Superparamagnetic Materials for Cancer Medicine (Nanomedicine and Nanotoxicology) 3031372867, 9783031372865

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Nanomedicine and Nanotoxicology

Nanasaheb Thorat Niroj Kumar Sahu   Editors

Superparamagnetic Materials for Cancer Medicine

Nanomedicine and Nanotoxicology Series Editor V. Zucolotto, Institute of Physics São Carlos, University of São Paulo, São Carlos, São Paulo, Brazil

“Nanomedicine and Nanotoxicology” is a book Series dedicated to the application of Nanotechnology to achieve breakthroughs in healthcare as well as its risks and impact on the human body and environment. This book Series welcomes manuscripts on in vivo and in vitro diagnostics to therapy including targeted delivery, magnetic resonance imaging (MRI) and regenerative medicine; interface between nanomaterials (surfaces, particles, etc.) or analytical instruments with living human material (cells, tissue, body fluids); new tools and methods that impact significantly existing conservative practices; nanoparticles interaction with biological systems, and their risk assessments; among others. To submit a proposal or request further information, please contact Dr. Mayra Castro, Editor Applied Sciences, via [email protected].

Nanasaheb Thorat · Niroj Kumar Sahu Editors

Superparamagnetic Materials for Cancer Medicine

Editors Nanasaheb Thorat Limerick Digital Cancer Research Centre, Department of Physics and Bernal Institute University of Limerick Limerick, Ireland

Niroj Kumar Sahu Centre for Nanotechnology Research Vellore Institute of Technology Tamil Nadu, India

Fellow of The Royal Society of Medicine London, Medical Science Division University of Oxford Oxford, Oxfordshire, UK

ISSN 2194-0452 ISSN 2194-0460 (electronic) Nanomedicine and Nanotoxicology ISBN 978-3-031-37286-5 ISBN 978-3-031-37287-2 (eBook) https://doi.org/10.1007/978-3-031-37287-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Cancer is a major problem and is the main cause of death worldwide. In the past few decades, several attempts have been made in the field of nanotechnology to counter this problem. In this direction, magnetic iron oxide nanoparticles have gained great attention because of their distinctive properties and biocompatibility. Various researches are still in progress to engineer the magnetic nanoparticles in such a way as to utilize them maximally for the treatment of cancer. Magnetic nanoparticles can be tuned according to need and they can respond to the alternating magnetic field. Among the various types of magnetic materials, superparamagnetic materials have been explored rigorously for their applications in biomedical fields such as cancer diagnostics and therapy. Till now, several superparamagnetic materials have been developed for their application in nanomedicine since they offer various advantages such as enhanced half-life, specific delivery of drugs in the presence of alternating magnetic field, and biocompatibility. Superparamagnetic materials don’t only increase the bioavailability of drugs, but they also enhance the drug’s solubility. They can be utilized as a hyperthermia agent to kill the cancerous tissue through necrosis at elevated temperatures. Traditional chemotherapy suffers from some major drawbacks such as unwanted side effects on the human body. These side effects can be reduced from specific targeting of drugs onto the tumor location. To target drugs, the superparamagnetic materials have to be functionalized in such a way that they should release the drugs at the specific location either through remote stimulation, pH stimuli or magnetic channelization. This book addresses advancements in the current superparamagnetic materials-mediated cancer therapy and diagnostics with a major emphasis on drug delivery, hyperthermia, and imaging techniques. Pharmacokinetic aspects of the superparamagnetic materials have also been included along with its toxicological effects. This book addresses the current ongoing clinical trials as well as their future perspectives. This book sums up the basics and fundamentals of superparamagnetic materials, their synthesis, functionalization, applications, in vitro and in vivo assessment, pharmacokinetics, toxicity, and preclinical to clinical translation. The book is designed to endorse some scientific knowledge in cancer theranostics (diagnosis + therapy)

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comprehensive approach and roadmap for the treatment of cancer by utilizing superparamagnetic materials. As of now, very few books are available in the market which can address a wide scope of superparamagnetic materials in a single document thus this book will hold a strong competition for the same. This book will be an eminent platform of required qualified expertise for the preparation of updated status documents for reference. Thus, this book will unify them together with the ultimate aim of eliminating a few major hurdles which hinder the translational aspects of superparamagnetic nanomaterials for cancer nanomedicine. The key features of the book are 1. Fundamentals and basics of superparamagnetic nanomaterials 2. Application of superparamagnetic nanomaterials in cancer theranostics with upto-date literatures and new modalities 3. In vitro, in vivo, pharmacokinetics assessment and toxicology of superparamagnetic nanomaterials 4. Information on the current clinical trials as well as preclinical and clinical translation. Oxford, UK Tamil Nadu, India

Nanasaheb Thorat Niroj Kumar Sahu

Acknowledgements

Dr. Thorat acknowledges funding received from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement “SUPERBRAIN” 840964 for the project leading to this work. Dr. Sahu acknowledges the funding receipt from Vellore Institute of Technology, Vellore under the scheme “Transdisciplinary Research Grant” and Department of Science and Technology, Govt. of India (DST-SERB Project Grant No. ECR/2016/ 000301) for the project leading to this work.

