction of liposomes. After preparation of miR139-5p-EpCAM Apt-HSPC/DOTAP/Chol/DSPE-PEG2000-COOH (MANPs), the inhibitory efficacy of MANPs was tested in HCT8 colorectal cell line in vitro and in vivo. The authors found the cellular uptake of miR-139 5b from MANPs was increased by up to 260% when compared to nontargeted liposomes. In addition, the MANPs displayed significant inhibition growth of HCT8 cells in vitro and showed a significant tumor suppressive effect on HCT8 colorectal tumor mice [112]. Mashreghi et al. utilized an anti-EpCAM DNA aptamer to functionalize Caelyx liposomes. The anti-Epcam aptamer -conjugated DSPE-mPEG2000 was attached to Caelyx liposome (ED-lip) via post insertion method. In vitro studies indicated the

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higher cell uptake of ED-lip in the C26 cell line resulted in a higher cytotoxic effect compared to Caelyx. Additionally, ED-lip significantly improved doxorubicin concentration in tumor site and extended survival time in mice bearing C26 tumor in compared to nontargeted ones [68]. They also studied the efficiency of anti-EpCAM RNA aptamers conjugated liposomes in mice bearing colon carcinoma tumor model. They fabricated an EpCAM aptamer conjugated to PEGylated liposomes for targeted delivery of Doxorubicin to cancerous cells. The authors demonstrated the enhanced accumulation of Dox in tumor cells expressing high level of EpCAMþCD44þ markers compared to Caelyx. The promotion of survival rate and reduction of tumor growth rate were observed in mice bearing C26 tumor treated with aptamer-Dox-liposome [67]. In another study, Moitra and colleagues employed an EpCAM-guided pHsensitive liposomal platform to deliver therapeutic siRNA to EpCAM overexpressing cancer stem cells. C5C/DOPE/TPHC at 8:24:1M ratio was used to prepare liposomes to entrap siRNA and then coated with anti-EpCAM RNA aptamers. They reported this targeted pH-sensitive liposomes has excellent efficiency to deliver siRNA to cancer stem cells [157].

6.4.9

Endoglin (ENG)

Endoglin (ENG, CD105), a type I homodimeric transmembrane glycoprotein, is a coreceptor for transforming growth factor beta (TGF-b). It is overexpressed on the surface of tumor neovascular endothelial cells; therefore, it is suggested as an appropriate marker of tumor vasculature, as well as an targeting ligand for cancer therapy [158]. In a study by Yang et al., liposome-based nanocapsules were engineered by incorporating DSPE-PEG2000-ENG-Aptamer on the surface of the liposome which encapsulated the mouse interferon-inducible protein-10 (mIP-10). Following treatment with ENG-Apt/mIP-10-LP NPs, a significant decrease in tumor progression in melanoma tumor-bearing mice was observed which demonstrated the CTLs enrichment in the tumor site [159]. In another study, Xie et al. developed nanoliposomes conjugated with hEndoglin (hEnd) aptamer and anti-CD3 antibody in order to faster encounter between T cells and tumors and render efficient tumor elimination. CRISPR/Cas9 technology was used to silence PD-1 in the T cells. PD-1- CTLs were stimulated with DC/tumor fusion cells followed by further functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1 CTLs [160].

6.4.10 Others Several studies have employed aptamers that target a specific cell line with no particular surface receptor in mind. These aptamers discovers from cell-based SELEX [38]. Harashima and colleagues developed AraHH001 DNA aptamers that specifically targets mouse tumor endothelial cells. They modified PEGylated liposomal rhodamine with AraHH001 for targeted delivery toward tumor vasculature. Targeted-liposomes showed a high selectivity for tumor vascular cells without binding to normal skin endothelial cells in vitro and increased tumor vasculature accumulation in vivo [161].

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Liu et al. modified liposome with IL-4Ra aptamer to deliver CpG to tumor cells [162]. As expected, IL-4Ra-liposomes improved cellular uptake of liposomes and distribution of liposomes in the tumor tissue. In vivo experiments showed IL-4Ra aptamer-liposomes had better antitumor efficiency compared with nontargeted liposomes in mice bearing CT26 tumor. PMN-MDSCs (polymorphonucleardmyeloid-derived suppressor cells) are a key component of the TME (Tumor microenvironment) which their selective modulation at tumor site could enable to regulate immune responses in various types of cancers [163] and provide a new avenue for cancer immunotherapy. In this regard, Liu et al. identified a novel DNA aptamer (T1) with high affinity for dual targeting of PMN-MDSCs and tumor cells and conjugated to liposomal Doxorubicin [164]. The present system resulted in triggering apoptosis of breast cancer cells and suppression of PMN-MDSCs which lead to enhancement of CTLs intratumoral infiltration. The transformation of the TME toward a more immunoactive state caused by T1-Doxorubicin consequence in the improvement of antitumor activity compared to nontargeted liposomes or free doxorubicin [164].

6.5

Future perspectives and conclusion

In 1990, three separate groups developed SELEX for selecting aptamers as targeting agents [40,165]. Since then, aptamers have gained increasing utility in active targeting of nanoparticles. The first FDA-approved aptamer is pegaptanib (Macugen) that is a 27-nucleotide RNA aptamer. Pegaptanib is used to treat age-related macular degeneration [166]. Some therapeutic aptamers for hemostasis and diabetes mellitus have also entered the clinical trial phase. AS1411 and Sgc8 aptamers are undergoing clinical trials for cancer treatment [167,168]. These aptamers have been described in previous sections. As mentioned before, AS1411 and Scg8 are short-length aptamers and may have less interference with the surface characteristics of nanoparticles. Currently, several ligand targeted-liposomes for cancer therapy are undergoing clinical trials, but none of them are aptamer-functionalized [3]. Despite the challenges in translational process, aptamer-targeted liposome is a growing interest in drug delivery and has the potential to improve the efficiency of cancer therapy. Therefore, it is important to continue the investigation in this field and refining the experiments to overcome the challenges to clinical translation. The high manufacturing cost of aptamer-liposomes is a notable limitation. Since the production of monoclonal antibodies is well-established setup technology; thus, many researchers prefer to work on antibodies. However, aptamer synthesis technology is improving, and aptamer patents will expire in the next few years [92]. We predict by falling manufacturing costs, the more widespread use of aptamer in ligand targeting systems and more clinical translation. By executing more detailed and careful aptamer characterization and target validation methods, we can in the future identify more efficient aptamers. The advances in SELEX technology allow developing more efficient aptamers as targeting ligand.

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The xenograft models have been used in preclinical studies of drug delivery systems for many decades. The wide gap between preclinical results and clinical efficiency of targeted-NPs reveals these models are not able to simulate the complex human tumor environment. Development of proper preclinical studies and more tailored animal models that allow us to successfully translate to human use are needed. Most of the in vivo experiments have used tumor-bearing animals with highly abnormal vascular in tumor tissue that nanoparticles could efficiently reach to tumor tissue by the EPR effect. This could lead to a false impression about the effectiveness of nanoparticles in clinical trials. Further investigation about the EPR effect in human cancer and development of more predictable preclinical model is deemed essential. Moreover, many in vivo survival studies have used single-dose regimens which are not enough to reach the tumor site and penetrate tumor tissue efficiently. In multiple-dose administration, a continuous efflux from the surrounding tissue into tumor provides enough time to overcome the high-density barrier [169]. In spite of the challenges, the effectiveness of aptamer-targeted liposomes as cancer-targeted drug delivery systems has already been demonstrated in a number of preclinical studies. By fabrication of aptamer-targeted platforms, design more predictable preclinical models, accelerate scale-up process the current shortcomings will be resolved and further clinical translations are still on the way.

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[152] R. Bavi, Z. Liu, Z. Han, H. Zhang, Y. Gu, In silico designed RNA aptamer against epithelial cell adhesion molecule for cancer cell imaging, Biochemical and Biophysical Research Communications 509 (2019) 937e942. [153] J. Zhong, J. Ding, L. Deng, Y. Xiang, D. Liu, Y. Zhang, X. Chen, Q. Yang, Selection of DNA aptamers recognizing EpCAM-positive prostate cancer by cell-SELEX for in vitro and in vivo MR imaging, Drug Design, Development and Therapy 15 (2021) 3985e3996. [154] G.S. Zamay, O.S. Kolovskaya, T.I. Ivanchenko, T.N. Zamay, D.V. Veprintsev, V.L. Grigorieva, I.I. Garanzha, A.V. Krat, Y.E. Glazyrin, A. Gargaun, I.N. Lapin, V.A. Svetlichnyi, M.V. Berezovski, A.S. Kichkailo, Development of DNA aptamers to native EpCAM for isolation of lung circulating tumor cells from human blood, Cancers 11 (2019) 351. [155] R.I. Gregory, R. Shiekhattar, MicroRNA biogenesis and cancer, Cancer Research 65 (2005) 3509e3512. [156] M. Song, Y. Yin, J. Zhang, B. Zhang, Z. Bian, C. Quan, L. Zhou, Y. Hu, Q. Wang, S. Ni, B. Fei, W. Wang, X. Du, D. Hua, Z. Huang, MiR-139-5p inhibits migration and invasion of colorectal cancer by downregulating AMFR and NOTCH1, Protein Cell 5 (2014) 851e861. [157] P. Moitra, S.K. Misra, K. Kumar, P. Kondaiah, P. Tran, W. Duan, S. Bhattacharya, Cancer stem cell-targeted gene delivery mediated by aptamer-decorated pH-sensitive nanoliposomes, ACS Biomaterials Science and Engineering 7 (2021) 2508e2519. [158] M.L. Nagpal, Y. Chen, T. Lin, Effects of overexpression of CXCL10 (cytokineresponsive gene-2) on MA-10 mouse Leydig tumor cell steroidogenesis and proliferation, Journal of Endocrinology 183 (2004) 585e594. [159] X. Yang, J. Zhao, S. Duan, X. Hou, X. Li, Z. Hu, Z. Tang, F. Mo, X. Lu, Enhanced cytotoxic T lymphocytes recruitment targeting tumor vasculatures by endoglin aptamer and IP-10 plasmid presenting liposome-based nanocarriers, Theranostics 9 (2019) 4066e4083. [160] S. Xie, X. Hou, W. Yang, W. Shi, X. Yang, S. Duan, F. Mo, A. Liu, W. Wang, X. Lu, Endoglin-aptamer-functionalized liposome-equipped PD-1-silenced T cells enhance antitumoral immunotherapeutic effects, International Journal of Nanomedicine 16 (2021) 6017e6034. [161] M.N. Ara, T. Matsuda, M. Hyodo, Y. Sakurai, H. Hatakeyama, N. Ohga, K. Hida, H. Harashima, An aptamer ligand based liposomal nanocarrier system that targets tumor endothelial cells, Biomaterials 35 (2014) 7110e7120. [162] Y.J. Liu, X.Q. Dou, F. Wang, J. Zhang, X.L. Wang, G.L. Xu, S.S. Xiang, X. Gao, J. Fu, H.F. Song, IL-4Ralpha aptamer-liposome-CpG oligodeoxynucleotides suppress tumour growth by targeting the tumour microenvironment, Journal of Drug Targeting 25 (2017) 275e283. [163] L. Sun, P.E. Clavijo, Y. Robbins, P. Patel, J. Friedman, S. Greene, R. Das, C. Silvin, C. Van Waes, L.A. Horn, J. Schlom, C. Palena, D. Maeda, J. Zebala, C.T. Allen, Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy, JCI insight 4 (2019). [164] H. Liu, J. Mai, J. Shen, J. Wolfram, Z. Li, G. Zhang, R. Xu, Y. Li, C. Mu, Y. Zu, X. Li, G.L. Lokesh, V. Thiviyanathan, D.E. Volk, D.G. Gorenstein, M. Ferrari, Z. Hu, H. Shen, A novel DNA aptamer for dual targeting of polymorphonuclear myeloid-derived suppressor cells and tumor cells, Theranostics 8 (2018) 31e44. [165] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (1990) 505e510.

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[166] E.W. Ng, D.T. Shima, P. Calias, E.T. Cunningham Jr., D.R. Guyer, A.P. Adamis, Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease, Nature Reviews Drug Discovery 5 (2006) 123e132. [167] NIH, The Clinical Application of 68Ga Labeled ssDNA Aptamer Sgc8 in Healthy Volunteers and Colorectal Patients, 2018. [168] NIH, A Study of AS1411 Combined with Cytarabine in the Treatment of Patients with Primary Refractory or Relapsed Acute Myeloid Leukemia, 2018. [169] G.M. Thurber, M.M. Schmidt, K.D. Wittrup, Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance, Advanced Drug Delivery Reviews 60 (2008) 1421e1434.

Aptamer-functionalized micelles for targeted cancer therapy

7

Fatemeh Salahpour-Anarjan 1 , Faraz Zare 2 , Farnaz Hosseini 3 , Sara Davari Ahranjani 4 , Mahnaz Alipour 5 and Elahe Gozali 6 1 Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran; 2Faculty of Medicine, Aalborg University, Aalborg, Denmark; 3Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran; 4Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran; 5Polymer Composite Research Laboratory, Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran; 6Department of Health Information Technology, School of Allied Medical Sciences, Urmia University of Medical Sciences, Urmia, Iran

7.1

Targeting

In recent decades, the development of targeted nanomaterials to target cancer cells and interact at subcellular scales with a high degree of specificity has been interested in precision medicine. Targeted therapy has been reaching a pivotal role in cancer therapy since we cannot count upon the enhanced permeability and retention (EPR) effect to penetrate into all tumor microenvironment (TME) types because recent analysis of tumor vessels from patient biopsies has been showing that vessels are sealed and continuous across different tumor types such as ovarian, breast, and glioblastoma. Even so, these sealed vessels express proteins associated with transcytosis that can be targeted by proper ligands and help to their uptake and internalization into the tumor interstitium by receptor-mediated endocytosis (RME) mechanism [1,2]. Targeted nanomaterials as targeting drug delivery systems are being translated into clinical applications and substantial efforts have been exerted for carrying the maximum therapeutic advantage and limited adverse effects. For this purpose, targeting drug delivery systems require (1) blood circulation stability, (2) having targeting specificity with high affinity against corresponding molecular targets that are exposed on cell surfaces or in tumor tissues, and (3) having sufficient internalization into the target cells in the target site. The most important of these properties is the second one that causes aptamers to be an appropriate targeting moiety for drug/gene carriers. Therefore, aptamers are being emerged as chemical antibodies and they are attractive alternatives to antibodies since synthesis and chemically modification of aptamers is relatively easier than antibodies or peptide ligands; moreover, aptamers’ stability is more than antibodies or peptide ligand counterparts.

Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00015-4 Copyright © 2023 Elsevier Ltd. All rights reserved.

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7.1.1

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Aptamer

Nucleic Acid aptamers (NA-Apts) are short single-stranded (20e100 bases long) DNA or RNA (ssDNA or ssRNA) oligonucleotides that are folded into different unique three-dimensional conformations characterized by stems, bulges, loops, hairpins, triplicates, pseudoknots, kissing stem-loop complexes, or G-quadruplex structures. Thanks to their three-dimensional folding, aptamers can bind with high specificity and affinity to their wide range targets and form strong complexes through conformational adjustments by interacting and accommodating with each other. Peptide aptamers (P-Apts) developed after NA-Apts. P-Apts are small combinatorial proteins that consist of the short amino acid loop (5e20 residues), typically this loop is embedded in a stable protein backbone (loop on a frame design). Due to the lower conformational entropy of the restricted peptide loop, the binding affinity of P-Apts could be as much as 1000 times higher than the free peptide [3]. Aptamer-target binding is affected by complementarity in the geometrical shape; therefore, investigating three-dimensional structures of aptamer/target interactions at the atomic and molecular levels can help structure-based drug discovery [4]. In high-throughput cancer drug screening, the compound that can compete with aptamer for binding to target molecules can be identified as a drug candidate [5,6]. Aptamers are able to bind to nonnucleic acid target molecules such as drugs, inorganic and organic molecules, metal ions, live cells, toxins, peptides, proteins, or even poorly immunogenic targets that are difficult to produce antibodies against, with high affinity and specificity and successful internalization of their cargos to target cells [7,8]. The interesting thing about aptamers is their application both as therapeutics and as targeting ligands. The most advantages of aptamers are simple synthesis, easy screening, low molecular weight (10e50 kDa), programmable designing, serum nuclease degradation resistance, stability in blood circulation, ease of cell internalization, rapid tissue penetration, the lack of immunogenicity, having long shelf life, low toxicity, useable in a variety of environmental conditions, and ease of modification that can facilitate the translation of aptamers’ functionality into clinical use. These advantages guarantee aptamers’ bioavailability and chemical integrity in physiological conditions. The most essential properties of aptamers that affect cellular uptake and efficient internalization are size and charge. Due to the negative charge of the cell membrane and aptamers’ negative phosphate backbone, there exists repulsion between aptamers and the cell surface. Size is a very critical parameter in targeting strategies; therefore aptamers because of their tiny size in comparison with the other ligands are proper targeting ligands. Also, aptamers’ stability depends on size, since has been shown longer than 25 bases oligonucleotides confront difficult internalization since longer oligonucleotides tend to self-hybridize [9,10].

7.1.2

SELEX

Systematic evolution of ligands by exponential enrichment (SELEX) is a known protocol to screen aptamers from 1012e1015 combinatorial oligonucleotide libraries with

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175

Figure 7.1 SELEX technique. (a) In vitro illustration of SELEX aptamer screening schematic. Strongly binding NA-Apts are amplified by PCR for subsequent rounds of selection and identified via DNA sequencing. (b) The oligonucleotide pool from the last round of SELEX was subject to sequencing and bioinformatic analysis of homology and frequency. Reproduced with permission from Ref. [11].

high purity and reproducibility to bind to a specific receptor with high affinity and sensitivity from the nanomolar (nM) to picomolar (pM) range. Over generally 6e18 rounds of selection, more than 1013 different sequences be sieved, and the few nucleic acid species with specificity to the target be isolated (Fig. 7.1). Polymerase chain reaction (PCR), sequencing, and bioinformatic analysis are beneficial technologies for aptamer screening as well as the isolation of aptamer candidate probes could be facilitated by technology platforms such as microfluidics, capillary electrophoresis, and flow cytometry. SELEX is able to produce aptamers for even unknown molecules, and this advantage has enabled the recognition of unknown surface biomarkers and their function on corresponding cells [11]. HT (High throughput)-SELEX allows screening aptamer candidates from a large number of oligonucleotide sequences within only a few days. Cell-SELEX has been developed to screen aptamers for many types of cancer cells by using whole live cells even without the previous information of their molecular signatures [12e14]. SELEX technique provides easy, timesaving, cost-effective in the large-scale manufacture of desired aptamers.