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Contents

Strategies for Synthesis of Superparamagnetic Nanoparticles . . . . . . . . . . Ashwini B. Salunkhe, Manohar S. Lad, and Vishwajeet M. Khot

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Functionalized Strategies of Superparamagnetic Materials . . . . . . . . . . . . Shubhangi D. Shirsat, Rajaram S. Mane, and Nanasaheb D. Thorat

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Ferrite- and Non-ferrite-Based Superparamagnetic Materials . . . . . . . . . Ashwini B. Salunkhe, Maithili V. Londhe, and Vishwajeet M. Khot

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In Vitro and In Vivo Assessment of Superparamagnetic Materials . . . . . . Ahmaduddin Khan, Chandunika R. Kalaiselvan, Shalmali Sudhindra, Mohammad Suhaan Dar, and Niroj Kumar Sahu

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Applications of Superparamagnetic Materials in Drug Delivery Toward Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jagriti Gupta, P. A. Hassan, and K. C. Barick

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Applications of Superparamagnetic Nanomaterials in Hyperthermia Toward Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Sandeep B. Somvanshi and Nanasaheb D. Thorat Superparamagnetic Materials in Cancer Imaging and Diagnostics . . . . . 139 Chandunika R. Kalaiselvan and Niroj Kumar Sahu Pre-clinical Practices of Functionalized Superparamagnetic Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Sougata Ghosh, Bishwarup Sarkar, Nanasaheb Thorat, and Sirikanjana Thongmee Pharmacokinetic Study of Superparamagnetic Materials . . . . . . . . . . . . . . 181 Ahmaduddin Khan and Niroj Kumar Sahu Toxicology of Superparamagnetic Materials . . . . . . . . . . . . . . . . . . . . . . . . . 199 Kinnari Parekh and Neeraj Jain

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In Vivo Mechanistic Study of Superparamagnetic Materials . . . . . . . . . . . 219 Rakesh M. Patil, Prajakta B. Shete, Prabhanjan S. Giram, Sandeep B. Somvanshi, and Nanasaheb D. Thorat Recent Status of the Current Clinical Trials Going on for Superparamagnetic Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Priyal Chikhaliwala and Sudeshna Chandra

About the Editors

Dr. Nanasaheb Thorat, M.Sc., Ph.D., (Gold Medal) MRSC, Lead Editor is an outstanding researcher currently working as Research Scientist and Marie Curie Fellow at the University of Oxford, Oxford, UK. Dr. Thorat has published more than 100 international publications including 7 books, ~80 peer reviewed journal articles and 20 book chapters (total citations ~4000, H index: 40), presented 5 Keynote Speech, 25 Invited Talks at prestigious scientific peer conferences, received international acclaims and awards for research contribution, supervised students/junior researchers and actively participated in outreach and scientific dissemination for the service of the wider community. He is also a recipient of various prestigious fellowships including the Marie Skłodowska Curie Fellowship (IF), Poland/Switzerland, 2018, Japanese Society for the Promotion of Science (JSPS) Fellowship, Japan, 2017, and Government of Ireland IRC fellowship, Ireland, 2015. In addition to his current research, he has been deeply engaged in collaborative work with the many eminent scientists from Japan, USA, Germany, Korea, Ireland, India, Poland, Saudi Arabia and Australia. He also has excellent collaboration and interactions with over 30 junior and senior colleagues in India, South Korea, Japan, Taiwan, and Ireland with whom he has co-authored his publications. Dr. Thorat is the awardee of the European Commission’s Innovation Radar “Grand Prix of the Innovation Radar Prize 2020” for developing ground-breaking technology for breast cancer theranostics, funded under EU’s Marie Curie IF Project ‘NANOCARGO’. He is the recipient of many prestigious awards such as xi

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Young Scientist Awardee through open competition by Lindau Nobel Laureate Foundation in the 67th Lindau Nobel Laureate Meeting, held from 25th to 30th June 2017 in Lindau, Germany, and European Materials Research Society (EMRS) Young Investigator Award in Biomaterials field. Dr. Thorat’s contribution to the area of nanobiotechnology and theranostics has been recognized by the Royal Society of Chemistry (RSC, UK), which admitted him as the Member of The RSC and entitled the designation MRSC in the year October 2017. Dr. Thorat is Management Committee member of 3 European Cooperation in Science and Technology (COST) actions such as COST Action CA17140, Cancer Nanomedicine-From the Bench to The Bedside (NANO2CLINIC), CA17104, New Diagnostic And Therapeutic Tools Against Multidrug Resistant Tumors and CA17115, European network for advancing Electromagnetic hyperthermic medical technologies. Dr. Thorat was also involved in an Enterprise Ireland innovation partnership research project that involved four world-leading pharmaceutical companies such as Jansen, GSK, Pfizer, and MSD (year 2017–2018) at pharmaceutical manufacturing technology centre (PMTC), University of Limerick. His work involved upskilling of spectroscopic techniques such as X-ray photoelectron spectroscopy and Raman spectroscopy to probe drug/material interface. Dr. Thorat has established his own educational start-up—REINSPIRE Research and Education LLP—in India (http://reinspire.in/). The REINSPIRE provides a conceptual framework as a starting point for the implementation and evaluation of effective consulting for systemic improvement in research, education, and institutional human resource (HR) policies in Indian institutes.

About the Editors

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Dr. Niroj Kumar Sahu, M.Sc., M.Tech., Ph.D. is currently working as an Associate Professor at the Centre for Nanotechnology Research at the Vellore Institute of Technology, India. His areas of research are focused on the development of functionalized nanomaterials, study of the physical and chemical properties of nanomaterials, and applications of nanomaterials in cancer therapy as well as energy storage. He is the author of 45 peer reviewed journal articles, 8 conference proceedings, 10 book chapters, and 1 book with total citations of 935 and an h-index of 18. He is the recipient of the “Award for Excellence in Thesis Work” from the Indian Institute of Technology Bombay in the year 2016. He has completed 5 funded projects as principal and co-principal investigators from the Department of Science and Technology, Government of India. He has supervised 2 Ph.D. students and 20 M.Tech. students for their degree certificates. Presently, he is guiding 5 Ph.D. students and 2 M.Tech. students. He is actively involved in research as well as teaching UG/PG students.

Strategies for Synthesis of Superparamagnetic Nanoparticles Ashwini B. Salunkhe, Manohar S. Lad, and Vishwajeet M. Khot

Abstract Superparamagnetic nanoparticles (SPNs) due to their resonant response to an externally applied magnetic field (AMF) and tunable intrinsic magnetic properties have been extensively considered for a range of biomedical applications. Among various SPNs, superparamagnetic iron oxide nanoparticles (SPIONs) have attained considerable interest in cancer diagnosis and treatments in recent years. Though there is a tremendous advance in this research field, synthesizing SPNs poses a challenge in a variety of nanoparticles. Superparamagnetism is a size-dependent phenomenon; synthesis of such SPNs is often achieved by choice of proper synthetic route and controlling the reaction parameters during the in-situ reactions. In this section, various synthetic routes for the synthesis of SPNs are reviewed in detail. Keywords Chemical synthesis methods · Iron oxide · Magnetic nanoparticles · Superparamagnetic nanoparticles · Superparamagnetism

1 Introduction The core of Magnetic nanoparticles (MNPs) is made up of magnetic elements, e.g. Iron, Cobalt, Nickel, and corresponding oxides and can be manipulated in the presence of an alternating magnetic field (AMF). This unique ability of MNPs has been utilized for biomedical applications that include the Magnetic Resonance Imaging technique (MRI) as a diagnostic tool in cancer detection (where MNPs are used as a contrast agent), bio-separation of proteins and DNAs, cell tracking and labelling, biosensors, transfection, targeted therapies such as gene and localized drug delivery, and Magnetic Hyperthermia Therapy (MHT) (Salunkhe et al. 2014).

A. B. Salunkhe Department of Physics, Rajaram College, Kolhapur 416004, India M. S. Lad · V. M. Khot (B) Department of Medical Physics, Center for Interdisciplinary Research, D. Y. Patil Education Society, (Deemed to Be University), Kolhapur 416006, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_1

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Reduction in the spatial extension results in novel effects for the properties of MNPs which have impacted the prior understanding of magnetism at the mesoscopic scale (Grünberg 2001; Magnetoelectronics 1998; Dormann et al. 1998). The existence of a particle of ferromagnetic materials in a superparamagnetic state that consists of a single magnetic domain structure below its critical size was first predicted by Frenkel and Dorfman (Himpsel et al. 1998; Frenkel and Doefman 1930). The spherical and single-domain nanoparticles of a typical ferromagnetic sample are predicted to have a radius of around 15 nm which falls within the superparamagnetic regime. These single-domain ferromagnetic particles can be thought of as giant magnetic units since the size of a particle’s magnetic moment, measured in Bohr magnetons, is proportional to its volume. It is typical to assume that the particles have an ellipsoidal shape, where the magnetic moments tend to align along the longest axis, defining the direction of the maximum “shape” anisotropy energy (Kittel 1946). The intriguing interactions between intrinsic features, the nanoparticles (NPs’) inhomogeneous size distribution, and particle–particle interactions give rise to the novel phenomena in nanomaterials. Each NP’s magnetic behaviour is dominated by finite-size effects, which become more significant as particle size decreases. Since particle anisotropy is often affected by the volume of the particle, finite-size effect has received the most attention. Owing to the distinct atomic coordination, concentration, and type of defects present in both areas, the magnetic behaviour of particles located at the surface and that corresponding to the core vary. Thus, a substantially larger magnetic disorder present at the surface gives rise to magnetic behaviour ranging from those of a magnetic dead layer to those of a spin glass-like material, while the spin arrangement of the core is similar to that of the bulk. The ground state of the particle is mostly determined by the magnetic ordering of the surface and core. It may depart greatly from the straightforward premise of a single domain. Moreover, the response of the NPs is significantly influenced by this significant finite-size effect (Corchero and Villaverde 2009). To successfully use MNPs in new technologies, it is essential to have a complete grasp over chemical stability, dispersion in various fluids, interparticle interactions, surface chemistry, and magnetic properties.