7.1.3

Aptamer internalization mechanisms

The most known aptamer internalization mechanisms are RME and macropinocytosis. Aptamer on the carrier and specific molecules on the plasma membrane form a

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receptor-ligand complex that causes RME. RME has known a standard internalization mechanism for most aptamers but the RME mechanism limits via aptamers’ inefficient endosomal escape, while the macropinocytosis overcomes this defect. It has been confirmed that macropinocytosis occurs often in the internalization of the nucleolin aptamers that shuttle between the cytoplasm and nucleus; therefore, nucleolin aptamer can directly deliver cargo molecules to the cell nucleus without endosomal trapping. This property makes them a perfect delivery tool for gene-based drugs. Although nucleolin aptamer internalization cannot be explained only by the standard model of macropinocytosis, the rate and mechanism of uptake are different between cancer cells and other cells, for example, AS1411 is an antinucleolin aptamer that macropinocytosis is the predominant mechanism of uptake in cancer cells such as prostate cancer cells (DU145); however, AS1411 may be taken up by clathrin- or caveolae-dependent route of entry in nonmalignant skin fibroblasts (Hs27) [15]. Two important points should be considered in using aptamers as a targeting ligand: (1) Temperature-dependent manner of binding and internalization; and (2) Cells membrane receptors; whereas aptamers’ internalization must occur at physiological temperature (37
C) in vivo, then it is very important to determine whether aptamer internalization occurs at 37
C or not! Because the secondary structure of aptamers depends on temperature [16]. Being an agonist or antagonist for aptamers’ function depends on the cellular localization and molecular functional of the target molecules that are involved in a cellular metabolic pathway. On the other, it was found that the Aptmicelle formation can improve the target-binding ability of low-affinity aptamers at physiological temperature. TDO5 is an aptamer that shows high affinity and selectivity for immunoglobin heavy mu chain receptors on the Ramos cells (a B-cell lymphoma cell line) surface at 4
C while at 37
C, TDO5 does not bind to its target protein, which could have hindered its potential in vivo applications. In a study [17] a research team has shown when TDO5 is used in Apt-micelle formation, the TDO5-micelle will have perfect binding selectivity at 37
C.

7.2 7.2.1

Aptamer-functionalized micelles Micelles

Self-assembling is the way to form micelles from amphiphilic blocks. Since most of the anticancer drugs are hydrophobic; therefore, micelles are proper nanocarriers for the delivery of anticancer drugs. In contrast, some anticancer drugs such as cytarabine suffer from high hydrophilicity that causes rapid plasma degradation, and significant side effects, loading in micelles can overcome these limitations [18]. Drug-loaded micelles form spontaneously in water after comixing the hydrophobic or lipophilic drug with an amphiphilic polymer. During self-assembling hydrophobic sections of amphiphilic blocks withdraw from the aqueous media and micelle core form based on hydrophobic and nonpolar interactions between the hydrophobic polymer chains. Improving micelle stability is crucial to ascertain keeping encapsulated drug molecules within the micelles before reaching the tumor site, at the same time, drug molecules’ release rate

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177

from the micelle must also be controlled to attain sustained drug release. Many strategies have been employed to enhance drug loading capacity, although all of them are not particularly effective in controlling the burst and early drug release from the micelles carriers. Strategies have been operated to enhance drug loading capacity including variations of hydrophobic structure, dimer drugs formation, increasing interactions between the drug and the core of micelles, pep stacking, and host-guest interactions. Complex aromatic pep conjugated structure of most anticancer drugs (e.g., paclitaxel (PTX), doxorubicin (DOX), and camptothecin (CPT)) increases pep stacking interaction between these drugs and polymeric micelles [19,20]. Block copolymer micelles are divided into multimolecular polymer micelles (selfassembled of some multiple amphiphilic block copolymers) and unimolecular micelles (selfassembling starts from individual hyper-branched amphiphilic block copolymers). The unimolecular micelles possess a uniform spherical shape with a diameter varying from 20 to 37 nm. It has been shown, in vivo, unimolecular micelles are more stable than multi-molecular polymer micelles [7,21]. Also, ABC triblock copolymer micelles have been developed to achieve sufficient drug encapsulation, proper drug release profile, and micelle stability. In these triblock copolymer micelles, the C block is a relatively drug-compatible polymer to increase the efficient solubility of the drug, B block is a drug incompatible polymer segment to provide a barrier against drug release, and the A block form micelle shell and interfaces the media [19]. Smart block copolymer micelles are designed to respond to chemical/physical variations in vivo. Physical stimuli such as using light, ultrasound, and magnetic field, and chemical stimuli such as pH, glutathione (GSH), and redox reaction [22].

7.2.2

Apt-micelles

The coupling of aptamers with micelles leads to being well qualified for the development of new biomedical devices for diagnostic, molecular imaging, and drug delivery systems [23e26]. Chemical modification is used for linking chemotherapeutic agents to aptamers through forming amide, stable ester, disulfide, and hydrazone bonds. Hydrophilic oligonucleotides and hydrophobic polymers can form RNA or DNAmicelles that are termed aptamer-functionalized micelles and have been shown the size and shape of the selfassembled structures can be fine-tuned by changing the length of the DNA sequence and spacers (Fig. 7.2). Spacer uses to link the hydrophobic tail and aptamer headgroup to create the aptamer-amphiphile. Type of spacer would impact the macromolecular assembly. Aptamer-amphiphiles with hydrophilic spacers or without spacers form spherical micelles while with hydrophobic spacers form bilayer nanotapes [7,27,28]. The successful attainment of Apt-micelles strategies requires attention to nonspecific adsorption issues that commonly control the toxicity to nontargeted cells in the drug delivery systems. Therefore, surface physicochemical characteristics of micelles must be controlled while coupling aptamers to reduce nonspecific binding and maintain the aptamers’ active structures. Furthermore, proper washing and surface blocking steps should be used to more optimize the performance of these bioconjugates. Aptamer’s binding affinities to target molecules are related to stacking interactions,

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Figure 7.2 Loading poor water-soluble drug molecules in Apt-micelles to achieve sustained drug release.

van der Waals force, and hydrogen bonding. To ensure the selectivity binding of aptamers, a negative screening using nontarget molecules performs to remove any unwanted ligands that bind to nontarget molecules. Significantly, many bioconjugations or chemical modifications of aptamers can be automated on a DNA/RNA synthesizer and handled under bio-friendly situations. Various chemical tags provide monitoring, controlling, and immobilization of Apt-micelles [11].

7.2.3

Apt-micelles in cancer treatment

The therapeutic index of anticancer drugs is often very narrow, and since the cytotoxic dose of the drug needs to be maintained in the desired tissues and on the other, lowdrug-loading capacity, lack of active targeting of tumor cells, multiple drug resistance issues, and unspecific drug release of nanocarriers hinder the success of cancer clinical therapy. It seems targeted drug delivery and controlled release by using engineered nano-based carrier drug delivery systems can be overcome these difficulties to some extent. The aim of targeted drug delivery in cancer is to enhance efficacy, moderate side effects, reduce off-target effects and increase ED50 (median effective dose), and decrease IC50 (half-maximal inhibitory concentration) of anticancer drugs. Smart micelles can respond to changes between TME and healthy tissue such as enzyme, pH, GSH levels, and redox interactions and accomplish a selective cytoplasmic release [22]. Micelle-based smart drug delivery systems are empowered by the use of systematically developed aptamers for cancer-associated biomarkers. In this way, mapping out the profile of overexpressed specific biomarkers in many types of cancers allows screening proper aptamers to guide therapeutic agents to the tumor site and to help their internalization into the cancer cells. Hence, aptamers have been screened for a wide variety of cancer-related biomarkers [29]. One of the most used aptamers in targeted-cancer therapy is AS1411, and many research groups have used it as a targeting agent to deliver nanoparticles, genes, and drugs into cancer cells. AS1411 (5ʹ-GGTGGTGGTGGTTGTGGTGGTGGTGG-3ʹ) also known as AGRO100, is a guanine-rich 26-base antinucleolin DNA aptamer with Gquadruplex 3D structure which it relatively stable relative to other aptamer (Fig. 7.3). The most advantages of quadruplex aptamers compared to nonquadruplexs are nonimmunogenicity, heat stability, improving cellular uptake, efficient cellular

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179

Figure 7.3 Gquadruplex (4stranded) structures of AS1411. Reproduced with permission from Ref. [32].

internalization, and resistance to nuclease degradation. AS1411 binds to the nucleolin protein with high affinity and specificity. Nucleolin (C23) is a multifunctional nucleolar phosphoprotein, not only found in the nucleolus but also found in the cytoplasm and on the cell membrane which binds to various ligands and affects many physiological functions. Overexpression of nucleolin is a potential molecular target for diagnosis and targeting certain cancer cells [30,31]. In recent years, many cancer treatment researches directed to using smart-targeted nanocarriers such as smart Apt-micelles. Some of the applications of Apt-micelles (normal or smart micelles) have been categorized in Table 7.1 that have been investigated on cancer and their methods and results have been described in the following. Normal cells express efflux pumps to protect against toxic metabolites. Tumor cells use this trick and overexpress drug efflux transporters on their membranes to resist chemotherapy agents. To date, many efflux pumps are reported as transporters that are involved in drug efflux to decrease intracellular drug concentration in cancer cells. Three main ATP-binding cassette (ABC)-superfamily multidrug efflux pumps named P-glycoprotein (ABCB1), ABCG2 (BCRP), and MRP1 (ABCC1) have an important role in multidrug resistance (MDR). MDR is turned to be a major obstacle in cancer therapy and causes the failure of chemotherapy. Based on many studies that have shown gene delivery to silence these pumps’ genes could be promising. Hence, the

180

Table 7.1 Some Apt-micelles in targeted cancer therapy.

Aptamer

Micelle platform

Therapeutic agents

Nucleolin

AS1411

Normal

Nucleolin PSMA

AS1411 PSMAa10

Normal Normal

Protein tyrosine kinase-7 (PTK7) HSP70

Sgc8 A8

Nucleolin Nucleolin

AS1411 AS1411

Redoxsensitive Redoxsensitive pH-sensitive pH/Redoxsensitive

DOX, miR519c Tetraoxane (T) TGX-221, BL05 Cytarabine (Ara-C) DOX PTX DOX, TLR4 siRNA

Cancer/Cell line

In vivo

In vitro

Refs.

Hepatocellular carcinoma

þ

þ

[33]

HepG2 Prostate

þ

þ þ

[34] [35]

Acute lymphoblastic leukemia (ALL) Fibroblast, breast

þ

þ

[18]

þ

[20]

Ovarian Lung

þ þ

þ þ

[36] [37]

Aptamers Engineered Nanocarriers for Cancer Therapy

Targets

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181

combination of chemotherapy and gene therapy can be considered as a reasonable approach to improving the efficacy of the anticancer drugs by suppressing the MDR effect [38]. In a study [33] an anticancer chemogene therapy based on Apt-micelles have been developed. PEG-PLA micelles functionalized by AS1411 aptamer were assembled for the codelivery of DOX and miR-519c to hepatocellular carcinoma in vivo and in vitro. Cellular uptake and tumor penetration of micelles in tumor cells increased to normal cells due to AS1411 tumor-targeting property via the specific recognition of nucleolin overexpressed on cancer cells and the efficient internalization of DOX/miR-519c loaded micelles. In this study inhibiting ABCG2-dependent drug efflux through miR-519c resulted in reversing the MDR effect then following increasing DOX concentration levels in targeted cancer cells. The therapeutic efficacy investigation showed that Apt-micelle-loaded DOX/miR-519c exhibited 65.45% and 78.35% cell cycle arresting at the G2 phase. The development of such micellar carriers with a targeting moiety can decrease the acute side effects of chemotherapy drugs such as DOX. Since intravenous administration of Dox could result in severe tissue ulceration, necrosis, and significant cardiac toxicity. Dox increases oxidative stress, down-regulates of cardiac-specific genes, and induces cardiac myocyte apoptosis. Acute cardiac toxicity of DOX occurs during days of the administration and emerges in approximately 11% of patients who receive the drug. These acute problems limit the long-term use of the drug and require the development of novel stable carriers [39]. Converting tumorous endogenous hydrogen peroxide (H2O2) into harmful reactive oxygen species such as radical highly toxic hydroxyl radicals (•OH) via classical Fenton or HaberWeiss chemistry is known as chemodynamic therapy (CDT) that causes cell apoptosis and necrosis in tumorous cells [40,41]. Even so, their anticancer efficacies under TME are limited by the low H2O2 rate, requirement of strong acidity conditions (pH 2e4), and tumorous upregulated antioxidants such as GSH in tumor cells [42]. Furthermore, for maximized therapeutic effects, minimize safety concerns, and spatially controlled chemical reactions of free radicals in CDT, tumor-targeted accumulation is essential to reduce the influence on normal redox signaling. In a study [34] a novel aptamer-prodrug conjugate (ApPdC) micelles have been assembled that is capable of TME dual-targeting for a new cancer-targeted CDT system. ApPdC micelles were composed of three moieties: (i) AS1411aptamer as the tumor-targeting part; (ii) tetraoxane (T) as the free radical generator prodrug that is activable by Fe2þ ions and also as a hydrophobic tail; and (iii) hemin (Fe3þ) that loaded into ApPdC micelles by hydrophobic interactions between hemin and T. The strong hydrophobicity of T motifs acts as a lipid-like tail along with the secondary structure of DNA sequences not only to trigger self-assembly into micelles but also could allow loading of hemin in ApPdC. In this study, ApPdC micelles are designed to target liver hepatocellular cell line (HepG2) via receptor-mediated targeting that recognized nucleolin on this cell line and enhanced cellular uptake. Loaded hemin (Fe3þ) reduced into heme (Fe2þ) by the high level of intracellular GSH, and then prodrug bases in ApPdC micelles activated by Fe2þ and triggered cascading bioorthogonal reactions and caused in situ self-circulation production of toxic C-centered free radicals to induce apoptosis in HepG2 cells. Unlike traditional CDT systems, this introduced CDT system has no

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dependence on either pH or H2O2, and even GSH depletion causes to reduce cancerous antioxidation ability. This group reported that this targeted CDT system is able to minimize side effects of CDT on normal cells since the ApPdC micelles after endocytosis could produce toxic radicals more efficiently than nontargeted micelles in cancer cells and effective oxidative damage to cancer cells caused improved antiproliferation effect in cancer cells. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a lipid-signaling second messenger that activates downstream effectors, such as Akt that in turn prompts cell growth, proliferation, and survival. The phosphatidylinositol 3-kinase (PI3K) is an enzyme phosphorylate phosphatidylinositol-3,4-diphosphate (PIP2) into the second messenger PIP3. In contrast phosphatase and tensin homolog (PTEN) is a phosphatase that dephosphorylates PIP3 back to PIP2 and suppresses cell growth and proliferation. PTEN-deficient is the index property of many cancer types. Many chemotherapeutic drugs are being developed to treat PTEN deficient cancer cells [43]. TGX-221 is a PI3K inhibitor agent that solves in organic solvents such as DMSO or propylene glycol which might cause cardiac toxicity in intravenous injection. Therefore nano-targeted drug delivery systems such as Apt-micelles could be the proper carrier of these agents in blood circulation to reduce their cytotoxicity and off-target effects. In a study [35] block copolymers, PEG-PCL (polyethylene glycol-polycaprolactone) micelles were developed by using PSMAa10 (prostate-specific membrane aptamer) as a targeting moiety to encapsulate the TGX-221 and its analog BL05. TGX-221 has antiproliferative activity against PTEN-deficient tumor cell lines such as prostate cancer. This group has shown that Apt-micelles dissolve into nontoxic degradation products and following slowly released the drug over several days that were resulted in significantly improved cellular uptake in a PSMA positive cell line. In vivo pharmacokinetics demonstrated that PSMA-targeted micelles significantly increased the area under the plasma concentration time curve (AUC) and decreased the total body clearance rate of the drug. The plasma concentration was 2.27-fold better than the naked drug, and the clearance rate of the drug was 17.5-fold slower. It has been shown the difference between the level of GSH in healthy cells with tumor cells can be used as a drug release trigger from micelles with disulfide bonds, inside tumor cells with the high level of GSH (2e10 mM), although these smart micelles can be stable in body physicochemical conditions. In a study [18] Sgc8 aptamers targeting, GSH-responsive, PCL-ss-Ara@Sgc8-BSA(bovine serum albumin) micelles were developed to overcome cytarabine (Ara-C) limitations in the treatment of acute lymphoblastic leukemia (ALL). ALL is a prevalent type of blood cancer that characterizes by uncontrolled proliferation and accumulation of abnormal white blood cells in the bone marrow and blood, which results in hematopoietic failures and a decrease in the production of blood matured cells. Ara-C is a clinical chemotherapy medicine used to treat ALL. Ara-C has high hydrophilicity that causes rapid plasma degradation and resulted in significant side effects. This research team has synthesized a prodrug via covalent bond formation between acryloyl chloride-terminal PCL-ss-PCL and Ara-C, and surface decoration with Sgc8-BSA. The cleavage of disulfide linkage (-ss-) in the presence of the GSH environment resulted in redox-triggered dissociation of the micelle’s structures and drug-releasing. Targeting ability and cellular uptake of

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these Sgc8 Apt-micelles was investigated on CCRF-CEM cells and apoptosis of these cells has been examined in vitro. Sgc8 Apt-micelles were recognized by CCRF-CEM cells and internalized through RME mechanism while free Ara-C molecules have passive diffusion. Cellular uptake of the micelles had an improved inhibition effect on CCRF-CEM cell growth in vitro. Then, this group made hemolysis and coagulation studies of these micelles and proven their biocompatibility for in vivo applications. Compared with free Ara-C, PCL-ss-Ara@Sgc8-BSA significantly enhanced tumor growth inhibition in mice bearing CCRF-CEM xenograft tumors, while causing little side effects, and improved the survival rate of CCRF-CEM tumor-bearing mice in vivo. Then PCL-ss-Ara@Sgc8-BSA micelles were administrated intravenously in nude mice bearing CCRF-CEM tumors and strong synergistic antitumor activity was observed. Since these Apt-prodrug micelles exhibited efficient targetability, good GSH-responsive drug release behavior, and effective antitumor on ALL, therefore, this team acknowledged that self-assembling Apt-prodrug micelles are a potential treatment for the targeted therapy of ALL. Heat shock proteins 70 (HSP70s) mainly reside in the endoplasmic reticulum (ER) lumen and collaborate with (co)chaperones in protein folding or degradation. In cancer, HSP70s can suppress apoptotic pathways, regulate necrosis, avoid cellular senescence programs, interfere with tumor immunity, promote angiogenesis, and support metastasis. The expression of HSP70 in malignant tumors and linking tumor survival to the HSP70 expression describe as the phenomenon of cancer addiction to HSP70. Therefore, HSP70 is a biomarker for poor prognosis. Clinically, tumor cells intensively release HSP70 in the extracellular microenvironment which can be a proper target in cancer targeting therapy [44,45]. In a study [20] a research team has constructed A8 aptamer-decorated, redox-activated, and DOX-loaded biodegradable polymeric micelles (DOX-loaded HPGssML) by self-assembling. Amphiphilic biodegradable poly (benzyl malolactonate-co-e-caprolactone) copolymer with disulfide linkage (as a redox-sensitive section) alongside p-conjugated moieties formed good stability redox-activated nanosized diameter (150 nm) micelles. PEG segment form the hydrophilic shell and the polyester segment with p-conjugated moieties form the hydrophobic core of micelles. Decreasing fluorescence intensity and redshift in UV adsorption of loaded-DOX micelles showed that DOX has loaded efficiently into the core of micelles via strong pep interactions. Due to the strong pep interaction between DOX and p-conjugated portions in hydrophobic blocks, HPGssML possessed fine stability and high drug loading content. After DOX-loading, polymeric micelles diameter was reported about 200 nm. Aptamer A8 is an 8-amino acid (SPWPRPTY) P-Apt that can actively guide the polymeric micelles and bind specifically to the extracellular domain of membraned HSP70. In this study 4T1, MDAMB-231, and MCF-7 cells that expressing HSP70 were selected to explore the tumor-targeting ability of DOX-loaded HPGssML. This group attributed the improvement of internalization efficiency of micelles into the tumor cells to the tumor homing ability of A8 aptamer via RME mechanism. After internalization into cancer cells, the breakage of the reductive-sensitive linker in HPGssML under redox conditions (10 mM GSH) in tumor cells resulted in accelerating the release of DOX into cancer cells, and DOX antitumor efficacy was observed by killing the cancer cells. Moreover,

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this team investigated DOX releasing under different pH levels and have been shown that at pH 5.0, the relating rate is fast because the protonation of DOX increased its solubility in water and resulted in easier diffusion from micelles. A sustainable profile of release behavior has been observed from HPGssML until 72 h and the final accumulative of DOX was near 60% and IC50 of DOX was reported two times less from the control group. The sustainable release behavior was attributed to the strong pep stacking interaction between the hydrophobic core and DOX, which limited dissociation of the micellar core. Therefore, the presence of 10 mM GSH at pH 5.0 can cause the breakage of the reductive sensitive linker and protonation of DOX respectively, and results the fast release of DOX. Poly(b-amino ester)s (PBAE)s are widely used as biodegradable pH-sensitive copolymers. The micelles composed of PBAE demonstrate a pH-triggered release profile in responding to acidic microenvironment inside tumor tissue (pH ¼ 6.8) or lysosome (pH ¼ 5.5) [46]. A research group in a study [36] designed a smart Apt-micelle system consisting of a pH-responsive copolymer D-a-tocopheryl polyethylene glycol 1000block-poly-(b-amino ester) (TPGS-b-PBAE, TP) that was loaded PTX, and decorated by AS1411aptamer-TPGS, which could recognize the overexpressed nucleolin on the plasma membrane of SKOV3 cancer cells. They have shown PTX/Apt-micelles are stable at physiological media (pH 7.4) then internalize in SKOV3 ovarian cancer cells by aptamer-receptor mediated interaction and dissociate in endosomes and lysosomes in response to the acidic environment (pH 5.5) and release PTX. This research team reported a synergistic effect of cancer cell recognition and pH-sensitive drug release. They publicized that intravenous administration of PTX/Apt-micelles for 16 days significantly increased tumor’s PTX accumulation, inhibited tumor growth (cell cycle arrest in the G2/M phase), lowered hematotoxicity, and reduced myelosuppression (cancer treatment side effect) on ovarian tumor-bearing mice compared with free PTX injection. This group used the endostatin for proving Apt efficiencies in the transmembrane ability of PTX through Apt-nucleolin interaction. The endostatin is a nucleolin inhibitor that blocks nucleolin activity. By pretreatment with endostatin, when the concentration reaches 0.02 mg/mL, the Apt-micelles cellular uptakes rate would be the same for micelles treated with or without Apt modification. In this study, the highest anticancer activity of PTX/Apt micelles was observed at pH 5.8 (IC50 ¼ 0.108 mM) that reveals the synergistic effects of Apt-mediated endocytosis and pH-triggered drug release. Negatively charged cell membrane and the risk of serum endonucleases degradation are two main extracellular obstacles to direct delivering genetic materials in vivo. The naked or unmodified half-life of genetic materials such as siRNAs, miRNAs, and shRNAs in vivo is between 5 and 10 min, and on the other, because of their negative charge, crossing cellular membrane is challenging. Rapid renal clearance and immunogenicity are other extracellular hindrances. Besides these obstacles, gene delivery is also faced with intracellular barriers such as endosomal trap and off-target effects [47]. To overcome, these problems, nonviral nanoscale delivery vehicles called nanovectors are being developed for the efficient transfection of genetic materials. It has been shown, these carriers are able to carry designed genes to the target cells and silence many types of target mRNAs, and destroy them [48]. In recent years,

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cancer researchers have been concentrating on combination therapy and this field is getting into cancer clinical therapy. The codelivery of chemotherapy drugs and genes with targeting function in a delivery system is a notable strategy for cancer treatment. In a study [37] AS1411-chitosan-ss-polyethylenimine-urocanic acid (ACPU) micelles with dual-pH/redox sensitivity have been synthesized to codelivery of TLR4 siRNA and Dox for anticancer chemogene therapy. Toll-like receptor four is a transmembrane protein that is encoded by the TLR4 gene. TLR4 belongs to the pattern recognition receptor family and TLR4-mediated signaling pathways play role in the process of survival, invasion, and metastasis in many cancers. Accordingly, invasion and metastasis are major concerns in cancer therapy that lead to treatment failure therefore silencing the genes are involved in cancer cell migration such as TLR4 is an important issue to prevent cancer recurrence. In ACPU micelles, urocanic acid as a hydrophobic core was used for Dox loading and siRNA was condensed into the PEI skeleton via electrostatic interaction. Cationic PEI has been widely used within nanovectors structures, which is a qualified exogenous genetic material delivery vehicle in vitro and in vivo. Negatively charged oligonucleotide strand can condense with PEI via electrostatic interactions and forms compact nanometer polymers that called as PEI-based polyplex NPs which can act as a “proton sponge” and promote the endosomal release of siRNA. Even so, the cytotoxicity of cationic polymers due to their ability to disrupt cell membrane and the mitochondrial membrane has restricted in vivo applications [49]. In mentioned above study, nucleolin highly expressed luc-A549 cells utilized as in vitro model and lungtumor-bearing BALB/c mice as in vivo model for nucleus-targeted cancer therapy. Proton-sponge effect of PEI in endosome acidic environment leads to osmotic swelling and resulted in endosomal membrane rupture. On the other, the protonation of urocanic acid in acidic environment leads to the instability of the micelles structure and rapid release. Another factor that causes the fracture of micelles structures is the rupture of disulfide bonds under reductive conditions (GSH ¼ 10 mM) and result in the imbalance of hydrophilicity/hydrophobicity within micelle arrangements and promote rapid intracellular DOX and siRNAs release. Thus Dox and siRNA were delivered by micelles in the cells and then released into the nucleus and cytoplasm, respectively. Dox intercalates within DNA base pairs and by inhibiting the enzyme topoisomerase II causes DNA damage and induces tumor cells apoptosis. After entering into the cytosol, siRNA incorporates into the RNA-inducing silencing complex (RISC) then its passenger strand releases and its guide strand targets the encoded mRNA of the interest protein and is cleavaged by endonucleases [50]. Silencing TLR4 expression by siRNA caused down-regulating the TLR4 protein level and subsequently, overcome tumor survival and invasion and enhanced the antitumor effect of Dox. This research team attributed the highest uptake rate and the lowest elimination speed of DoxsiRNAACPU micelles to the interaction of AS1411 and nucleolin, which increased cellular uptake through receptor-mediated pathway.

7.3

Conclusion

In recent decades, aptamer-functionalized nanomaterials such as Apt-micelles have been shown good potential application prospects in medicine and can be used as a

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powerful tool for the treatment of any type of cancer. The development of Apt-micelles needs both creating nanomaterials with proper physicochemical properties and increasing the performance of the aptamers. In cancer research, the ultimate aim is to develop aptamers and attach them to efficient carriers such as micelles that can target each pathogenic cell type and effectively deliver theranostic agents to the target organelles. To achieve this goal, reaching the practical internalization mechanisms and situations, selecting efficient receptor molecules and improvement of cell-SELEX is necessary. Studies in nucleic acid chemistry could extend the library diversity for aptamer screening and enhance the biostability of NA-Apts. Also, bioinformatics, the science of collection of data, can propose proper linkers that do not interfere with the conformation and function of aptamers as well as introduce fit binding positions on cargo molecules to develop new therapeutic systems. On the other, DNA origami box technique can generate new powerful aptamer-conjugated nanomaterials that could produce more outstanding sensors and drug delivery systems that provide new perspicuity into the design of targeted cancer therapies.

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Ananya Kar 1 , Smruti Rekha Rout 1 , Lopamudra Giri 1 , Amirhossein Sahebkar 2,3,4 , Prashant Kesharwani 5,6 and Rambabu Dandela 1 1 Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indian Oil Odisha Campus, Samantapuri, Bhubaneswar, Odisha, India; 2Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan, Iran; 3Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 4Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; 5Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 6University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India

8.1

Introduction

Nanotechnology has brought significant developments in a variety of sectors, including communication, electricity, manufacturing, material science, medical care, and life sciences, over the last few years. Nanomaterials and biomolecules combination have resulted in a revolution in the detection and treatment of certain diseases, such as cancer. Cancer is defined as uncontrolled cell proliferation that has the ability to infiltrate or migrate to other parts of the body. This is a common disorder caused by epigenetic and genetic changes. When cancer is at its early stage, its detection is very difficult because of the low number of cancerous cells and molecular markers. Moreover, tumor identification could be to some extent have an adverse effect on cancer therapy. In conclusion, understanding the molecular features of the tumor, especially the distinctive protein involved with certain kinds of cancer, could be advantageous for medicinal therapy. The drug used for optimal cancer treatment must be proficient in destroying cancerous cells causing little harm to normal tissue. Most anticancer medications are harmful to both healthy and cancerous cells. It is because they mainly damage cancer and those cancerous cells divide and grow rapidly so during the process of killing cancerous cells they can attack normal cells. Moreover, when taken orally, most antitumor medications are widely distributed throughout the body, resulting in just a very tiny proportion of the drugs reaching the target site. As a result, it is critical to pick the best transporting agent for targeted specific delivery of antitumor medications to cancerous cells. Biologically compatible

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compounds such as peptides, RNA, and DNA allow for precise imaging and targeting. Furthermore, nanomaterials’ distinct thermotherapeutic and spectroscopic capabilities give significant benefits for therapeutic, imaging, and sensing applications. Nanoparticles do have the capacity to more efficiently enclose and deliver anticancer drugs to the tumor site [1e5]. Nanoparticles have emerged as the best intriguing sensor materials because of their customizable surface properties, unique shape- and size-dependent optical characteristics, and high surface energy and surface-to-volume ratio. However, nanoparticles will not exhibit cancerous cell selectivity so it gets aggregated primarily in cancer areas mostly because of tumor tissue’s enhanced permeability and retention (EPR) effects [6e11]. On the other hand, when nanoparticles can be fused with ligands, they could able to precisely identify cancerous cells, it would be able to attack the cancerous cells, and transport drugs to them. Therefore, considerably enhancing their therapeutic index (reducing toxicity while increasing therapeutic efficacy). A variety of compounds were researched even further to modify nanoparticles that enable particular targeting, and aptamer represents just one of them [12e19]. Furthermore, to execute those applications, nanoparticles must be equipped with target identification capabilities. Antibodies are indeed very effectively targeting molecules that may be fused with nanoparticles that can recognize targets [20e26]. Aptamers, which were initially discovered mostly in the early nineties, have emerged as a new class of compounds that may be employed in association with nanoparticles in biomedical uses [27]. Aptamers are oligopeptides or nucleic acids with elevated selectivity and sensitivity for a variety of desired molecules, along with cells, peptides, nucleotides, small molecules, antibiotics, and proteins. Smaller nucleic acid aptamers are stable in harsh environments, while peptide aptamers possess appropriate configurations which react with target biomolecules. Aptamers are mostly made up of customizable binding region that binds to the particular compartment of target biomolecules. Aptamers offer several benefits as compared to antibodies, such as relatively high stability in severe chemical and physical conditions, ease of modification, and small size, as well as their quick and cost-effective manufacture, minimal immunogenicity, minimum processing variation, and great versatility [28,29]. As a result, aptamers are considered ideal alternatives for antibodies in biomedicine throughout imaging and targeting, and they are employed in a variety of domains. Although nanoparticles could be utilized as therapeutic and diagnostic agents, but lack the ability to be specifically targeted. As a result, a variety of aptamerenanomaterial combinations were developed and employed in a variety of fields [30e32]. Nanoparticles that are aptamer-functionalized are being investigated as prospective frameworks enabling specialized diagnostic and therapeutic utilization as there is progress in aptamer selecting techniques and nanomedicine. The subsections that follow provide a full description of the preparation of several kinds of aptamer-functionalized nanoparticles, as well as diverse uses of aptamer-functionalized nanoparticles in cancer therapeutics and applications other than cancer therapy.

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8.2

193

Preparations of different aptamer-functionalized nanoparticles

Emerged at the beginning of the 1990s, many distinct groups described the systematic evolution of ligands by exponential enrichment (SELEX) approach for isolating particular nucleic acid strands which have high affinity and specificity for binding to targeted entities. Since then, this topic of aptamer studies has continued to stimulate the research society’s attention, and this has evolved into a significant and frequently utilized method in biochemistry. The aptamer is derived from the Latin term “aptus,” which means “suited,” and the Greek term “meros,” which means “particle,” underlining the lock-and-key interaction of this category of molecules with its target [33]. Aptamers (Apt), which are small RNA or single-stranded (ss) DNA, were utilized for greater binding affinity to peptides, proteins, viruses, parasites, and cells. Moreover, the biological compatibility and minimal immunogenicity of nucleic acid aptamer make them suitable drug-targeted carriers, functional substances, and therapeutic agents for in vivo biosensors. Such active nucleic acids have the ability to fold into the 3D frameworks in order to achieve binding clefts and pockets for the unique efficient binding and recognition of any certain target molecule. These could be synthesized and generally recognized in vitro through large combinatorial databases containing trillions of distinct strands by employing an approach called SELEX which was lately been made completely automated. Apt are produced according to molecules on the cell membrane in cell-based SELEX despite the need for background information on the molecular objectives. Using cell-based SELEX techniques, and in vitro strategy for separating highly affinitive aptamers, particularly toward a complicated combination of possible targets was developed. As a result, protein extraction is not required previously for selections. The SELEX method is outlined in (Fig. 8.1) [30]. This automated process has shortened the time it would take to choose aptamers in vitro between months down to days. After 5e10 cycles of the SELEX procedure, the collection is often limited just to a few sets of aptamers with especially high affinities to a particular target. Aptamer-based heterogeneous and homogeneous biosensor devices were used to identify proteins, tiny organic molecules, metal ions, and nucleic acids due to their remarkable affinities. Within those sensing devices, popular detection techniques include electrochemistry, colorimetry, and fluorescence. In recent times, the incorporation of active aptamers onto nanomaterials has emerged as novel multidisciplinary research with the goal of developing unique hybrid smart sensors for sensitive and selective chemical identification [34]. Likewise, with the emergence of revolutionary nanotechnology for clinical applications, also known as nano-medicine. It has become clear how nano-medicine has the potential to significantly improve treatment, diagnosis, and disease elimination. It is getting more practical to create nano-medicines that could have the following properties: enhance the drug’s therapeutic and pharmacological characteristics, target drug deliverance in a cell- or tissue-specified way with improved therapeutic safety and efficacy, allow drug transportation through a variety of biological boundaries, particularly endothelial and epithelial, enhance easier drug transport to intracellular areas

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Figure 8.1 Depicts the SELEX method. (a) A standard SELEX schematics depiction. It comprises four stages: incubating targeted molecules with libraries, bound libraries are eluted, libraries amplified, and libraries separated for the next cycle. (b) A schematic representation of cell-based SELEX. Every stage involves critical selection.

of action, provide delivery of a variety of medications having possibly varying physicochemical characteristics, provide a synergistic combination of diagnostic and therapeutic agents enabling actual observation of treatment efficacy, overcome multidrug tolerance pathways using proteins of cell surface pumps since nanoparticles (NPs) penetrate the cell through endocytosis, and possibly create widely distinctive therapeutics that have a specific range of characteristics.

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Aptamer (Apt)-functionalized NPs can pave the way toward new and complex design ideas for the bio-medical field by combining the benefits of nano-medicine having the cell-targeting capacities of Apt. Farokhzad et al. released a paper on the synthesis of Apt-NP conjugates for the site-specific transport of NPs to cancerous cells. Ever since, several unique Apt-NP systems have been prepared to enable targeted therapy, directed imaging, and high cell sorting, in addition to the identification of novel cell-specific Apt out of consistently growing SELEX technology [35]. Aptfunctionalized structures had been widely used and show promising outcomes in a variety of areas because of their particular abilities like targeting, ease of modifications, minimal immunogenicity, and high binding affinity. This is predicted that the particular identification and incorporation of therapeutic molecules to specific tumor tissues would be enhanced by functionalizing cell-specific, integrating aptamers with a drug or delivering vehicle. Recent studies concerning the synthesis of NPs with Apt tagging rely upon that NPs’ efficient surface functionalization. The aptamers could be conjugated to the NPs by covalently bonding as well as noncovalently bonding of the aptamer on the NPs surface, or by encasing the NPs outer layer with a biologically compatible amphiphilic component and afterward conjugating the aptamer to the encasing. Furthermore, remedies might be systematically constructed to counteract the interactions among Apt and their specific targets, resulting in regulated targeted therapeutic systems. Considering the latest scientific advancements, designing selecting methods to identify new Apt capable of detecting antigens of specific diseases and using these to differently target disease cells would become more viable. In developing such structures, we have to keep in mind that “Apt-functionalized NPs” are fused entities with a discrete series of characteristics that could differ from Apt or NPs. Apt-drug moiety is utilized for efficient drug administration, for instance, the aptamer-fluorophore moiety is usually employed to image cancer cells or identify certain analytes. Furthermore, nanomaterials having their distinct physicochemical features paired with aptamers (Apt-nanoparticle moieties) particularly caught the interest of researchers in biomedical utility. Despite the fact that numerous studies have been published about the uses of aptamers, they only describe the specific properties of aptamers that have little biological importance [36]. In addition here in this book chapter only five types of aptamer-functionalized nanoparticles preparation are discussed but there are many more other types also which are prepared for specific purposes.