2 Types of Magnetic Nanoparticles Nanostructured materials contain random and conflicting interparticle interactions, which may significantly alter the dynamic behaviour of these systems. The energy barrier’s height, which controls each particle’s relaxation time, has an impact on how interactions change. The interactions depend on the nature of magnetic material, surface spin order/disorder, and sizes in the case of MNPs. In general, MNPs are categorized as diamagnetic (no response to an external magnetic field, negative susceptibility), paramagnetic (poor response, low positive susceptibility), and ferromagnetic (high positive susceptibility) based on the magnetic response. MNPs may represent a distinct kind of magnetism known as single-domain MNPs (characterized

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by the high coercive field with high stability) and superparamagnetism (characterized by zero remanences, zero coercivity, and unstable) depending on their size.

3 Important Properties of MNPs Superparamagnetism (SPM), one of the important phenomenon related to MNPs, find application in healthcare. When particles are tiny enough, thermal turbulence can flip magnetic moments at random, leading to SPM. In the absence of an AMF, random alignment of these magnetic moments produces an average magnetization to zero. The Néel relaxation time is the typical time between moment flips (Walkey et al. 2012) given by  τ = τ 0 ex p

KV kBT



where T is the temperature, K is the magnetic anisotropy energy, V is the particle volume, k B is the Boltzmann constant, and τ 0 is the interval between flip attempts, which is typically between 10–9 s and 10−12 s depending on the material. As can be observed from the equation, the relaxation time is proportional to the particle volume/particle size. In the case of iron oxide crystals, the size limit needed to produce superparamagnetism might change depending on the composition of the core material. This criterion indicates that instead of a single big crystal, MNPs with sizes more than 20 nm often consist of clusters of numerous small particles. Attraction forces between adjacent MNPs could generate huge aggregates that are easier to remove from the circulation and increase the risk of vascular embolism. When subjected to an external magnetic field, SPNs have additional desirable features such as greater magnetic susceptibility and magnetic saturation (Salunkhe et al. 2015). An individual’s reorientation is the cause of their high magnetic susceptibility. Due to their unique qualities, MNPs in particular, iron oxide-based spinel ferrites became the preferred option for biological applications. The requirement for specific MNPs to have the intended therapeutic effect is a feature shared by all MNPs-based therapies (Thanh et al. 2014). Depending on the precise role that the NPs performed in therapy, each diagnostic or therapeutic procedure calls for a particular chemical or physical attribute of the particles involved(e.g. Vector, contrast agent, heater, moment carrier for magnetic fields, etc.). An external parameters that interacts with the MNPs, such as magnetic fields, and light, may occasionally be used to activate the particle function. Thus, a wide range of innovative materials, synthesis techniques, and study areas are needed for MNPs to function as biological agents.

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4 Synthesis of SPIONs As the magnetic characteristics of MNPs being dependent on the synthesis techniques, later play a significant role in producing SPNs with homogeneous sizes and uniform shapes. The development of synthesis techniques that allow for adequate control over particle size, distribution, and defects has long been a difficulty in material science research. Due to MNPs’ aggregation nature, their synthesis is a difficult process. The generation of monodispersed nanostructures and their reproducibility present two major chemical hurdles. The typical strategy for synthesizing monodisperse MNPs recommends avoiding nucleation during the growth phase or separating the two processes of nucleation and growth (Thanh et al. 2014). Many physicochemical and biological techniques have been developed with this goal in mind. Physical techniques like ball milling, Laser plasma deposition, lithography, etc. are mostly utilized in engineering and electronics; hence, they are outside the scope of this subject. The synthesis of nanomaterials is favoured by chemical and biological processes because they provide good control over size and shape.