8.2.1

Aptamer-functionalized Au nanoparticles

Gold nanoparticles (Au NPs) are frequently employed in a variety of applications, such as surface accelerated Raman, bioassay, and biomarkers because of their exceptional optical characteristics, catalytic capabilities, and biological compatibility. Because of the characteristics of surface plasmon resonance (SPR), Au NPs may be utilized as visual sensing devices, which has piqued the interest among researchers. These are frequently employed in biosensors. Since Au NPs possess particular optical and electrical characteristics and thus are simple to functionalize. The latest research with aptamer-functionalized gold nanoparticles (Apt-Au NPs) based on signaling

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enhanced colorimetric, SPR, and electrochemical tests indicated how Apt-Au NPs might be employed for efficient signal amplification [37]. Au NPs are excellent materials for research since they are nontoxic, most stable, and simple to manufacture NPs and display a variety of intriguing features such as assembling into different kinds and show quantum size effect. The optical behavior of Au NPs is governed by its SPR which is situated in a broad portion of the spectra extending from visible to infrared. The spectra’s range is determined by several properties of Au NPs, such as shape and size. Techniques for reproducibly synthesizing those materials, which may then be changed with a variety of biochemical functional groups, were established. Based on gold nanoconjugates, several novel specific and sensitive tests have been developed. Au NPs could be considered as a promising possibility for use in delivering diverse contents to the targeted region. Some pharmaceutical molecules could be chemically linked with Au NPs by and without requiring alteration of a monolayer of Au NPs. Gold nanoparticles, on either hand, require functionalization such as PEGlyation, amino acid, and peptide conjugation with oligonucleotides, in order to carry additional loads [38]. Lately, DNA aptamers were created as extremely sensitive and selective biosensors and image analysis sensors, and also promising candidates for targeted cancer therapies. Due to their strong binding capacity for tumor cells’ nuclei, the ss DNA aptamer AS1411 was already proven to work like an anticancer agent. The above aptamer (Apt) was coupled to Au NPs in order to develop 2-component nanoconjugates of Apt-packed Au NPs capable of interfering with cancerous cell nuclei [39]. Au NPs are suggested to be adequate drug transporters because they could be customized utilizing bio-related compounds to increase tumor-targeting selectivity. From standpoint of application and engineering, ligandbonded gold nanoparticles offer a potent framework for targeted recognition, diagnosis, and therapy. Ding et al. created antitumor NPs with targeting characteristics which are (AptamerAu)-mutated Morin pH-sensitive liposomes (MSL). They discovered that it has good monodispersity qualities and cancer targeting and therefore it may be discharged in an acidic medium via dialysis. Once it was tested on cancerous cell models, it was discovered to efficiently decrease the growth of cancerous cells. Morin Hydrate, an antitumor medicine, was employed in the investigation. The hydrate form of Morin is a naturally existing active ingredient derived from Chinese plants or herbal remedies. Within the plant, this is a progesterone component and secondary metabolites of phenol. Morin is extensively dispersed in the environment and has significant anti-inflammatory, antitumor, and antioxidant properties [40]. Ding et al. initially generated Au NPs and then created Apt-Au NPs from them. In the manufacture of Au NPs, HAuCl4.4H2O was mixed in Milli-Q water, and polyvinylpyrrolidone was put into the aforementioned solution. The produced mixture was then finally put into a flask and microwaved for some time while being constantly agitated. As a result, a 1e2 mL solution of Sodium Citrate was immediately given to the solution and reheated for a few minutes. The mixture then was centrifuged for 5e10 min to extract Au NPs. The disulfide bonds of Apt were first broken utilizing tris (2-carboxyethyl) phosphine. The produced Apt solution mixture was then added up to the Au NPs solution after 30 min and incubated for the day to create Apt-Au NPs. Over 1 day, the

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combined mixture was salted two times using NaCl spaced by 4 h. The produced Morin-liposome lipid layer was then saturated with Apt-Au NPs in dextrose solutions, proceeded by ultrasonic treatment, resulting in Apt-Au@Morin pH-sensitive liposomes. Furthermore, a strategic preparation of Apt-Au@Morin pH-sensitive liposomes is described in (Fig. 8.2) [39]. Apt-Au@MSL demonstrated superior tumor-targeting and monodispersity characteristics. Morin’s polarity has been altered, and its anticancer activity has been increased. There in characterization studies, the solution pH was 5, and the rate of release of Morin from Apt-Au@MSL remained indeed the highest. According to tumor weight tests, in vivo studies indicate that it can reduce tumor development in xenograft rat models.

8.2.2

Aptamer-functionalized liposome nanoparticles

The development of targeted deliverance systems for cancer therapy is a growing area in the pharmaceutical companies. The majority of tumor treatment drugs have low bioavailability and high off-target toxicities. As a result, specific localization of the tumor tissue is critical for improving therapeutic efficacy and reducing the adverse effects of antitumor drugs. The utilization of nanomaterials as deliverance systems was the topic of substantial research over the last several years [41]. Liposomes were utilized to deliver a wide range of small genes, pharmaceuticals, even nanoparticles, and imaging agents, making them the most effective nanomedicine deliverance systems. Liposomes exceed alternative deliverance systems in terms of biodegradability, biological compatibility, control over size, toxicity, and

Figure 8.2 A diagrammatic representation of the Apt-Au@MSL production.

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surface activity. These are tiny lipid bi-layers with an aqueous center that may transport either hydrophobic or hydrophilic medicines [42]. Because liposomes had previously been licensed by the US Food and Drug Administration (FDA) for a variety of therapeutic treatments, therefore on starting with this framework and introducing DNA aptamers could enhance targeting capabilities and might possibly speed up the translations to medical practice. A PEGylated liposomal doxorubicin formulation (Doxil-R), would be the earliest FDA-approved liposomal composition for the medication of firm tumor cells. Doxorubicin encapsulated in PEGylated liposomal composition enhances its pharmacokinetic characteristics. Liposomal encapsulating lengthens the lifetime of doxorubicin and slows its elimination. Furthermore, the cardiotoxicity from the liposomal composition is lower than that of the free pharmaceutical. Further liposomal formulations allowed for tumor treatment include vincristine, daunorubicin, cisplatin, and cytarabine. Although doxorubicin has lower cardiotoxicity, researchers have revealed that using Doxil-R could result in adverse effects like mucositis, myelosuppression, and hand-foot syndrome that can be caused by the gradual releasing of the entrapped drug [43]. Furthermore, passive-targeting is ineffective for antitumor drug deliverance in hypo-vascular regions such as hepatic, pancreatic, and prostatic types of cancer. The effectiveness of a drug during passive targeting is primarily proportional to its circulating time. It is accomplished by covering the NPs with a coating. Despite the constraints discussed above, there would be an increasing consideration in developing unique kinds of liposomes with enhanced properties of sustained drug deliverance and clinical efficacy, such as ligand-targeted liposomes, ultrasound/magnetic responsive liposomes, and photo-sensitive/temperature/pH liposomes. The frequently utilized technique is dependent on using particular ligands with a higher affinity for cancerous cells but no or minimal affinity for healthy cells. The addition of particular ligands onto liposomes increases tumors aggregation and drug administration. There are diverse kinds of ligands that might be employed to interact with these liposomes, however, aptamers are preferred due to their distinct characteristic enabling controlled delivery. Additionally, the peculiar targeting of cancerous cells is an important stage in the earlier identification of cancer. To meet this demand, DNA Apt had earned a lot of interest as a possible targeting ligand. These are easy to produce and easily combine with nano-medicine systems. Furthermore, complementary DNA (cDNA) segments can hinder the targeting activity of DNA Apt, therefore, acting as a particular antidote to avoid overdosing on the drug during the deliverance of the drug. Furthermore, as compared to earlier described RNA Apt, DNA Apt exhibited lower liability to biodegradation, making them better suitable for therapeutic trials [44]. This utilization of aptamer-functionalized liposomes nanoparticles for tumor treatment is a potential breakthrough in the targeted deliverance of drugs. Aptamertargeted liposome significantly increased cancer cell absorption, according to in vitro and in vivo observations. Directed deliverance of cytotoxic drugs through aptamer-functionalized nanostructures is a suitable technique that has proven to be versatile in its applicability [45].

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Xing et al. produced a liposomal drug deliverance system for directed anticancer therapy. They created a nanosized liposome that was altered using the 26-mer guanosine-rich DNA aptamer AS1411. AS1411 does have a greater affinity for nucleolin (NCL). Nucleolin is a bcl2 mRNA-stabilizing protein that is mainly present in the membrane of plasma in many kinds of tumor tissues, particularly breast cancer and leukemia. They are multipurpose proteins that are over-expressed in different kinds of tumor cells. Nucleolin suppresses tumor cell apoptosis and is involved in its angiogenesis. This nucleolin-specific Apt AS1411 is the best accomplished in targeted deliverance and cancer therapy. AS1411 Apt potentially suppresses cell growth in a range of tumors [46]. AS1411 is the anti-cancer aptamer to reach the clinical first phase [47]. They created AS1411 Apt functionalized liposomes carrying doxorubicin (Dox), an antitumor pharmaceutical. DOX is a highly efficient chemotherapy drug. DOX incorporated into nanoparticles is favorable due to its higher effectiveness and lower cardiac risk because of its sustained-release effects. They prepared directed liposomal doxorubicin (Apt-Dox-Lip), showing specific administration and increased mortality of MCF-7 breast cancerous cells. When xenograft MCF-7 tumors containing nude mice is injected with Apt-Dox-Lip that showed an early outset of tumor suppression and higher antitumor efficacy, owing to greater tumor absorption and internalize cellular manner as compared to nonaptameric DNA altered constituents. They prepared NCL aptamer-functionalized liposomes having a radius of 100 nm by utilizing a polycarbonate membrane proceeded via lipid extrusion. The total lipid content was kept constant in a particular synthesis. It was centrifugated to condense the Dox-restraining Apt-liposomes. A mixture of dye-restraining liposomes was spotted as distinct luminous spots underneath a fluorescence microscope, proving the development of liposome nanoparticles. After studying the morphology and structure of aptamer-modified liposomes it was found that these liposomes were circular in shape, having a lipid bilayer covering having width in the nanometre range. The normal width is between 190 and 200 nm which corresponds to the pore diameter of the polycarbonate membranes employed in synthesis. In order to illustrate the functionalized liposomes using the DNA Apt and to assess the DNA content, it was dyed with OliGreen a coloring ssDNA dye to quantify the DNA densities across every liposome. Liposomes that are not functionalized with DNA Apt exhibited just a minimal amount of basic fluorescence. They developed doxorubicin-incorporated AS1411 Aptfunctionalized liposomes as drug deliverance systems that are capable of targeting NCL. It was shown that the MCF-7 cells shows targeted efficacy of AS1411 Apt-functionalized liposomes was excellent as NCL was highly expressed. In vivo observations on MCF-containing rats revealed that employing regulated liposomes containing AS1411 Apt-functionalized liposomes increased anticancer efficacy and cytotoxicity within MCF-7 cancerous cells. The anticancer efficacy is boosted because the Apt-functionalized liposomes have a greater capacity to penetrate tumors. On comparing to nontargeting liposomes, AS1411 Apt-functionalized liposomes improved cytotoxicity and cellular incorporation to A375 malignant carcinoma cells and MCF-7 breast cancerous cells [48].

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Aptamers Engineered Nanocarriers for Cancer Therapy

Aptamer-functionalized polymeric nanoparticles

Chemotherapy together with surgery has been the leading treatment technique for many tumors. Unfortunately, nonspecific transport of chemotherapy drugs has a negative impact on healthy cells and restricts drug dose to tumor cells, reducing the medication’s therapeutic potential. For instance in polymer NPs, biological compatible particles of different sized could be prepared, and their hydrophobic center could be used to encapsulate drugs having low permeability and solubility while having a greater packing capability. The simplicity of preparation of polymer NPs is its desirable characteristic. Furthermore, releasing rate of drugs out of these nanoparticles could be modified by selecting biodegradable polymers having an ideal degrading rate and interacting affinity to the enclosed drugs. Variations in temperature and pH conditions could act as a trigger to begin the delivery of the drug. Notwithstanding most of these enticing characteristics, polymeric NPs had showed a medium circulating lifetime to date [49]. Chemically synthesized nanomaterials could be employed in a range of biological and medicinal applications enabling molecular payload transfer as well as imaging. Polymeric NPs could be made from a variety of widely viable synthesized substances or monomeric organic molecules, comprising basic polymer components like poly(D,L-lactic-co-glycolic acid) (PLGA), polystyrene (PS), and Poly(ethylene glycol) (PEG), to produce 3D amphipathic frameworks [50]. PLGA is particularly most often utilized for the preparation of polymeric NP-Apt bioconjugates. Such polymers could encapsulate therapeutic drugs in their hydrophobic centers and they could be efficiently modified for deterioration. And consequent regulated drug delivery by changing its content, enabling for medium sustained doses throughout extensive durations of time [51]. Apt are presently being studied as a viable replacement to antibodies in vivo as targeted treatments independently or as directed deliverance vehicles for molecular imaging probes, small interfering RNAs, and chemotherapy drugs. Furthermore, nanocomposites have come out as critical technique for delivering smaller compounds throughout the body with improving bioavailability. Whenever the exterior of therapeutic-packed nanocomposite is functionalized, that is, coupled to cell-explicit targeting ligands like aptamers then the distribution of the nanocomposites is improved. Because its safety for clinical utilization is well recognized, NPs produced out of PLGA as the sustained releasing polymeric system are indeed an ideal option. PEG-functionalized-PLGA NPs are principally appealing therefore PEGylated polymeric NPs show considerably lower circulatory elimination than equivalent nanoparticles missing PEG [52]. PEGlyation of nanoparticles compositions expands the circulatory period and easily bioavailable in the blood system, lowers renal clearance, and even offers a validated influence over safety in both commercial and clinical situations. Polymer and copolymers are hydrolyzed onto metabolites monomers, which are rapidly absorbed by tissues via regular respiratory processes including the citric acid cycle [53]. Different aspects must be regarded when selecting appropriate NP drug carriers and target specific ligands; significant factors include the charge and size of the particles in addition to that of the ligands, binding affinity, and specificity

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of the ligands, ligand, and particle sustainability, and poisoning hazards. Moreover, it also includes criteria like production cost, simplicity of production and scaling-up process, consistency during the production environment, and the simplicity of necessary upgrades and combinations as well can be regarded [54]. Docetaxel, cisplatin, and other anticancer medicines might be delivered to specific carcinoma cells using Aptfunctionalized polymeric NPs. Docetaxel is a chemotherapeutic drug that is utilized in treating a vast range of tumors. Breast cancer, gastric cancer, prostatic cancer, neck and head cancer, and lung carcinoma are among the several examples. And can either utilized solo or in conjunction alongside other chemotherapeutic drugs. Cisplatin has been employed to cure a vast range of tumors, although dose-limiting toxicity or acquired and inherent resistance hinder their efficacy throughout various cancers, especially prostatic cancer. Farokhzad et al. produced docetaxel (Dtxl)-encapsulated nanomaterials made of biologically compatible and bio-degradable poly(D,L-lactic-co-glycolic acid)-blockpoly(ethylene glycol) (PLGA-b-PEG) copolymer and their outer layer functionalized with A10 20 -fluoropyrimidine RNA Apt which recognize the extra-cellular site of the prostate-specified membrane antigen (PSMA). Such Dtxl-encased NP Apt bioconjugate (Dtxl-NP-Apt) attach to the protein of PSMA expressed along the exterior of prostate epithelial cells LNCaP and are absorbed by such cell lines, leading to considerably higher in vitro cell toxicities than non-targeting NPs lacking the PSMA Apt. The nano-precipitation technique of creating nanomaterials has numerous significant benefits, including reproducible carrier size and easily adjustable within nanometre scale and the employment of compounds with minimal harmful risk, particularly essential for intravascular deliverance [55]. Farokhzad et al. used nanoprecipitation to encase Dtxl inside a PLGA-b-PEG block copolymer containing a terminally carboxylic acid group (PLGA-PEG-COOH) and created Dtxl-encased, PEGylated PLGA NPs (Fig. 8.3). The PEG group hydrophilic enables carboxylic acid expression on the exterior of NPs. Furthermore, the PEG group reduces the non-specific in vivo accumulation of nanoparticles and reduces nanoparticle absorption by healthy cells, particularly premature clearing via the mononuclear phagocytic system. Because of this inclusion of carboxy altered PEG upon the NPs surface, leading to negative surface charged potential, which might reduce Apt interactions with NPs, retaining Apt conformation and binding properties. The exterior of such NPs is subsequently functionalized by the Apt A10PSMA, allowing for selective absorption by specific PCa cells. Scanning electron microscopy (SEM) was utilized to check the surface size and shape distributions of NPs. They investigated the in vivo and in vitro effectiveness of the NP-Apt bioconjugate toward tumor tissues. Furthermore, the ingredients utilized in the production of the bioconjugates are recognized by the FDA before using it for clinical purposes. Because of directing compounds utilized in its synthesis are nonimmunogenic, tiny, reasonably stable, and simple to manufacture, such bioconjugates could be easier to translate into medical applications [56].

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Figure 8.3 Dtxl-encased PEGylated PLGA NP-Apt bioconjugates are being developed. (a) Diagrammatic depiction of the PLGA-PEG-COOH co-polymer production and encapsulating technique of Dtxl. NHS (N-hydroxysuccinimide); EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG).

8.2.4

Aptamer-functionalized hybrid nanoparticles

The principal purpose of the present chemotherapeutic is to impart an effective and safe dosage of therapy drugs in reaching the cancer region while causing no harm to healthy tissue. Nanotech-based drug deliverance systems are acquiring popularity as a method of improving drug deliverance efficacy since these possess the ability to lessen adverse reactions, lower toxicity, and increase antitumor therapeutic efficacy. Using the EPR effect, NPs could transport medications to tumors selectively. Lipidpolymer hybrid nanoparticles (LPNs) are central NPs made up of a lipid exterior

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and a polymeric center. The polymeric center may encase hydrophilic and hydrophobic medicines, and the lipid coating covers the polymeric center’s exterior surfaces, producing a wall to protect immediate drug leaking and extending drug-releasing duration. The latest research indicates suggested LPNs offer a considerable benefit during coupled person-centered therapies. Scientists used a variety of ligands to generate ligand-functionalized LPNs for cancer therapy, out of which Apt-functionalized LPNs receive the most consideration [57]. Bio-degradable LPNs which make use of the beneficial properties of polymeric NPs had recently turned out as a more appealing type of drug deliverance vehicle. The hybrid NPs could be a good drug deliverance system that uses a one-step nano-precipitation and self-assembly technique to reach optimal drug-encasing efficiencies, simplicity of preparation, the adjustable and persistent release of drugs, and have better serum stability [58]. Li et al. synthesized a hybrid of Curcumin NPs (CUR-NPs) in combination with soybean lecithin, PLGA, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Ncarboxy(polyethylene glycol) 2000 (DSPE-PEG2000-COOH) (or DSPE-PEG2000Apt) by employing a slightly modified nano-precipitation process. At the start, PLGA and CUR have been dissolved with CH3CN and diluted to a particular concentration. Lecithin and DSPE-PEG2000-COOH were heated for the next few minutes after being dissolved into ethyl alcohol aqueous with a particular percentage content of PLGA polymers. The above PLGA-CH3CN solution was then add on in drops to the lipids aqueous system to enable self-assembling for half an hour. Rotavap has been used to discard the organic solvent at lower temperature and pressure. The leftover organic solvent was subsequently then centrifuged, and the NPs were washed several times using water. As a result, PLGA-lecithin-curcumin-PEG NPs were created. The Apt-Functionalized Hybrid NPs were then prepared by hybridizing Apt with the PLGA-lecithin-curcumin-PEG NPs via an amide bond involving the carboxylic group of DSPE-PEG2000-COOH and the amide group at terminus of the Apt over 2 phases. During the first phase, DSPE-PEG2000-COOH is stimulated, and then in the second phase, it is covalently coupled to NH2-modified Apt. The resulting solution then was wash out repeatedly with water by using centrifugal equipment, and unbounded Apt were thus can be eliminated [58]. Chen et al. created Apt in a similar manner as Li et al. developed coupled PLGAPEG (APT-PEG-PLGA). Cabazitaxel (CTX) and curcumin (CUR) are both anticancer compounds that might be employed for the therapy of tumors. During the manufacture of LPNs, they employed L-Phosphatidylcholine from soybean (SPC) as lipids. SPC had first been diluted in C2H5OH and heated to 60e65 C, then CTX, CUR, and APT-PEG-PLGA were mixed in CHCl3 and introduced dropwise into the SPC solution while agitating and gyrating over some time. Apt-functionalized solo CUR or CTX releasing LPNs self-assembled to generate Apt-functionalized CUR and CTX LPN co-delivery. Such LPNs exhibit persistent CUR and CTX discharge. These additionally possess strong cell inhibiting capabilities, exceptional cancer elimination efficacy, and great tumor aggregation, leading to the possibility for synergistic tumor combinational treatment [57].