4.1 Chemical Synthesis of SPIONs Chemistry has been crucial in the manufacturing of advanced materials with significant and innovative properties. Chemical synthesis has the advantage of being flexible in the design and synthesis of novel materials that can be improved into the final product. Good chemical homogeneity is the main advantage of chemical processes over other processes since chemical synthesis allows mixing at the molecular level. The recent advances in biomedical fields such as MRI, and adjuvant cancer therapies involving Starvation, Chemodynamic, Photodynamic, Radiotherapy, and MHT involving SPNs have attracted the attention from researchers. The optimization of preparative parameters solely depends on the synthesis method to achieve SPNs with desired physicochemical properties. The chemical method of producing SPIONs in the liquid phase is the most preferred bottom-up approach for the synthesis of the preferred shape and size. It entails the reduction of atoms or molecular components of the raw materials in the liquid or gaseous state. The chemical process for fabricating SPIONs can be divided into distinct categories depending on the synthesis medium, such as wet-chemical (liquid-phase) and gas-phase approaches (nucleation of SPNs in the gas-phase medium). The production of SPIONs through chemical reactions in a solvent environment requires the liquid-phase approach. For the synthesis of SPIONs, several chemical liquid-phase techniques have been documented in the literature. Because the parameter optimization for fine SPIONs can be done in a variety of ways, several wet-chemical techniques are used. Such methods include (A) Co-precipitation, (B) Polyol, (C) Solvothermal, (D) Hydrothermal, (E) Sol–gel, (F) Microemulsion, (G) Sonochemical, and (H) Electrochemical process.

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These techniques allows to synthesize particles with restricted size distribution and homogeneous content. By adjusting various synthetic parameters during the nucleation and particle formation phase, wet-chemical approaches can be used to generate nanoparticles of the ideal size.

4.2 Aqueous Co-Precipitation of Inorganic Salts The chemical co-precipitation method is most likely the quickest and most straightforward process to fabricate MNPs. Because of its simplicity, quick production process, uniformity, low cost, high product purity, and absence of need for organic solvent, surfactant, or high-temperature treatment, the co-precipitation approach has typically been selected. In a typical synthesis of SPIONs, inorganic metal salts/precursors (Fe3+ :Fe2+ :2:1) are added into a beaker in respective stoichiometric ratios. To prepare MNPs, a homogeneous aqueous mixture magnetically stirred for some time. The pH of the solution is adjusted by adding bases like NaOH, KOH, and NH4 OH slowly or vigorously reducing the salt solution while maintaining the pH around 11–12 to form a solid insoluble precipitate. After mixing for a few minutes, the reaction is continued for several minutes along with heating if necessary. Such a simple approach has been carried out for the synthesis of the nanoparticles and followed by postsynthesis processes, e.g. multiple times washing with DDW for removing unreacted material and excess ions, centrifugation, magnetic separation, and finally drying at 40–80 °C (Fig. 1). The reaction mechanism can be understood as follows: Fe2+ + 2Fe3+ + 8OH− ↔ Fe(OH)2 + 2Fe(OH)3 → Fe3 O4 + 4H2 O The pH of the solution should be between 9 and 14, to produce magnetite effectively. After the reaction, the precipitate solution still has a black colour. The co-precipitation process consists of two steps: The first is co-precipitation, which produces solid metal hydroxides in the form of colloidal particles, and the second is ferritization, which involves heating the hydroxides produced during co-precipitation to convert them from a solid metal hydroxide solution to ferrite. The ability of the co-precipitation technique to produce a significant amount of NPs is its key benefit. Since only kinetic parameters are in control of the crystal’s formation, the control over particle size distribution is restricted. The co-precipitation process involves two steps: First, a brief burst of nucleation occurs once the species concentration exceeds critical supersaturation, and then the nuclei gradually increase as the solutes diffuse to the crystal’s surface. To obtain monodispersed iron oxide nanoparticles (IONPs), these two phases must be separated, i.e. nucleation must be controlled. Controlling the particle size and, consequently, the narrow size distribution presents an experimental challenge in the co-precipitation synthesis of MNPs. The size and size distribution of the particles has a major impact on the magnetic properties of MNPs, such as the blocking temperature. The range of blocking temperature

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Fig. 1 Schematic of the Co-precipitation method