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Aptamers Engineered Nanocarriers for Cancer Therapy

Aptamer-functionalized mesoporous silica nanoparticles

Owing to such unique properties as massive packing capacity, biological compatibility, excellent thermal stability, tunable pore sizes (210 nm), inertness, homogeneous porosity, and ease of functionalization of the exterior and interior surfaces, mesoporous silica nanoparticles (MSN) were often chosen as a favorable carrier for designing an “on-command” deliverance system. Furthermore, MSNs undergo endocytosis via acidic lysosomes and are noncytotoxic, making these an appealing choice for intracellular stimuli-responsive deliverance of drugs. Many research have been conducted on the usage of MSN for sustained release systems. Various stimuli-responsive systems are now being developed in order to develop sensible MSN-based controlled systems that control the mobility of guest biomolecules. There are many different type’s poreblocking agents, such as supramolecular assemblies, organic molecules, and inorganic NPs were used as covers in such gating systems to protect guest biomolecules from escaping the porous system. Due to this, it can be released in particular surroundings by responding to various stimuli like competitive binding, pH, enzymes, temperature, redox activation, and photo-irradiation. For regulating the porosity of MSN, such systems are often adopted for smaller organic molecules, NPs, supramolecular assemblies, polymers, and biomolecules as the capped agent. Chemical or physical stimuli, including pH, antibodies, light, enzymes, temperature, and nucleotides, could cause the discharge of biomolecules [59,60]. Z. Chen et al. developed MSN via a base-catalyzed sol-gel technique, leading to highly porous NPs with hexagonal-shaped organized pores. Using distilled water, Cetyltrimethyl ammonium bromide (CTAB) got saturated. After that, a particular proportion of NaOH was gently introduced to the above CTAB solution while vigorously agitating for over half an hour and heating at 60 C to obtain a transparent and clear solution. Subsequently, tetraeth-oxysilane (TEOS) is then introduced to the aboveprepared mixture dropwise while vigorously agitating until precipitates are produced. The residue was cleaned using methyl alcohol and distilled water prior to air-dry. To get customized MSN using chlorosilane template (MSN-Cl-CTAB), a combination of trimethyl chlorosilane (TMCS) and synthesized MSN undergo refluxing for nearly 1 day under the presence of dehydrated toluene. MSN-Cl-CTAB were distributed in methyl alcohol containing HCl and the resulting mixture was then refluxed over one more day to extract CTAB. The residue produced upon centrifugation was cleaned using dehydrated ethanol and double-distilled water. For extracting solvents off the pores of the substance lacking a template was subsequently heated at 60 C. For creating N3MSN, MSN-Cl was distributed in dehydrated DMF, then slowly added NaN3 while agitating for several hours in icy water. The N3-MSN pores were packed with fluorescein isothiocyanate (FITC). The thrombin Apt was then coupled into cDNA over 60 min and maintained the temperature at 37 C in phosphate-buffered saline (PBS). For ensuring all cDNA was completely coupled to Apt, the Apt quantity was maintained greater than the cDNA quantity. N3-MSN was first distributed in PBS including FITC, then the resultant solution was subsequently agitated in a thermomixer at ambient temperature over 15e17 h. FTIC was distributed into the pore of the N3MSN throughout this procedure. The solution was then treated with tris [(1-benzyl-

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1H-1,2,3-triazol-4-yl)methyl] amine (TBTA), CuI, and dsDNA. At 37 C, the solution was slowly agitated over a few hours. Subsequently, the dsDNA-capped N3-MSN packed with FITC (dsDNA-FITC- MSN) were distributed in the buffer of PBS and stored at 4 C for later use. To demonstrate that it might be employed for thrombin analysis, they took dsDNAFITC-MSN and introduced varying quantities of thrombin into the buffer of PBS, then subsequently agitating the mixture upon thermomixer for some time at ambient temperature. The solution was then agitated, and the fluorescence of the leftover was measured. It was discovered that these Apt-functionalized mesoporous silica NPs were extremely sensitive and may be employed as an effective fluorescence biosensor in thrombin detection. This suggested biosensor has reproducibility, high selectivity, and good stability and it would be used effectively to detect thrombin in human serum samples with satisfactory outcomes. Moreover, the suggested biosensor provides a novel technique for the specific identification of targets as those biosensor contains aptamers that could act as sustained release systems.

8.3

Applications of aptamer-functionalized nanoparticles

Aptamers (Apt) are sequences of oligonucleotides, such as RNA or ssDNA, or segments of amino acids with a particular 3D framework that were determined in vitro from a huge arbitrary library employing a method called SELEX. This possesses a significant affinity and specificity for nonnucleic acid substrates including peptides, pharmaceutical medicines, proteins, inorganic and organic compounds, or an entire cell. Apt derived from nucleic acids have developed as interesting therapeutic treatments and detection aspects. Aptamers’ chemical characteristics are mainly owing to nucleic acid components, which have benefits such as easy chemical modification with various functional groups, small size, nontoxicity, ease of synthesis, and analysis of a diverse range of objectives, minimal immunity, and simple surface adsorption in biological applications. Apt is constantly being used in chemistry, biological sciences, and other disciplines such as diagnostic and therapeutic aids, drug design and distribution, and biosensing assays. Between these, Apt studies from the perspective of biological sciences are primarily focused on disease detection and therapy. To reach the full capability of nanoparticles in biomedical applications, they should be biologically compatible and capable of targeting particular molecules for selective imaging, sensing, and pharmaceutical delivery in complex surroundings like humans, animals, tissues, and living cells. Generally, nanoparticles could be easily functionalized by noncovalent action or noncovalent interaction. This improvement intends to increase detecting sensitivities and selectivity, decrease time consumption, and enhance binding specificity to targeted entities. In conclusion, the application of nanoparticles in healthcare offers a milestone in material design since the substances are developed to react with the body of a human at a cellular level with extreme accuracy. A variety of nanomaterial-Apt conjugates has demonstrated its value across a number

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of fields by integrating the intrinsic properties of nano-materials alongside the particular identification capabilities of Apt and hence could be utilized for several purposes. Their uses are divided into two categories: Firstly, Apt offer particular prospective in biological applications like drug deliverance and the production of novel therapeutic devices due to their precise interaction with target biomolecules. Secondly, the adaptor’s characteristic allows these to perform a vital part in the preparation of novel biosensors. Hydrogels, magnetism, silica, metals, and carbon-based substances have generated significant breakthroughs in biology till date [61].

8.3.1

Biosensors to detect cancerous cells

Innovative colorimetrical biosensors centered on Apt-gold nanoparticle (Au NP) probes were produced throughout the last 10 years because Au NPs have distinct optical and electrical characteristics as well as a large molar extinction coefficient, allowing these to act as effective probes for colorimetrical tests. Apt-functionalized AuNPs are frequently utilized in colorimetric molecular identification in which targeting molecule-induces AuNP aggregation could be detected via a color shift between red to blue. Apt having a particular sequence are deposited on the surface of gold nanoparticles (Au NP) by a self-assembling method. Integrating the superior molecule identification characteristics of Apt with the specific optical features of Au NPs colorimetric test for detecting proteins and antigens, thus functions as a biosensor. As a result, it could be employed as a carcinoma biomarker. The assay’s operating concept combines Apt-target identification and Apt-regulated synthesis of Au NPs. Whenever the targeted molecules are associated with the Apt, the quantity of Apt strands deposited onto the exterior of Apt-functionalized Au NPs is regulated by desorption of the Apt strands. Relying on the Apt covering, Au NPs form structurally diverse nanoparticles that lead to the production of a range of colored mixtures. Au NPs having limited Apt covering build into globular NPs that yield red solutions, while Au NPs with great Apt covering build into branched NPs that yield blue solutions [62]. AuNPs are frequently employed in colorimetrical sensors because of their great stability, ease of production, high biological compatibility, and intense SPR. Colorimetric biosensors have gotten a lot of interest because of their simple viewing with the bare eyes which don’t need any costly equipment and technical operation. These NPs may assemble based on the changes in the surface. NP accumulation could be influenced by a variety of reagents, including proteins, enzymes, and possibly bacteria and viruses. Appropriate immobility of Apt on the surface is critical in the preparation of Apt-based biosensing assays. These contain appropriate surface densities for giving structural diversity to the Apt enabling overlapping, which is required for the development of interaction in the area of paralleling target [63]. The depiction of several methods for colorimetric bio-sensors is given in (Fig. 8.4). The accumulation of AuNP is a simple and inexpensive technique having great sensitivities [64]. Qin et al. employed lateral flow strip biosensors (LFB) as they are very important to build an ultra-sensitive sensor with key characteristics like rapid response, simple design, high sensitivity, and minimum material utilization. Such LFB based upon

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Figure 8.4 An overview of several techniques to colorimetric bio-sensors, as well as a brief description of respective procedure.

Apt-functionalized AuNPs were utilized to assess bio-recognition processes like detection of single-base variations in DNA, carcinoembryonic antigen (CEA), histone methylation, thrombin (TB), human stem cells, salmonella, natural toxins, metal ions, microRNA, and so on. They suggested that strip biosensors depending on Apt-connected AuNPs accumulates that would break with the interaction of Apt and target. TB is utilized to show the principle and provides experimental results for this case. The unique interactions of TB with its interacting Apt were already studied. Because of the SPR, the aggregation remains stable around ambient temperatures when TB is absent. Whenever the Apt attaches to the TB molecule there is a structural shift. The suggested LFB was competent for quantitative and qualitative thrombin detection having excellent efficiency and accuracy under ideal circumstances. Furthermore, this technique is likely to be effectively suitable for strip sensing of a diverse scale of analytes. Furthermore, the Au NPs could be substituted with alternative metallic NPs (such as magnetic nanoparticles, quantum dots, nanotubes, or polymer beads) to generate unique changes in color with respect to various analytes. As a result, these basic bio-sensors would be used in near-patient testing, environmental monitoring, medical diagnostics, clinical diagnosis, and home testing [65]. Moreover, because of its increased stability, better resistance to chemical changes, and the potential for chemical production, nucleic acid Apt offer a far more practical platform enabling the construction of multifunctional structures. For conjugating Apt modules with one another and with other biomolecules of interest, a vast range of chemical methods is known, allowing nearly any required criteria for specific studies

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to be accomplished. A broad range of bioanalytical techniques for cell imaging, affinity capturing, and biosensing have been produced on integrating various Apt modules with reporting groups, NPs, or analytical systems [66]. Apt-based colorimetric biosensors are typically made by covalently bonding Apt with Au NPs. Thiol-derived Apt are most often utilized Apt for producing functionalized Au NPs via the strongest AueS interaction. Thiol-modified Apt are considerably more costly than original Apt (having no thiol fixation). Urmann et al. developed a tag-free optical bio-sensor based on Apt-conjugated porous Silicon, which showed low interaction of nontargeted proteins also in complicated biofluids, validating the exceptional selective nature of such Apt-based bio-sensors for its targeted analytes. Significantly, the bio-sensors are extremely stable and may be easily reproduced for numerous bio-sensing tests with a simple purification step. The disclosed optical tag-free bio-sensor approach shows considerable potential for the development of adaptable bioanalytical studies, enabling the easy and effective quantification and detection of analytes [67]. Due to a major shortage of viable indicators, the majority of research relating to breast cancer identification is constrained within limited ranges. Jo et al. developed new diagnostic complexes, double Apt-customized Si NPs, owing to the observation that MUC1 and HER2 are overexpressed in breast cancer cell lines, for the simultaneously targeted breast tumors comprising MUC1(þ) and HER2(þ) cells. Except for a standard Apt-customized probe, these probes detect cancerous cells more efficiently. Additionally, the synthesized particles are tested via selectivity, binding, and cytotoxicity assays, and the findings indicated that these nontoxic probes are extremely sensitive to MUC1(þ) and HER2(þ) breast cancerous cells [68].

8.3.2

Targeted drug delivery and cancer therapy

Even though polymeric nanomaterial- and NP-based treatment techniques are extensively used for the delivery of drugs and anti-cancer therapeutics and diagnostics, there are still issues in biological compatibility, complexity, and efficacy. The introduction of RNA/DNA nanobiotechnology had also enabled the exquisite self-assembling of 1D nucleic acid biomolecules into 2D and 3D nanomaterials via particular molecular identification and customizable molecular structure, like H-bonding and p-stacking, to prepare the next-generation of drug deliverance systems. Such 3D nucleic acid nanomaterials, which undergo self-assembling by foreseeable and customizable nucleic acid biomolecules with varied functional groups, have aroused interest in disease diagnosis and bio-sensing, particularly as possible biological carriers for the deliverance of drugs and diseases therapy. The capability of RNA/DNA nanotech in self-assembling 1D DNA basic elements into 2D and 3D nanomaterials has vast applicability in bio-imaging, fundamental biological mechanism investigations, disease diagnostics, and drug deliverance. Moreover, the majority of nucleic acid nanomaterials’ cellular absorption is based on passive deliverance or the improved retention effect and permeability that might not be ideal for some kinds of cancer, particularly during in vivo therapy. They developed multi-functional Apt-based DNA nanoassemblies (AptNA) enabling targeted malignancy treatment to fulfill the above demand. This Apt-based nanoassembling system

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had many notable characteristics like-it has specific functional areas that consist of targeting Apt, entrapped anti-cancer drugs, and medicinal antisense oligonucleotide that helps these nanoassemblies serve as a viable system for targeted cancer therapy. And also these systems have a simple modular structure, easy assembling, and synthesis, and undergo photopolymerization to generate size-regulated nanomaterials. The essential building block allows for exact regulation of the functional component ratio and also customizable construction of functional domains depending on the treatment objective. A high capacity loading of antitumor drugs or bio-imaging reagents solo nanoassembling is also possible with the hybrid ssDNA structure. The hybrid ssDNA arrangement, therefore, enables the packing of antitumor pharmaceuticals or bioimaging reagents thousands of times in one nanoassembly and that nanoassembly possess a high bio-stability due to their enzyme tolerance and packing stability throughout physiological circumstances. And also those nanoassembly exhibit low cytotoxicity whereas showing selective cytotoxicity once altered with the proper Apt, implying great biological compatibility [69]. Apt-functionalized NPs have a vast ability as a novel form of drug deliverance device for cancer cell-targeting chemotherapy. During the last several years, Apt-based treatment techniques for reversing multi-drug resistance (MDR) were produced. Because of the high selectivity of Apt-functionalized NPs in the targeted deliverance of therapeutic medications could be transferred selectively to the cytoplasm as well as the nucleus of cancerous cells, disrupting MDR-related pathways and reversing them [70]. Advanced Apt-NPs conjugates were thoroughly investigated for the study of targeted deliverance of drugs in vivo and in vitro. Apt could be changed and coupled to several biomolecules like therapeutic drugs, siRNAs, imaging agents, and NPs depending on their pharmacokinetic characteristics, therefore acting as the best reagents in targeted cancer treatment [71]. Furthermore, self-assembling nucleic acid nanomaterials are present difficulties that supposed to be overcome. To begin, the majority of 3D nucleic acid nanomaterials penetrate cancerous cells or tumors by passive deliverance. Since this passive deliverance could be enough for breast or prostatic tumors but with leakage in vasculatures, this might not be appropriate for other kinds of tumors like leukemia and lymphoma, limiting wide in vivo applicability. Apt moiety could act as a reference system for specifically targeting cancerous cells via combining all functional domains into a single nanoassembling system. Several of these fundamental building blocks also are photo-cross-linked to form multi-functional and customizable nanoassembling frameworks having variable radii. The massive nanoassembling offers multiple numbers of sites enabling greater loading capacity of drugs or bio-imaging agents. Furthermore, AptNAs have good bio-stability in physiological settings (pH 7.4), preventing unwanted drug leakage throughout delivery.

8.3.3

Targeted photodynamic therapy

Photodynamic therapy (PDT) had attracted interest as a better potential cancer treatments method because of its minimal infiltrative nature and innate specificity over

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focused irradiation at tumor regions. PDT needs a mixture of radiation, a photosensitizer (PS), and cellular oxygen to produce cytotoxic singlet oxygen (1O2) which could harm cancer cells. While porphyrin derivatives are commonly used as PS in PDT but its hydrophobicity makes efficient transport of porphyrinic agents to tumor regions difficult. Likewise, nanostructures were used as vehicles for PDT treatments, however, those examples frequently demonstrate disadvantages such as inadequate regulation of packing, enhanced toxicity, leaching, and instabilities. Controlled deliverance of intracellular stimuli-activated photosensitizers (PSs) onto the cancerous cells has enabled targeted imaging and desired PDT of cancers. Therefore, it presented a significant chance for exact cancer detection and treatment [72]. PSs which exhibit simultaneously increased fluorescence and PDT among the cancerous cells had provided a major chance for genuine tumor diagnosis and their particular treatments, which is greatly desired for targeted tumor therapy and diagnostics. In contrast to standard PSs, which are frequently fluorescent and photo-toxic for healthy tissues, simulatable PSs could only generate fluorescence and photo-toxicity to cancerous cells when they engaged with a particular stimulus that is present inside cancerous cells. This could increase the accuracy and sensitivity in differentiating cancer tissues from healthy tissues in basic terms. Furthermore, targeted stimulation of PSs to regulate the generation of reactive oxygen species (ROS) inside cancerous cells could be used to selectively destroy cancerous cells while causing minimal adverse effects in healthy tissues. As a result, a variety of simulatable PSs which respond to various biological stimuli, such as enzymatic, biothiols, pH, and metal ions, were extensively characterized, with the goal of improving cancer therapy. A best simulatable PS for tumor cell PDT does have properties like targeted delivery and aggregation in cancerous cells, precise initiation by intra-cellular biomolecules, poor fluorescence and minimal dark toxicity previous to the initiation, and better fluorescence and enhanced photo-toxicity upon initiation by biomolecule [73]. In directed PDT, an Apt-functionalized metal-organic framework (MOF) could be potentially utilized. Such nanostructures are simple to make and could be employed for directed PDT having remarkably improved therapeutic efficacy in vivo and in vitro. Meng et al. designed an advanced cancer-specific imaging and photodynamic treatment platform by inserting a cell-specific Apt to target domains and a G-quadruplex as PS carrier domains to the surfaces of Zr-based nanoscale metalorganic frameworks (Zr-NMOFs) via stronger ZreOeP bonding. The Aptfunctionalized MOFs nanostructures they created have the following distinguishing features: increased 5, 10, 15, 20-terakis (1-methylpyridinium-4-yl) porphyrin (TMPy4) packing effectiveness, improved deliverance of TMPyP4 to targeted cancerous cells and regulated PDT in vivo. Furthermore, the NMOFs exhibit great stability, strong biological computability, and the capacity to preserve nucleic acids against nuclease digesting, suggesting their possible use in biomedical research. As a result, these advanced Apt-functionalized MOFs nanostructures might be investigated subsequently as a multifunctional therapeutic means for earlier diagnostics and targeted cancer treatment [74].

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211

Thermo-chemotherapy

Throughout the analysis of cancers, nonetumor-directed distribution of chemotherapy medicines has major consequences for healthy tissue cells that lower the dosage of drugs accessing cancerous tissues, and this would reduce drug medicinal value. As a result, developing newer drugs and therapy alternatives for cancers is a problem. The deliverance of cytotoxic drugs to cancerous cells by active or passive targeting has proven to be an efficient strategy for reducing the effect on healthy cells while enhancing the treatment. Thermo-chemotherapy had gotten a lot of interest as a possible cancer combination therapeutic technique. Wang et al. synthesized gold nanorod (AuNR) having properties like biocompatibility, superstability, and layered fluorescent carbon then prepared AuNR@carbon centre-shell nanostructure that is again packed with cell-specified Apt and remedial molecules by p-p interaction. Therefore, act as a beneficial device for tumor-targeted cellular therapy and imaging. Furthermore, innate and stable fluorescent characteristics of the AuNR@carbon facilitate concurrent tracing of delivered therapeutic molecules and nanostructures undergoing thermo-chemotherapy. They used Doxorubicin (DOX), an anthracycline chemotherapy drug that was efficiently packed onto the carbon exterior of AuNR@carbon nanostructures. By utilizing DOX-packed nanostructures, the intra-cellular intake of AuNR@carbon-DOX composites can perhaps be activated by irradiation of NIR laser, leading to regulated drug distribution in the cell inside. Moreover, the AuNR@carbon nanostructures fluorescent exterior can be used for tracing the drug carrier NIR laser exposure before and after. Therefore, these in vitro findings show that the AuNR@carbon nanostructure is a unique and effective therapeutics and diagnostics framework that not only delivers anticancer drugs but additionally traces drug nanocarriers, indicating its viability for in vivo applicability. Because of stronger absorbance in the NIR area, integrated with the special characteristics of carbon and AuNR, AuNR@carbon was described as the most stable having outstanding photothermal warm-up capacity. In comparison to AuNR, the AuNR@carbon might have more resistance to disintegration on undergoing photo-activated regional heating due to the presence of a stable carbon exterior. These photothermal warm-up effects are then studied. To about 348 K the solution temperature of AuNR@carbon was increased previously to a few minutes of laser irradiation. These outcomes were just like AuNRs, however a lot greater than that of water. The massive carbon-confined exteriors imparted the AuNR@carbon superstability from photothermal heating without obstructing its heating effectiveness. The photothermal in vitro analysis was additionally observed with MCF-7 cancerous cells. Higher the quantity or duration of laser irradiation would significantly enhance photothermal effectiveness. The majority of cells were discovered dead after the introduction of a definite proportion of AuNR@carbon solution and laser irradiation for a few minutes, suggesting the higher effectiveness of AuNR@carbon as a suitable material in photothermal therapy. Combining photothermal and chemotherapy treatment will significantly improve therapeutic efficacy [75]. In order to demonstrate the synergistic effect of chemo-photothermal treatment, the cell viability of various therapies was recognized quantitatively by K. Wang et al. with the use of the Live-Dead Kit. They prepared HB5 Apt-functionalized mesoporous silica-carbonebased doxorubicin (DOX)-packed nanostructure (MSCN-PEG-HB5/

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DOX) and examined their utilization in chemo-photothermal combination treatment of human epithelial growth factor receptor 2 (HER2)- þVE breast cancerous cells. When these packed nanostructures are introduced to the NIR laser (chemo-photothermal treatment) then it gives simultaneously lower cell viability in comparison to the photothermal treatment and chemotherapy unaccompanied. Simultaneously, these packed nanostructures do not show detectable cytotoxicity of cancerous cells specifying that they have higher biological compatibility. Furthermore, the eliminating efficiency of the cancerous cells was enhanced with the amount of these packed nanostructures for combination treatment [76].

8.3.5

Other applications besides cancer therapy

Integrating nanomaterials with Apt could efficiently increase the sensitivity and selectivity of Apt-functionalized NPs. There are several uses of Apt-functionalized NPs other than cancer therapy. It could be used as a catalyst, for instance, catalytic DNA is functional DNA having catalytic characteristics. So they could catalyze various types of biological and chemical reactions, comprising splitting of nucleic acid substrates, tiny biomolecule disintegration, ligation of RNA/DNA, reorganization of the nucleic acid framework, and porphyrin metalation. Catalytic DNA was already frequently utilized in biosensors, disease diagnostics, and nanomaterials synthesis due to its distinctive catalytic characteristics and extended catalytic action [77]. The Apt-functionalized NPs for detection of pesticides i.e., as a biosensor could have faster detection and higher sensitivity to pesticides that don’t require synthesis of any complex samples. Pesticide contamination has sparked huge growing concerns, due to the widespread use of pesticides in agriculture for boosting crop yield and enhancing agricultural product qualities. A considerable amount of effort was added to develop quantitative or qualitative approaches for the detection of chemical pollutants in food during the recent few years. The Nishima Wangoo group published a work that employed Apt, cationic pesticides, and unaltered Au NPs for detecting Maldison, a deadly organophosphorus pesticide extensively utilized in agricultural purposes [78]. The Aptfunctionalized NPs could be used as Lab on a chip (LOC) that acts as a system that allows the combination of those components, giving a stronger automated system for the applications like controlling processes, protection of the environment, effluent treatment, and conducting life science discovery study [79]. Since LOC systems could do a comprehensive analysis of samples, the constituents of every system are chosen depending upon this analysis. Based on this problem analysis, the microfluidic systems and LOC systems are coupled. This technology can be a potential genuine therapeutic tool for medical and agricultural diagnostics, as well as bio-safety. The Aptfunctionalized NPs also show Chemiluminescence properties that could be used for detection. CL is an efficient and significant analytical method having very high sensitivity and a simple mechanism. This doesn’t require an outer light source for generating signals. In those fluorescent studies, Apt could be utilized as detection moiety and also regulates the distance between quenchers (materials having the ability to absorb energy from fluorophore). Several Apt-based biosensors techniques utilizing CL detection were proposed lately [61].

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213

Conclusion and future perspective

The current progress in tumor nanotechnology has highlighted the ability of molecular selection both in the treatment and diagnosis of disease. A great number of aptasensors were prepared, which have applications in biosensing and cancerous cell imaging. These apta-sensors could identify a little number of cancer-associated biomarkers or analytes with high sensitivity and specificity. Its high binding specificity, sensitivity, smaller size, and ease of detection with in vitro selection procedure (SELEX) suggest aptamers as potential options for various utilization in molecular targeting. Despite significant improvements in aptamer screening technologies, the fundamental aspects of aptamers, like production costs, resistance to nucleases, and flexibility of design, cannot be ignored. Additionally, aptamers and antibodies share many features that make aptamers potential candidates for widespread therapeutic and diagnostic applications. Therefore, more attempts should be made for making aptamer-functionalized frameworks to be applicable in medical practice. With extensive utilization of aptamer-functionalized frameworks, there is an increase in the number of researchers and pharmaceutical industries understanding their potential application and it could be guaranteed that aptamer-based items would start accessing markets in the future.

Acknowledgment Rambabu Dandela thanks DST-SERB for Ramanujan fellowship (SB/S2/RJN-075/2016), Core research grant (CRG/2018/000782) and ICT-IOC start-up grant. The authors acknowledge ICTIOC Bhubaneswar for providing necessary support.

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Aptamer-functionalized PLGA nanoparticles for targeted cancer therapy

9

Atena Mansouri 1 , Thozhukat Sathyapalan 2 , Prashant Kesharwani 3 and Amirhossein Sahebkar 4, 5, 6,7 1 Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran; 2Department of Academic Diabetes, Endocrinology and Metabolism, Hull York Medical School, University of Hull, Hull, United Kingdom; 3Department of Pharmaceutics, School of Pharmaceutical Education and Research, New Delhi, Delhi, India; 4Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 5Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 6School of Medicine, The University of Western Australia, Perth, WA, Australia; 7Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

9.1

Introduction

As described by the World Health Organization, cancer is an abnormal growth of cells that can invade and spread to other parts of the body resulting from genetic and epigenetic alterations. Due to the low number of tumor cells and molecular markers, early detection of cancer is limited at the precancerous stage and the early stage of cancer. Knowing the molecular characteristics of cancer, such as the specific proteins associated with a particular type of cancer, could be valuable in determining the management [1]. The term chemotherapy describes cancer treatments involving the use of chemotherapeutic agents (anticancer drugs) in a specified regimen. This method, however, comes with major limitations, including side effects, lack of specificity, and the development of drug resistance [2e7]. Multiple aspects of drug design have to be optimized simultaneously to create a safe and effective cancer drug. These include toxicity, side effects, targeting, delivery, and controlled release. The Food and Drug Administration (FDA) has approved a large number of anticancer drugs [8]. Since most of these agents are not molecularly targeted for cancer cells, they are toxic to normal cells as well. A major goal for cancer therapy is to develop new drugs with higher efficacy, but fewer toxicity and side effects. In addition to systemically delivering small molecules, nanocarriers can significantly improve the bioavailability of various therapeutic agents [9,10]. Nanocarriers with therapeutic-loaded surfaces are enhanced when they are conjugated, for example, with aptamers, a ligand that targets specific cells. Nanoparticles can deliver different molecules (e.g., proteins, chemotherapeutics, inhibitors, vaccines, and miRNAs) to target cells [11e14]. In addition, these particles show an increased Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00005-1 Copyright © 2023 Elsevier Ltd. All rights reserved.

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ability to tolerate high doses, the bioavailability of hydrophobic agents, and biodistribution to metastatic sites beyond the primary tumor. Nanoparticles can be synthesized using a variety of materials, among which polymer-based carriers have been highly sought after because they are biodegradable, biocompatible, and FDAapproved. The use of these technologies facilitates their future transition to clinical trials. Because PLGA is a proven, reliable controlled release polymer, NPs derived from this material are an excellent choice for clinical applications [15e18]. It is essential to target cells specifically when developing new strategies for diagnosis, prognosis, treatment, and imaging. Compared to other candidates, antibodies are interesting and widely used candidates in medicine; however, their high cost, immunogenicity, temperature sensitivity, reoptimization for each product, and production costs limit their application. An aptamer is a sequence of DNA or RNA that binds to a certain target specifically. They are highly chemically and thermally stable and are less toxic. A process known as systematic evolution of ligands by exponential enrichment can be used to screen Apts for high affinity. Compared with antibodies, aptamers show little or no immunogenicity [19]. As functionalized nanocarriers, they can increase the local doses, the effectiveness of cell death, and reduce the number of treatment cycles and relapse rates, thereby overcoming the limitations of conventional systemic anticancer treatments. Aptamers with nanoparticles are incorporated into aptamer-nanoparticle conjugates to demonstrate active-controlled delivery of drugs to disease sites [20e22]. Aptamer-based anticancer systems can be utilized in novel ways by combining nano-vehicles with antineoplastic agents [23]. In this chapter, we present data exploring the development of aptamer-based polymeric nanoparticles (NPs), which utilizes polymeric nanoparticles as anticancer delivery systems.

9.2

Aptamers

The aptamers are oligonucleotides consisting of RNA (ribonucleic acid), deoxyribonucleic acid (ssDNA), or peptide molecules. The specific structure of these molecules makes them capable of binding to their targets with high specificity and affinity. Even though RNA and ssDNA aptamers bind to the same target, they often differ in their sequence and folding pattern. As a result of studies on human immunodeficiency virus (HIV) and adenovirus in the 1980s, the idea of this fusion emerged. There were several small, structured RNAs present in these viruses that bound to viral or cellular proteins with a high level of affinity and specificity [24]. Short RNA ligands called transactivation response (TAR) elements of HIV promote transactivation, which in turn leads to viral replication by binding to viral Tat proteins [25]. RNA aptamer in adenovirus called viral-associated (VA)-RNA controls its translation [26,27]. A substantial amount of progress has been made on aptamers since Gold’s group and Szostak’s group first reported the system for selecting aptamers known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX) in 1990 [15]. In recent years, SELEX has increasingly become a technique for isolating aptamers. It is

Aptamer-functionalized PLGA nanoparticles for targeted cancer therapy

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Figure 9.1 Aptamer and target interaction. Reproduced with permission from Ref. [30]. License code: 5173000545583.

possible to select aptamers against a variety of targets directly in vitro, ranging from small biomolecules to proteins and even cells (Fig. 9.2) [28]. Several studies have used aptamers as biomaterials in the development of new drugs and delivery systems, as well as diagnostic and therapeutic tools. Various attempts have been made to find aptamers designed specifically to inhibit targets involved in various diseases, including cancer and viral infection. Researchers have developed aptamers primarily for diagnostic or therapeutic purposes. Aptamers have become more popular and are being used in more studies as aptamers have been considered more effective and more reliable. In general, aptamers often contain defined structures because complementary base pairs are often formed. Several secondary structures can be folded into them (such as the stem, loop, kissing hairpin, G-quadruplex, and pseudoknot). Hence, it is possible to form unique three-dimensional (3D) structures from these secondary structures and they can recognize specific molecular targets. There are several interactions in 3D, including hydrophobic and electrostatic bonds, hydrogen bonds, van der Waals forces, and shape modification. The affinity and specificity of aptamers are determined by these factors (Fig. 9.1). Aptamers, also known as chemical antibodies, Similar to the way antibodies bind to antigens, aptamers form complexes with their targets via specific 3D interactions [29] (Fig. 9.1).

9.3

Role of aptamers in cancer therapy

Despite its importance as a cancer treatment, chemotherapy is not perfect and can have undesirable side effects, including systematic toxicity. Targeted delivery systems of anticancer drugs may be less prone to systematic toxicity due to their enhanced delivery of chemotherapy to tumor tissue. As a result of enhanced permeability and retention (EPR), nanocarriers can substantially increase the effectiveness of chemotherapeutic agents against tumors [31,32]. In addition, targeting agents may be conjugated to the nanoparticle surface to activate the targeting effect. Monoclonal antibodies were previously used as targeting ligands [33]. A new generation of targeted agents has been introduced in the past decade, including peptides, aptamers, and chemical molecules [34]. DNA or RNA aptamers are small strands that bind specifically to the target molecule due to a specific folding that they possess. The

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Figure 9.2 A principal of aptamer generation by conventional SELEX. Selection starts with the construction of an initial oligonucleotide library, which is directly followed by selection. Reproduced with permission from Ref. [30]. License code: 5173000545583.

SELEX method, which involves selecting and amplification steps sequentially in vitro, makes aptamers with a high affinity for targets [35,36]. The elegant structural and molecular properties of aptamers are combined with the ability for them to be easily modified and for the fact that they induce minimal immune responses compared with antibodies [37]. There have been previous studies of several forms of aptamerconjugated carriers as potential targeted therapies for cancer [38,39]. The aptamer conjugated nanoparticles (NPs) targeting mucin-1 (MUC-1), HER2/neu, epithelial cell adhesion molecule (EpCAM), prostate-specific membrane antigen (PSMA) and nucleoin overexpressed on the surface of different tumor cell lines have shown promising results [36,40e43]. Several studies have shown that aptamer-targeting drug delivery can effectively enhance the therapeutic effect of anticancer agents on prostate cancer, breast cancer, colorectal cancer, and other cancers such as leukemia [44e47] (Table 9.1). Aptamer conjugated nanoparticles have now opened the door to developing targeted drug delivery systems. As a result of the new strategies for attaching specific ligands to the surface of nanoparticles [34,44] and because of successful experiments in aptamer

Table 9.1 Aptamer in cancer therapy.

Cancer type

Nanoparticle

Drug

Aptamer

Main effect

Refs.

In vitro

Lung cancer

PLGA

Gemcitabine

AS1411 aptamer

[51]

In vitro

Breast adenocarcinoma

PEGePLGA

Doxorubicin

MCF-7 cancer cells

PLGA

Paclitaxel

19-mer EpCAM RNA aptamer AS1411

The conjugation of AS1411 aptamers onto the surface of NP may be a potential treatment strategy for A549, which overexpresses nucleolin. This suggests that AS1411-GEM-NPs might be useful for treating NSCLC. Doxorubicin should have an improved therapeutic index when delivered with EpCAM-targeted nanopolymersomes.

[53]

GI-1 cells

PLGA

Paclitaxel

AS1411

MCF7 (human breast carcinoma cell) and C26 (murine colon carcinoma cell) Prostate cancer

PbAE and PLGA

Epirubicin

MUC1 aptamer

Since these NPs offer several perspectives, including hyperthermia therapy, targeted drug delivery, and MRI imaging in a single platform, they can be used for many applications. Apt-PTX-PLGA NPs were identified as a potential targeted therapy delivery vehicle for cancer treatment. When compared to Epi alone, MUC1 aptamer-modified nano complex showed remarkable tumor-suppressing effects.

PLGAePEG

Docetaxel and paclitaxel

A10 PSMA

MDA-MB-231

PLGAePEG

Paclitaxel

HPA aptamers

In vitro and in vivo In vivo

In vitro and in vivo

[54]

[55]

[51]

[56]

223

When NP size can be controlled in conjunction with targeted delivery, favorable biodistribution and targeted therapies can be developed. Pharmacotherapy for TNBC can be improved by combining nanoparticles with HPAaptamer bioconjugates. Bioconjugates of nanoparticles with HPA can be promising targets for drug delivery.

[52]

Aptamer-functionalized PLGA nanoparticles for targeted cancer therapy

Invitro/ invivo

Continued

Table 9.1 Aptamer in cancer therapy.dcont’d 224

Invitro/ invivo

Cancer type

Nanoparticle

Drug

Aptamer

Main effect

Refs.