for MNPs is typically narrow and depending upon the particle size. Smaller particles tends to have lower blocking temperature, while larger particles have higher blocking temperature (Tartaj et al. 2006; Yallapu et al. 2011). According to Ayyappan et al. (2009) nucleation rate and particle size have been shown to affect material properties significantly due to the change in the solvent’s dielectric constant. The coprecipitation approach typically tends to produce polydisperse particles. By separating or controlling the nucleation and growth, process factors including reaction temperature, reaction duration, ageing time, and pH can results in monodispersed MNPs. By using an aqueous co-precipitation technique, Beeran et al. synthesized monodispersed spherical iron oxide-embedded hydroxyapatite (HAIO) nanocrystals. SPIONs demonstrated the opposite tendency than HAIO particles’ capacity to generate heat declined with increasing concentration. With compared to SPIONs, the higher stability and presence of surface modification in HAIO can be used to explain this phenomenon. Nonmagnetic hydroxyapatite (HA) crystals were deposited on the surface of SPIONs in the HAIO nanocomposite. Moreover, the magnetic dipolar interactions and particle surface potential offset one another to lesser the aggregation. According to research, HAIO produces controlled hyperthermia that encourages death in HeLa cells, but SPIONs produce uncontrolled temperature rise that seriously damages cellular membranes (Ereath Beeran et al. 2019). The Brazilian group investigated the dual behaviour of doxorubicin (DOX) encapsulated into vesicle-like supramolecular nanocrystals as a magneto-thermal agent and as a nanocarrier for drug delivery/release. Carvalho et al. developed biocompatible and water soluble biopolymer carboxymethylcellulose (CMC) encapsulated magnetic iron oxide nanoparticles MION@CMC, a natural cellulose derivative, that serves as both a stabilizing ligand and a biodegradable organic coating layer to produce ultra-small iron oxide-based SPIONs (7 nm) in an aqueous medium. Moreover, these MION@CMC nano colloids capable of forming magneto polymersomes with core–shell hybrid structures by covalently coupling them with anticancer medication DOX (MION@CMC-DOX). The outcomes showed that SPIONs, upon incubation with MION@CMC-DOX and application of an alternating magnetic field, had exceptional killing ability against brain cancer cells in vitro (U87 cells) (Carvalho et al. 2019).

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To explore magneto-chemotherapy doxorubicin loded and polymer grafted SPIONs DOX@PM@SPIONs were synthesized using weak base alkanolamine (DIPA) by the group of Salunkhe et al. This group synthesized spherical-shaped SPIONs (Fe3 O4 ) of ~12 nm, with a high magnetic moment and high magnetization (92 emug−1 at room temperature) by a simplified co-precipitation method. In this synthesis, a weak base Diisopropylamine (DIPA) was used for surface stabilization as well as reducing agent. Double-stranded pluronic block copolymer chains are used to enhance the biocompatibility that results in enhanced dispersion stability and allows bio-functional groups to bind drugs and biomolecules. The cytotoxic effect of DOX@PM@SPIONs (measured in terms of cell viability) increased with increase in NPs concentration (Salunkhe et al. 2020). With the aid of hydrophilic SPIONs-based ferrofluids with carboxyl (terephthalic acid-TA) functionalization, Kandasamy et al. detect the improved killing efficiency of MCF-7 cancer cells. The team discovered that in their in-vitro MFH tests, TA functionalized hydrophilic SPIONs revealed greater killing efficacy towards MCF-7 cancer cells and very good colloidal stability. The enhanced magnetic response was attributed to a high magnetization value (74.3 emug−1 ) and the short chain TA molecules (length 1.2 nm) incorporate on the surface of nanoparticles via π–π conjugations. The maximum specific absorption rate (SAR) value, which is attributed to the prominent Néel relaxation and causes therapeutic temperatures to be attained at a much faster rate at lower (0.5 mg/ml) concentrations within Hergt’s safety limit, is shown by a dispersion investigation of SPIONs in viscous media, such as triethylene glycol (TEG) (Kandasamy et al. 2018). Xie et al. investigated the co-precipitation-based synthesis and characterization of ultra-small superparamagnetic Janus NPs for cancer adjunct chemotherapy and MHT. The structural stability in a solvent provided by asymmetric functionality of J-NPs (simultaneous incorporation as hydrophobic as well as hydrophilic) plays a dual role as the drug carrier and also the magnetic heat mediator for cancer chemotherapy, MHT, and MRI modality (Xie et al. 2020). The group of Cazares-Cortes et al. studied superparamagnetic (γ-Fe2 O3 ) synthesized by co-precipitation of inorganic salts of Iron like Fe2+ and Fe3+ according to Massart’s process. It produces nanogels with high content of uniformly distributed MNPs and ensures good properties of the two distinct colloidal systems (Cazares-Cortes et al. 2017).

4.3 Hydrothermal Synthesis Hydrothermal synthesis is an environment-friendly method that makes it possible to quickly and easily produce pure SPIONs that depend on the stability of the precursor in hot water. The experiment is generally performed in aqueous media in a device made of a steel pressure vessel (autoclave). Heating aqueous iron precursor solution to a critical temperature (374 °C) and applying water pressure (22.1 MPa) were used to manufacture ultra-fine powders and MNPs. The controlled size and shape of

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IONPs which truly depend on the reaction conditions such as reaction time, temperature, and other additions (e.g. surfactant, anion, and metal ions) significantly affect the properties of MNPs. Two types of hydrothermal synthesis method have been stated; one method involve the use of surfactant and the other without surfactant. The hydrothermal process has been further developed to generate SPIONs using microwave and electromagnetic radiation (Chen and Xu 1998). This method has successfully produced crystals of a variety of materials with high magnetic saturation values. When water is in a supercritical condition, the hydrothermal technique generate SPIONs that are hydrophilic and extremely crystalline (high temperature and pressure). Two steps can be used to explain how magnetite forms in supercritical water (SCW): (1) Dehydration follows the hydrolysis of the Fe2+ ions, at which point an intermediate hydroxide is produced. (2) SPIONs are produced as the hydroxide slowly dehydrates. To synthesize SPIONs using SCW, the precipitating cations’ solubility is essential. SCW serves as the reaction medium as a result, producing a favourable environment for nanoparticle production. The quality of the SPIONs formed, however, is improved by the addition of reducing agents such as NaOH, glycerol, NH3 , and citric acid to the SCW, according to several studies. Hydrothermal synthesis is used to produce non-agglomerated particles, in which distilled water and surfactant, e.g. Sodium salt of Acralic acid (PAANa) can function as both a reaction medium and a capping agent. Hydrothermal procedures can be utilized to quickly generate in-situ functionalized SPIONs with organic moieties such as Polyacrylic acid (PAA), thiols, amines, carboxylic acids, alcohols, or gluconic acids, according to research. Through steric stabilization, the hydrothermal technique uses surfactants to stop particle aggregation. Additionally, it aids in the synthesis of particle distributions with narrow sizes that are homogeneous. The concentration of the precursor and solvent composition both have an impact on the mono-dispersivity, magnetic properties, shape, and size of SPIONs produced using this method. The formation of small particle size and narrow size distribution are possible when a higher initial precursor concentration is used to produce a large number of seed nuclei as the system undergoes supersaturation. When nucleation and growth are isolated sufficiently from each other by a temperature window, the high concentration of nuclei produces a large number of particles that easily yield smaller particles. Shorter reaction times resulted in monodisperse particles; however, reaction time had a greater impact on average particle size. One disadvantage of this method is that it requires more time due to slow kinetics at any given temperature. With the other variables held constant, spherical particles are produced by increasing the precursor’s concentration. The hydrogel of xanthan gum and nanoparticle composite (XG/Fe3 O4 ) was demonstrated by Ribeiro et al. utilizing a hydrothermal synthesis method. With a saturation magnetization of 61 emug−1 , which is much lower than that of bulk IONPs with a diameter of 40 nm have been produced utilizing a hydrothermal approach (140 °C) (Ribeiro et al. 2021).

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4.4 Solvothermal Method The solvothermal synthesis method is a facile and one-pot synthetic method of preparation of a variety of inorganic nanomaterials like metal oxides and polymers. In contrast to the hydrothermal approach, using a solvent (as reaction medium) rather than SCW produce IONPs at moderate to high temperature, e.g. 100–1000 °C and cavity pressure of about 1–10,000 atm (Fakurpur Shirejini et al. 2023). Precursor contact/interaction during synthesis is facilitated by high temperature and pressure. The process is known as “hydrothermal synthesis” if water is utilized as the solvent. Under hydrothermal conditions, the synthesis is typically carried out below the water’s supercritical temperature (374 °C). Thin films, powders in bulk, single crystals, and nanoparticles can all be prepared using this method (Xu et al. 2020). Moreover, by adjusting solvent supersaturation, precursor selection and concentration, and kinetic control, one may regulate the shape of the produced nanoparticles as well as their phase purity (Sanna Angotzi et al. 2021). As nanocrystals have dominated the majority (80%) of the literature on solvothermal synthesis over the past ten years, this review will focus on notable developments in this area. For biomedical applications like targeted drug delivery, MRI contrast, and hyperthermia agents nanomaterial is required with a large surface-to-volume ratio, tiny particle size, and high saturation magnetization. Both magnetic as well as structural properties of Oleate-Capped Mnx Co1 − x Fe2 O4 considerably depend and vary with the chemical composition which have been studied by Angotzi et al. Oleatecapped Mnx Co1 − x Fe2 O4 with a size less than 10 nm exhibits moderately higher magnetization value around 99–98 Am2 /kg−1 due to specific chemical composition, comparative larger single-domain structure, and citation distribution (Sanna Angotzi et al. 2021). Thermal energy can break through the anisotropic potential barrier of a single nanoparticle of superparamagnetic nature at ambient temperature. Aggregation among SPIONs is hence a typical phenomenon. Furthermore, magnetic materials undergo rapid oxidation when exposed to humid air. To address this issue during the preparation of SPIONs, surfactants, capping agents, and stabilizers are commonly added to the reaction solvents to functionalize the particle surfaces. By improving the synthesis processes and adjusting the surface energy, it is possible to produce Fe3 O4 nanoparticles with ideal particle size, high stability, and uniform dispersion. Undoubtedly, factors such as the concentration of surfactants, synthesis temperature, and choice of solvents significantly influence the solvothermal method’s capacity to produce optimized Fe3 O4 nanoparticles (Ni et al. 2022). In the typical solvothermal reaction mechanism, solvents like glycols act as reducing agent and sodium acetate (NaOAc) acts as the electrostatic stabilizer providing the OH ions in the system (Chen et al. 2019). Researchers Xu Ni and colleagues investigated the growth kinetics of magnetic IONPs synthesized using the solvothermal process under various preparation time conditions ranging from 4 to 8 h. Studies have shown that nonporous morphology, potent magnetization, large specific surface area, a good degree of crystallinity, and thermal stability can be