In vitro

Prostate cancer

PLGAePEGApt

Docetaxel

Apt Wy5a

[51]

Pancreatic cancer

PLGA-ORM NP

Ormeloxifene

In vitro and in vivo

Breast cancer

PLGAePEGApt

Docetaxel

AS1411(Apt)

In vitro

Lymphoma

PEG-PLGA

Doxorubicin

In vitro and in vivo

C26 cancer cells

PLGA-PEG

Doxorubicin

CD30 aptamer AS1411

In vitro

Hepatocellular carcinoma

PLGA

Doxorubicin

TLS11a

In vitro

Breast cancers

PLGA

Sorafenib tosylate (SFB)

In the treatment of CRPC, targeted nanoparticles might prove to be an effective drug delivery system. In the future, PLGA-ORM formulation is likely to be valuable for treating pancreatic cancer because it is highly effective at inhibiting pancreatic tumor growth. By targeting MCF-7 human breast carcinoma cells with synergistic chemo-photothermal therapy, a novel promising treatment strategy for cancer was provided in vitro and in vivo. C2NP-functionalized nanoparticles may have therapeutic potential for ALCL Apt-NPs could be considered as a powerful tumor-targeted delivery system for their potential as dual therapeutic and diagnostic applications in cancers. Using aptamer-functionalized, drug-loaded nanoparticles as a targeted drug delivery system for HCC tumors, the aptamer nanoparticles significantly increased doxorubicin delivery and cell killing capacity. This study demonstrated the potential efficacy of SFB-loaded particles against ErbB3.

[57]

[58]

[60]

[61]

[62]

ALCL, anaplastic large cell lymphoma; CRPC, castration-resistant prostate cancer; DTX, docetaxel; EpCAM, epithelial cell adhesion molecule; HCC, hepatocellular carcinoma; NSCLC, nonsmall cell lung cancer; ORM, ormeloxifene; PbAE, poly (bamino ester); PLGA, poly (D, L-lactide-co-glycolide); SFB, sorafenib tosylate; TNBCs, triple-negative breast cancer cell lines; TMBC, triplenegative breast cancer.

Aptamers Engineered Nanocarriers for Cancer Therapy

[59]

Aptamer-functionalized PLGA nanoparticles for targeted cancer therapy

225

Figure 9.3 PLGA nanoparticles and liposomes prepared for bone delivery. Reproduced with permission from Ref. [50]. License code: 5171340643798.

targeting, aptamer-guided nanoparticles may be developed for targeted delivery of anticancer agents in vitro and in vivo [48]. Researchers have demonstrated that a 19-mer RNA aptamer specifically binds to the extracellular domain of epithelial cell adhesion molecule EpCAM. To deliver doxorubicin directly to MCF cells, other investigators successfully conjugated this targeting ligand with doxorubicin directly [49]. The biocompatible and degradable properties of PLGA make it an important material for the construction of bone-targeted nanomedicine. It is possible to prepare PLGA nanoparticles that contain drugs through emulsion or nanoprecipitation. When PLGA is fabricated, drugs are encapsulated within it. By functionalizing PLGA with PEG, it is possible to prolong blood circulation by avoiding reticuloendothelial capture. To modify PLGA nanoparticles with bone-targeting ligands, follow the following steps: PLGA nanoparticles can be modified postprocessing, or they can be modified beforehand and then coassembled with bone targeting lipids (Fig. 9.3) [50].

9.4

PLGA and nanoparticle

Recent advances in nanotechnology have made drug delivery more effective [63e65]. A variety of nanoparticles and antibodies/peptides/aptamers have been developed as drugs vehicles as well as targeting agents to reduce the side effects and toxin impact of anticancer agents [66,67]. The US FDA has approved the clinical use of polymer nanoparticles such as poly (D, L-lactide-co-glycolide) (PLGA), which have biocompatible and biodegradable properties, among many other delivery systems [68,69]. It has been demonstrated that PLGA is easy, hydrophilic, and rapidly metabolized by the human body [70]. During random ring-opening copolymerization of two different monomers, (1, 4-dioxane-2, 5-diones) of glycolic acid and lactic acid cyclic dimers are used to

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Aptamers Engineered Nanocarriers for Cancer Therapy

synthesize poly (lactic-co-glycolic acid). It is usually prepared using tin (II) 2-ethyl hexanoate or tin (II) alkoxides or aluminum isopropoxide as the catalyst. During polymerization, successive monomeric units (lactic acid, glycolic acid) are bound together. As a result, linear, amorphous aliphatic polyester products are obtained through ester linking in PLGA [71,72]. The monomers ratio is a way to identify the forms of PLGA. For example, PLGA 50:50 refers to a copolymer composed of 50% lactic acid and 50% glycolic acid. In PLGA, biocompatibility, longevity and durability are the key benefits. Getting continuous drug release for weeks, months, or even years, compared to conventional devices with a track record and well-documented utility, and easy injection of parenteral drugs are other positive benefits [73,74]. For the development of nanomedicines, PLGA is an effective biodegradable polymer because when hydrolyzed in the body, lactic acid and glycolic acid are formed, which are biodegradable [74]. Carbon dioxide and water are removed from these monomers as a result of metabolizing them in the Krebs cycle [75e77]. So they are minimally toxic to the system [74]. Human drug delivery systems using PLGA have been approved by the US FDA and European Medicine Agency. There is a variety of polymers available with different molecular weights and copolymer compositions. The degradation time will vary depending on the ratio of molecular weights in the copolymer [78,79]. The lactic acid in PLGA copolymers is less hydrophilic than glycolic acid, absorbing less water, and degrading less rapidly. In general, polymers with a lower molecular weight, those that are hydrophilic, and those that are amorphous, along with those with a higher glycolide content, degrade more quickly [77]. In vitro and in vivo polymer degradation is affected by a number of factors, including the method of preparation, the presence of low molecular weight compounds, morphology, size and shape [75], Liquid phase pinocytosis and clathrin-mediated endocytosis are part of the way PLGA nanoparticles enter cells. PLGA nanoparticles enter the cytoplasm in 10 min after escaping the endo-lysosomes. Nanoparticles are then enabled to interact with the membranes of the vesicles, resulting in temporary and localized deterioration and escape of the nanoparticles into the cytosol [80]. Nanoparticle surface charges also play an important role in their interaction with cells and in their uptake [81]. To increase the rate and extent of internalization of nanoparticles, a cationic surface charge is desirable, since it promotes their interaction with the cells [82,83]. Negative charges on PLGA nanoparticles can be neutralized or positively charged by appropriate surface modifications, such as by PEGylation of the polymer or by coating with chitosan [84e86].

9.5

PLGA nanoparticles for drug delivery to tumors

Cancer is a major public health problem with tens of millions of victims around the world [87]. Among economically developed countries, cancer is the leading cause of death, while it accounts for the second-highest number of deaths in developing nations [88,89]. A rigorous scientific approach is taken to investigate cancer to identify the causes and formulate precise strategies for prevention, diagnosis, and treatment.

Aptamer-functionalized PLGA nanoparticles for targeted cancer therapy

227

The current therapies for cancer, including chemotherapy, radiation therapy, and immunotherapy, can treat many types of the disease [72,90]. Chemotherapy is generally nonspecific in its approach to cancer treatment, which is its biggest weakness. Most therapeutic medicines (generally cytotoxic) are administered intravenously, which causes their systemic distribution. This method is not specific, which results in the common chemotherapy negative effects because of the cytotoxic drug attacks both healthy and nonhealthy cells at the same time [91e93]. Nanoparticles are useful for tumor targeting because: (1) NPs can deliver sufficient dose loads of the drug in the tumor area, which is due to their EPR effect, or their active targeting by ligands on the surface and the ability of NP to limit drug distribution to target organs while minimizing exposure to healthy tissues [94,95]. Nanoparticles are defined by their size, size distribution, morphology, surface chemistry, surface charge, as well as their shape as precursors to good nanomedicine. The following characteristics include surface adhesion, surface erosion, and inner porosity, drug diffusivity, drug release kinetics, and hemodynamic parameters [83,96]. An effective NP system reduces the number of carriers required for administration by having a large loading capacity. There are two methods for loading drugs into NPs. In the first method, drugs can be incorporated into NP production and in the second method when NPS is formed and incubated in drug solution, then adsorb the drug into them. Several drugs can be safely entrapped by the product by an incorporation method compared with adsorption [97e99]. Nanoparticles of PLGA are commonly used to encapsulate several cancer drugs and deliver them successfully to the targets [83]. A microsphere or a microparticle is the most common form of PLGA-based drug delivery system [100]. A variety of nanoparticles, films, cylinders, microparticles placed in situ, scaffolds, and foams are available [101]The synthesis of PLGA nanoparticles has been optimized and the synthesis of many cancer-related drugs has been incorporated into PLGA nanoparticles [83,102] Nanoparticles loaded with these payloads protect poorly soluble and unstable molecules from the biological milieu, and they are small enough to penetrate capillary walls, internalize, and escape endosomes [95,99,103]. The surface of those cells is modified to allow molecules to be targeted to tumors and other tissues [94,104]. Due to factors such as biodegradability, pH, ions, and/or temperature sensitivities, they may possess controlled-release properties [77].

9.6

Nanoparticle surface modification with PLGA

It is common for cancer drugs not to differentiate healthy cells from cancerous ones. To reach the tumor site, these drugs must be administered at high doses. As a result, optimum drug concentrations in the tumor are achieved at the expense of high drug concentrations in other organs, resulting in serious side effects. Nanoparticles offer the possibility of a targeted approach that can greatly improve cancer treatment. As a result of the surface characteristics of nanoparticles, they may get absorbed into the liver, spleen, and other parts of the reticuloendothelial system (RES), depending

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Aptamers Engineered Nanocarriers for Cancer Therapy

on their functions [105,106]. To avoid the body’s natural defenses, surface modification of nanoparticles is essential for passing through the body’s bloodstream [107]. If the nanoparticles have a long circulation time, they have a greater chance of reaching their target. To escape the molecular phagocytic system, nanostructures with hydrophilic surfaces smaller than 100 nm are preferred [108]. Nanoparticles in the hydrophobic environment will be primarily absorbed by the RES. Studies have shown that hydrophilic particles can stay in the circulatory system longer and are to a lesser degree absorbed by the liver [106,109,110]. Researchers have employed multiple strategies to make a hydrophilic cloud surrounding the nanoparticles, which reduces the uptake of the particles by RES organs. Nanoparticles are coated with Tween 80, PEG (polyethylene glycol), PEO (polyethylene oxide), polysorbate 80, poloxamers and poloxamines and polysaccharides like dextran to achieve these strategies [111e114]. It is possible to utilize hydrophilic polymers at the surface of NPs either through the adsorption of surfactants or the use of blocks copolymers or branched copolymers [95,107,113]. Xray photoelectron spectroscopy is used to measure surface chemistry [95,115,116] Nuclear magnetic resonance spectroscopy and Fourier transforms infrared spectroscopy [117,118]. In conclusion, the development of aptamer-based polymeric NPs, which utilizes polymeric nanoparticles as anticancer delivery systems offers a promising tool in our therapeutic armamentarium for managing various cancers.

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Aptamer-functionalized silicon nanoparticles for targeted cancer therapy

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Mohammad Banazadeh 1, 2 , Mohammad Mohajeri 3 , Kiarash Saleki 4, 5 , Behzad Behnam 6, 7,8 , Yong Teng 9 , Prashant Kesharwani 10 and Amirhossein Sahebkar 11,12,13, 14 1 Pharmaceutical Sciences and Cosmetic Products Research Center, Kerman University of Medical Sciences, Kerman, Iran; 2Students Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; 3Department of Pharmacology and Toxicology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; 4 Student Research Committee, Babol University of Medical Sciences, Babol, Iran; 5USERN Office, Babol University of Medical Sciences, Babol, Iran; 6Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran; 7Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; 8Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; 9Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, United States; 10Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India; 11Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 12Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 13School of Medicine, The University of Western Australia, Perth, WA, Australia; 14 Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

10.1

Introduction of silicon nanoparticles (SNP)

Pharmaceutical nanotechnology is known as one of the most emerging fields in pharmaceutical sciences. This branch of science is rapidly growing with new opportunities, tools, and scopes that have unique applications in the treatment and diagnosis of disease. One of the advanced and specialized fields of nanotechnology is the development of novel nanoparticles, and in this regard, SNP have been studied in various reports. SNP have the potential capability and are applied in nanomedicine due to their controllable structure and morphology, particle size, and pore diameter, while they can be modified to be biocompatible [1]. There are several types of SNP, including the

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conventional nonporous SNP, hollow mesoporous silica nanoparticle (HMSN), mesoporous silica nanoparticle (MSN), and core-shell SNP [2] (Fig. 10.1). These families of SNP have been widely used in various biomedical studies (Fig. 10.1) and are reviewed in the following paragraphs.

Figure 10.1 Working process and biomedical applications of SNP. (A) (1) Photodynamic therapy by UV enhances ROS synthesis and subsequently leads to cancer cell apoptosis. This process is achieved by photosensitizers, which are agents that can absorb light and transfer the energy from the incident light into another nearby molecule (2). Another approach to promote cancer cell death is photothermal therapy that utilizes near infrared light (3). SNP can facilitate drug loading and selective drug release (4). SNP can aid in imaging techniques. (B) Four main types of SNP, including HMSN, MSN, core-shell SNPs, and conventional nonporous SNP with or without surface modifications. HMSN, hollow mesoporous silica nanoparticle; MSN, mesoporous silica nanoparticle; ROS, reactive oxygen species; UV, ultraviolet. The figure was created with BioRender.

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10.1.1 Mesoporous SNP (MSNs) This type of nanoparticles is intended to deliver proteins due to their high stability, excellent biocompatibility, well-defined pore structure, rigid framework, tunable surface chemistry, and easily controllable morphology. The protein delivery is usually limited by the tiny pores utilizing traditional MSNs. MSNs featuring novel pore structures and large pores enormously expand their utility for delivery of therapeutic proteins [3]. Ordered mesoporous silica synthesis comprises segments of hydrolysis and condensation of an alkoxide on the surface of cetyltrimethylammonium bromide (CTAB) micelles. The condensation reaction continues either via water condensation or alcohol condensation. The whole reaction is summarized as follows: nSi(OC2H5) þ 2nH2O ¼ nSiO2 þ 4nC2H5OH. The synthesis process for ordered mesoporous silica that was described above is conducted by various concentrations of CTAB in a range of 0.5%e3.0% (w/w), to monitor shape and size of the particles. The yield is calculated for ordered mesoporous silica by the following formula: Y (yield) ¼ WOMS/ (WTEOS  28.4%)  100%. WOMS and WTEOS are the weight of ordered mesoporous silica gathered after calcination and tetraethoxysilane, respectively [4].

10.1.2 Conventional nonporous SNP The “gatekeeper” method or altering the inner surface of the pores could be used to control drug release and affinity from mesoporous silica NPs. In contrast, chemical linkers or the degradation of the silica matrix control the drug delivery of nonporous silica NPs. The shape and size of nonporous silica NPs could be greatly modulated [5]. The silica precursor, tetraethyl orthosilicate (TEOS) that is affected by hydrolysis followed by a polycondensation reaction in the presence of ethanol and ammonium hydroxide (NH2OH), produces nonporous silica particles with sizes tinier than 200 nm [6].

10.1.3 Hollow mesoporous silica nanoparticle (HMSN) HMSN comprising a large crater in each main mesoporous SNP has enormous potential as a more useful nano-platform to perform imaging and treating cancer [7]. Ge et al. proposed a mechanism for Synthesis: Polystyreneemethyl acrylic acid (or polystyrene) latex patterns should be ultrasonically treated in water to obtain a welldispersed suspension. The suspension would be moved into a flask. Then the aqueous solution of CTAB would be added and stirred for 1 h. The suspension would be tweaked to a pH value of 10 by adding an aqueous ammonia solution. To prepare a silica-coated latex sample, tetraethoxysilane should be added to the suspension mentioned above and stirred for 4 h at room temperature. Then the suspension would remain at room temperature without stirring. The yielded suspension would be moved into a Teflon-lined stainless-steel autoclave and heated to evaluate the impact of the reaction temperature on the silica-coating extent. The precipitate will be filtrated and washed using distilled water. Then the washed precipitate would be dried in an electric oven. The prepared materials would be heated under the atmospheric condition to oxidize the organic core materials [8].

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10.1.4

Aptamers Engineered Nanocarriers for Cancer Therapy

Core-shell SNP

Core-shell nanoparticles are biphasic materials with an inner core structure and an outer shell made of different composites. These particles are interesting as they show distinctive qualities arising from combining core and shell geometry, material, and design. Furthermore, they have been created so that the shell material improves the thermal stability, reactivity, or oxidative stability of the core material or utilizes a cheap core material to carry a thin, more expensive shell material. Core-shell particles are mainly synthesized using solution methods and usually comprise two steps: Synthesis of the core structure and coating the core structure with the shell material [9]. For the synthesis of core-shell SNP, first, a solution of water, ammonium hydroxide, and ethanol should be prepared. Then, heated to 40 C and mixed with a similar amount of TEOS solution in ethanol, while being stirred for a 30-min period. After that, the solution of vinyltriethoxysilane should be added and finally, the resultant precipitate should be centrifuged, washed with water and ethanol, and dried [10].

10.2 10.2.1

Biomedical applications of SNP Drug delivery

Various nanoparticles, including organic ones such as polymers, liposomes, micelles, carbon nanotubes and dendrimers, and inorganic ones such as magnetic, gold and SNP have been evaluated as drug delivery systems [11]. MSNs have received incremental attention from the initial report of their synthesis in the 1990s [12] and their first use in drug delivery in 2001 [13,14]. Showing a large surface area, good biocompatibility, a high pore volume, controlled pores, and varying surface chemistry, MSNs have shown advantages as nanocarriers for drug delivery, such as increased drug loading capacity for both hydrophilic and hydrophobic drugs, genes, and proteins [15e17]. Since 2001, MSNs began to be used for drug delivery from when huge growth has taken effect in developing various delivery systems based on MSNs and their biomedical uses [13]. The utilization of MSNs for drug delivery to deliver proteins, drugs, and genes has increased significantly in recent years, utilizing in vitro and animal studies. If clinical utilizations are considered, the biological safety of MSNs must carefully be taken into consideration [18].

10.2.2

Imaging

In various clinical fields, nanomaterials-centered bio-imaging methods have developed enormously by advancing diverse operational nanoparticles. Since MSNs demonstrate exceptional qualities, an imaging agent backed by MSNs could be an appropriate system for developing directed bio-imaging contrast vehicles showing superior structural stability and improved functionality, enabling imaging modalities of various types. These bio-imaging utilities comprise magnetic resonance imaging (MRI), optical

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imaging, computed tomography (CT), positron emission tomography, multimodal imaging, and ultrasound imaging for initial diagnosis [19].

10.2.3 Photodynamic therapy (PDT) This therapy utilizes photosensitizing agents by oral or intravenous administration, followed by exposure of tissues to light to trigger photosensitizers for eradicating anomalous cells and tissues [20,21]. The exposure of photosensitizers to light produces reactive oxygen species (ROS) known as singlet oxygen that demonstrate cytotoxic qualities. By encapsulating photosensitizers in advanced nanoparticles, we could improve the targeted delivery toward tumor cells and the effectiveness of PDT would be promising. SNP are an abundance of nanovehicles being suggested for PDT [22e31]. In 2009, MSNs were initially suggested to be the extremely promising vehicles for PDT. Porphyrins were incorporated into the MSN pores [32,33] or inside the MSN walls utilized as photosensitizers [34]. Ligands like mannose [34] or RGD peptides [35] were used to vectorize nanoparticles applied for antitumor purposes [31].

10.2.4 Photothermal therapy (PTT) technique This technique has recently captured incremental global attention and in this method, malignant cells could be eradicated through the heat-induced from near-infraredabsorbing chemicals caused by laser irradiation. PTT is fundamentally more efficient in tumor ablation because of its low onset of resistance, high spatiotemporal selectivity, and noninvasiveness in comparison with conventional surgical therapy, radiotherapy, and chemotherapy [36,37].

10.3

SNP for cancer therapy

Cancer therapy has had significant challenges through previous decades. Nevertheless, in some instances metastases are problematic, surgical removal is extremely hard, and radiotherapy and chemotherapy cause nontolerable side effects. As a result, we need more specific and effective therapies [38,39]. The application of drug delivery agents (DDA) based on nanomaterials is one method. MSNs were used for the first time as DDA in 2001 and achieved significant consideration in this area. The popularity of MSNs Because of their exceptional qualities; for example, their ability to capture cargo molecules MSNs, easy surface modification and functionalization, high pore volume and surface area, high stability, and adjustable pore and particle size have become so popular [7]. A variety of studies described the functionalization of folic acid on the surface of MSNs to target pancreatic cancer [40], leukemia [41], lung cancer [42,43], and breast cancer [44e46]. Several studies have described the functionalization of hyaluronic acid on the surface of MSNs to function as targeting agents [47e50]. Redox potential is used for developing a stimulus-reacting drug delivery factor. ROS production is increased due to different metabolism, genetic mutation, and

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mitochondrial dysfunction in a cancerous cell. The production of ROS scavengers (such as tripeptide glutathione (GSH)) is increased to overcome oxidative stress. Zhang and colleagues described the utility of hollow MONs for delivering cisplatin and doxorubicin molecules. As a result, the anticancer efficacy was increased [51]. MSNs were covered with folic acid-altered PEGylated lipid bilayer membranes to transfer tanshinone IIA and paclitaxel to cure acute promyelocytic leukemia [41]. MSNs were used to deliver tariquidar and doxorubicin, aiming at overcoming the multidrug resistance (MDR) of malignant stem cells [52]. The MSNs are not only used to treat malignancy as drug delivery agents but also served as other platforms of cancer diagnosis and therapy. MSNs have functioned as both imaging and controlled released delivery agents, respectively. Luminescent MSNs were produced by integrating aggregationinduced emission (AIE) molecule, 10-phenylphenothiazine, and covered by a pHsensitive polymer to create a pH-responsive carrier [53]. Another fascinating study described by Mira and coworkers applied MSNs for ultrasound therapy and imaging [54]. In conclusion, MSNs are multifaceted nano-molecules that could be utilized by various mechanisms to target and treat cancer.

10.4

Aptamer-conjugated SNP

SNP are new and encouraging carriers for biomedical applications. Their modifiable diameter and pore size, biocompatibility, and easy functionalization help make efficient nanostructures. Ligands could be easily glued to their surface, and stimuliresponsive (redox, pH, heat, light) gatekeepers could be incorporated to avoid early drug release and transfer the active agent exclusively at the site of disease. Like other nanoparticles, targeted drug delivery by SNP could be achieved by incorporating antibodies, magnetic nanoparticles, and also aptamers. In 2011, aptamer-conjugated paclitaxel-activated SNP were first investigated for the targeted treatment of breast cancer cells. In this study, SNP loaded with AS1411 aptamer-conjugated paclitaxel were able to target cancer cells with high specificity and to destroy them [55]. In 2012, Miranda S
a et al. studied labeled cells using mesoporous silica SBA-15 with aptamer [56]. In the same year, Li et al. investigated the anticancer effects on MCF seven breast cancer cells by loading doxorubicin in activated SNP with DNA aptamer AS1411, which were able to target cancer cells with high specificity and to destroy them [57]. In 2016, Xie et al. evaluated the anticancer effects of doxorubicin-loaded on activated SNP with aptamer. In this research, human colon cancer cells and B lymphoma were evaluated [58]. Bagheri et al. developed a targeted delivery system of doxorubicin with a dual receptor based on mesoporous SNP modified by mucin-1 and ATP aptamers (DOX @ MSNs-Apts). Aptamer and mucine-1 were covalently anchored to the surface of MSNs containing carboxylate. ATP aptamers were then immobilized on the surface of MSNs. By Adding doxorubicin, DOX @ MSNs-Apts showed higher cell uptake in MCF-7 and C26 cancer cells instead of CHO cells. The anticancer effect (in vivo) in C26 tumor-bearing mice

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revealed that DOX @ MSNs-Apts had higher tumor accumulation and a more inhibitory effect on tumor growth than DOX alone. These findings illustrate that the prepared intelligent platform could provide new insights into cancer treatment [59]. Wang et al. developed an efficient method for human breast cancer based on aptamers labeled with biotin and streptavidin-conjugated magnetic fluorescence silica nanoprobes (FITC @ Fe3O4 @ SiNPs-SA). SNP (FITC @ Fe3O4 @SiNPseCOOH) were first synthesized using the Stober method. This study reveals that the constructed aptamers have the potential to identify target cells in human breast cancer cells with high affinity and specificity and attach to them. Moreover, the prepared FITC @ Fe3O4 @ SiNPs-SA was used with optical stability and superparamagnets, which is useful for simultaneous labeling and isolation of cancer cells in a complex biological environment. They also showed that silica nanomaterials with good biocompatibility served as essential and useful supplements for the supersensitive diagnosis of cancer cells in various fields [60]]. Sakhtianchi et al. synthesized SPION/mesoporous silica core-shell SNP coated with PEG and aptamer AS1411 for simultaneous cancer treatment and MRI. In this study, nanoparticles called MMSNs were successfully loaded with doxorubicin and precoated with D-carboxylic acid-functionalized polyethylene glycol (PEG-MMSNs). After that, the AS1411 aptamers were covalently attached to the synthesized nanoparticles (APT-PEG-MMSN) at the end. The average diameter of the synthesized nanoparticles was about 89 nm. The results of this study indicate that APT-PEGDOX/SPION-MMSN can increase the specific translocation of DOX to MCF7 cells effectively. In addition, APT-PEG-DOX/SPION-MMSN had high potential as an MRI contrast agent due to high-rate cell uptake and SPION. APT-PEG DOX/SPION-MMSN represents an attractive choice for simultaneous targeted imaging, drug delivery, and monitoring in the treatment of cancers with the overexpression of nucleoli [61]].

Study Near-infrared light-triggered, targeted drug delivery to cancer cells by aptamer gated nanovehicles Polyvalent mesoporous silica nanoparticleaptamer bioconjugates target breast cancer cells

Aptamer and nanomaterial

Payload

Cell

Level

Refs.

AS1411, DNA Mesoporous silica

Gold nanorods

MCF 7

In vitro

[62]

AS1411, DNA Mesoporous silica

Doxorubicin

MCF 7

In vitro

[57]

Continued

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Aptamers Engineered Nanocarriers for Cancer Therapy

Continued

Study Targeting cancer cells with controlled release nanocapsules based on a single aptamer A vitamin-responsive mesoporous nanocarrier with DNA aptamermediated cell targeting Specific aptamerconjugated mesoporous silicaecarbon nanoparticles for HER2-targeted chemophotothermal combined therapy EpCAM aptamerfunctionalized mesoporous SNP for efficient colon cancer celltargeted drug delivery MUC1 aptamerconjugated mesoporous SNP effectively target breast cancer cells Aptamer/ photosensitizer hybridized mesoporous MnO2-based tumor cell activated ROS regulator for precise photodynamic therapy of breast cancer

Aptamer and nanomaterial

Payload

Cell

Level

Refs.

AS1411, DNA Mesoporous silica

Doxorubicin

MCF 7

In vitro

[63]

Sgc8, DNA Mesoporous silica

Doxorubicin

CEM

In vitro

[64]

HB5, DNA Mesoporous silica-carbon

Doxorubicin

SK-BR-3

In vitro

[65]

No name, DNA Mesoporous silica

Doxorubicin

SW620

In vitro

[58]

No name, DNA Mesoporous silica

Epirubicin

MCF 7

In vitro

[66]

No name, DNA Mesoporous MnO2

HMME

MCF 7

In vitro þ in vivo

[67]

Aptamer-functionalized silicon nanoparticles for targeted cancer therapy

245

Continued

Study Aptamerfunctionalized SNP for targeted cancer therapy Targeted delivery and controlled release of doxorubicin to cancer cells by smart ATPresponsive Y-shaped DNA structure-capped mesoporous SNP MUC1 aptamercapped mesoporous SNP for navitoclax resistance overcoming in triple-negative breast cancer Targeted rod-shaped mesoporous SNP for the codelivery of camptothecin and survivin shRNA into colon adenocarcinoma in vitro and in vivo MUC-1 recognitionbased activated drug nanoplatform improves doxorubicin chemotherapy in breast cancer Pegylated magnetic mesoporous SNP decorated with AS1411 aptamer as a targeting delivery system for cytotoxic agents

Aptamer and nanomaterial

Payload

Cell

Level

Refs.

No name, DNA Mesoporous silica Mucine-1 and ATP aptamers Mesoporous SNP

Paclitaxel

MCF 7

In vitro

[55]

Doxorubicin

C26 MCF 7

In vitro þ in vivo

[59]

No name, DNA Mesoporous silica

Navitoclax

TNBC

In vitro

[68]

AS1411 DNA Mesoporous silica

e

C26

In vitro þ in vivo

[69]

No name, DNA Mesoporous silica

Doxorubicin

MCF 7 Hs578bst

In vitro

[70]

AS1411, DNA SPION/ mesoporous silica core/ shell

Doxorubicin

NIH-3T3

In vitro

[61]

Continued

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Aptamers Engineered Nanocarriers for Cancer Therapy

Continued Aptamer and nanomaterial

Study A dual-functional HER2 aptamerconjugated, p H-activated mesoporous silica nanocarrier-based drug delivery system provides in vitro synergistic cytotoxicity in HER2-positive breast cancer cells Photo-responsive magnetic mesoporous silica nanocomposites for magnetic targeted cancer therapy Targeted delivery of anti-miR-155 by functionalized mesoporous SNP for colorectal cancer therapy Breast cancer cells synchronous labeling and separation based on aptamer and fluorescencemagnetic SNP

10.5

Payload

Cell

Level

Refs.

HApt aptamer Mesoporous silica

Doxorubicin

MCF 7

In vitro

[71]

S6 aptamer Au@SiO2

Doxorubicin

A549 HepG2

In vitro

[72]

AS1411 aptamer Mesoporous silica

e

CRC NCM460

In vitro

[73]

MUC-1 aptamer FITC@ Fe3O4@ SiNPs-SA

e

MCF 7

In vitro

[60]

Biocompatibility and toxicity of SNP

Having a sound understanding of the toxicity and biocompatibility of MSNs is critical for effective and secure clinical utilization because MSNs comprise inorganic nanoparticles. Thus it is hard to have them degraded in the body [16]. MSNs could simply enter the majority of normal and malignant cells without obvious negative impacts on cellular proliferation, differentiation, and growth [74e76]. Cellular experiments on

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MSNs showed that particle diameter and dose were vital for the cytotoxic impact of MSNs [77]. Results of MTT assay demonstrated that MSNs that vary from 30 to 300 nm demonstrated no toxic effect on HeLa cells. Furthermore, larger MSNs fit medical utilizations for hemolysis studies more as smaller MSNs have higher toxicity due to a higher cell absorption and even more silanol groups that exist for cell contacting [78]. There are studies that have focused on the cytotoxic effects of MSNs [58,74,76,79e81], but the genotoxic effects of MSNs have not been thoroughly studied. It has been demonstrated that MSNs with a medium size of 25 and 100 nm cause minimal genotoxic effects on HT-29 cells following 24 h of exposure [82]. Blood biocompatibility plays a key role in the utilization of medicine-loaded transporters for venous injection. For IV administration, we should evaluate thrombogenicity, hemolytic activity, and surface adsorptive activity of blood proteins for various MSNs [83]. The hemolysis induced by MSNs could reduce or even completely avoided by superficial modification. Activated partial thromboplastin time and Prothrombin time have been evaluated to assess the thrombus inducing activity of MSNs in which no thrombogenicity was observed. The superficial alteration of MSNs leads to a promising improvement in blood biocompatibility. Exposing functionalized MSNs to human serum gamma globulins and albumin showed no protein surface adsorption on the ionic-functionalized MSNs. No hemolysis was detected after exposing red blood cells (RBCs) to the carboxylate-altered MSNs. Exposing the TA-MSN-carboxylateCP complexes to RBCs resulted in no hemolysis under the efficient treatment dose [84]. In a study, no detectable histological damage or serious or pathological defects were seen in the lungs, muscle, stomach, intestine, heart, kidney, spleen, or liver. Kidneys play an important role in MSNs clearance [85]. To evaluate the toxic impacts of MSNs, kidneys were histologically evaluated for injury, inflammation, or histological damage, 48 h after injection with MSNs. No steatosis, inflammatory effects, hyperplasia, hydropic degeneration, necrosis, or fibrosis had been demonstrated in kidneys. Nevertheless, after IV injection of MSNs, local renal glomerular atrophy and bleedings were detected in the mice kidneys. The toxicity and biocompatibility of MSNs have been examined, while components such as aggregation and charge of MSNs in the blood, shape, ranges of particle diameter, surface chemistry, dose or administration and selected differences of physicochemical factors, have not been extensively studied for evaluating the biocompatibility of MSNs [86].

10.6

Conclusions

SNP comprising four main types are potential agents for cancer therapy and diagnosis that four primary mechanisms could mediate. SNP can be functionalized with aptamers to achieve better treatment efficacy. Aptamer-conjugated SNP should receive special attention due to their functional flexibility. However, one should carefully evaluate the safety of this mixed therapy for preclinical and clinical applications.

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Aptamer-functionalized dendrimers for targeted cancer therapy

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Afsana Sheikh 1 , Harsimran Kaur 2 , Mohammed A.S. Abourehab 3,4 , Mohammad Sarwar Alam 5 and Prashant Kesharwani 1,6, 7 1 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 2Department of Pharmaceutics, Delhi Pharmaceutical Sciences and Research University, New Delhi, Delhi, India; 3Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; 4Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt; 5Department of Chemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, Delhi, India; 6University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India; 7Assistant Professor & Ramanujan Fellow, Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India

11.1

Introduction

In terms of the number of incidences reported worldwide, cancers are demonstrated as the most anguishing condition. The progression of cancer cells is a result of oncogenic proteins that promote the development of cancerous lesions [1]. Genetic alteration of oncogenes is promoted by the tumor perceptive gene encoding protein causing DNA damage, initiating cancer progression [2e9]. Thus, so far, cancer is considered a major health concern, globally, that demands efficacious treatment that (a) increases the sensitivity of drugs by neoplastic cells, (b) would be nontoxic and highly efficacious, and (c) should improve the life expectancy and quality of life during and posttreatment. Chemotherapy is one of the most accepted cancer treatments that reduce the multiplication of tumor cells; however, tumor cells’ nonsensitivity, high toxicity and incompetent delivery overpower the therapeutic response [10e16]. Considering such points, the researchers found a new scope in targeted cancer therapy that decreases the delivery of drugs to the noncancerous areas thereby improving the therapeutic profile of the desired treatment. The strategical approach relies upon the understanding of cancer cell biology that reveals a high expression of particular antigen or receptors which essentially takes part in cell proliferation and metastasis. However, such an overexpression renders the drug delivery scientists to target them and regulate their growth and expression. Aptamers are short-length oligonucleotides bearing approximately 20e100 nucleotides with a unique 3D structure having high specificity toward targeted Aptamers Engineered Nanocarriers for Cancer Therapy. https://doi.org/10.1016/B978-0-323-85881-6.00011-7 Copyright © 2023 Elsevier Ltd. All rights reserved.

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structures [17e23]. Due to its versatility, the aptamers fit into the pockets of the target by electrostatic interaction. Aptamers are selected through a unique tool called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that screen the library amalgamating 1012
15 nucleotide bases having enormous target binding affinity. The selection process involves the binding and partitioning of nucleotides based on the binding affinity toward the target followed by amplification which is further separated, washed and amplified to get an anticipated aptamer [20,24e26] (Fig. 11.1). The inherent merits of aptamers such as excessed stability, high binding affinity, greater sustainability and magnificent selectivity make aptamer an ideal candidate for targeted therapy against cancer. Aptamers have a short half-life that is rapidly eliminated through the body via the renal route. On the other side, aptamers are highly degraded by nucleases challenging the fine production of the therapeutic aptamer [27,28]. To date, only a single aptamer made its way to reach the clinical market and was approved by US FDA in 2004 called Macugen [29]. This is a double modified aptamer as the first modified form contains 20 -O-methyl purine (to protect against endonucleases) and the second modification was the addition of poly(ethylene glycol) at the 50 position and attachment of 30 dT through the 30 -30 linkage (for improving the pharmacokinetic profile). Over the last few years, functionalized nanomaterials have been in the area of research due to their wide application in the biomedical field [30e33]. It extends its potential in the pharmaceutical application which must not be immunogenic and

Figure 11.1 The process of selection of desired aptamer using SELEX tool.

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ensure high selectivity and targetability toward complex microenvironments such as living animal and human cells and tissues. The modification of aptamers toward the nanorange could be the best tactic in the synthesis of biomaterials with special characteristics. Toward this, nano drug delivery system could be functionalized via noncovalent or covalent interaction or even bio-related conjugation. Aptamer functionalized nanoparticles are a novel approach as they are designed to specifically target tumor cells which is essential for early diagnosis and therapy. Besides target based cancer treatment, aptamer functionalized nanomaterials also unlocked a new horizon in tumor detection [34e37]. The engagement of nano drug delivery system with aptamer extended its selectivity and acceptability in cancerous lesions in addition to the reduction of adverse effects due to selective therapy. Moreover, its exceptional sensitivity encourages the splendid substrate meant for legible signal transduction making the way for biosensing approaches [38e42]. Undauntedly, dendrimers are one of the carriers which due to the highly organized structure and high functional groups that enable easy uptake by the cancer cells. Due to the numerous peripheral functional groups, the dendrimers are conjugated or modified with various ligands or even antibodies. Even the highly efficacious but challenging drugs which still face stumbling blocks on their way to clinical trials and clinical platforms could be delivered through dendrimer [30,43e47]. The structure of dendrimers is a major reason for this. The hydrophobic core encourages the encapsulation of lipid-loving compounds which the peripheral branched structure helps to conjugate with the hydrophilic drug. The extraordinary structure and low pk value of dendrimer make them excellent carriers in high drug transfection [8,48e52]. The invention by Tomalia, opened a new way of therapy by PAMAM dendrimer [44,53e57]. The positive surface potential of PAMAM dendrimer aids for high transfection of genetic material, in addition to this, the low polydispersity index (