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achieved by optimizing reaction conditions including precursor concentration and solvent choice at a reaction temperature around 200 °C and a proceeding time of 8 h. According to different scientific reports, SPIONs have been synthesized using solvents such as ethanol, methanol, ethylenediamine, diols and glycol derivatives, and hydrazine solution (Pachfule et al. 2011). The intermediate complex formation between the reactants and surfactant is responsible for the consistency in the shape and size of the nano-crystallites produced by this method. As a result, the solubility of the precursor in appropriate solvents, solvothermal temperature, and ageing duration affect the nucleation, growth, and size distribution of nanoparticles generated using this approach. Recently, IONPs were synthesized using poly (acrylic acid) (PAA) to stabilize the surface of IONPs by Jeong et al. to address the disadvantage in the mass production of SPIONs using in-situ environment. By adjusting the coordination of the stabilizer, it is possible to modify the size of the IONPs and the entanglement of PAA on the particle surface to lower their high surface energy. To boost the population of the nuclei in the synthetic reaction, multiple reducing agents were employed. When NaOAc is employed as the base, it generate iron hydroxides with complex structures and hydroxyl ions, which subsequently condense to produce Fe3 O4 . When a strong basic solution, such as NaOH, is added to the reaction solvent, alkoxides are produced that may also coordinate onto the surfaces of the nanoparticles (ethylene glycol). Consequently, by adjusting shape anisotropy and coordination chemistry, it was determined that the utilization of particles (25 nm) with high SAR (1250 W/g), which was 3 times more than that of the isolated IONPs, was achieved (Jeong et al. 2021).

4.5 Sonochemical Method Sonochemical method is one of the straightforward method of synthesizing nanostructures. The application of ultrasonic irradiation during the production of SPIONs has been suggested to enhance the uniform dispersion and hydrophilic characteristics of the magnetic particles. The shape and size distribution of the particles generated through the sonochemical method is influenced by specific ultrasonic irradiation factors, such as the temperature and strength of the ultrasound. This technique first proposed in 1980 uses ultrasonic energy irradiation to cause the disintegration of aqueous binary solutions of inorganic or organometallic iron precursors in deoxygenated water. The ultrasonic irradiation generates a huge temperature (5000 K) and pressures around 2–3 kPa in an acoustic cavitation process where intermediate formation and their growth, and collapse of bubbles take place with enormous cooling rates of 1000 K/s. These cavitation and high-temperature hot spots encourage the generation of oxidizing radicals, which triggers the process that generate the final stable phases. Improved solute diffusion is encouraged by ultrasonic irradiation, responsible for the decreasing width of the induction time, the width of the metastable zone, and the increase in the nucleation rate, as well as the crystallization process. High-energy ultrasonic irradiation can break down ferrous salt solutions without the

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need for a reducing agent at room temperature and atmospheric pressure. By adding a suitable stabilizer, SPIONs colloidal stability can be increased. The crystallinity of nanoparticles is impacted by the localized high temperature and pressure caused by the sonic cavitation process. Synthesis is followed by heat treatment (sintering) of the particles to increase the crystallinity of nanomaterials. In addition, intense mixing at high frequency generate turbulence and deters agglomeration (particularly for MNPs). The produced nanocrystals are smaller than the resonant size of the bubbles as a consequence of particle collisions caused by the shockwave produced by acoustic cavitation. The key factors affecting the quality of the final product are frequency, intensity/amplitude of ultrasonic energy, and the influence of solvent qualities in a solution. (like vapour pressure, solvent viscosity or surface tension, resonant size of the bubble, etc.) Depending on how the synthetic particles are formed, their cytotoxic profile may be affected more by factors other than particle sizes, such as shape, surface chemistry, oxidation states, or aggregation. On the other hand, Fuentes-García and coworkers examined that crystalline SPIONs may be synthesized at very low ultrasonic temperatures (50–52 °C) with a low reaction time and optimum reagent concentrations for controlling the size and morphology. According to the study done by this group, the size of the IONPs grows as the precursor Fe3 O4 concentration grows, and the MNPs continue to have a cubelike shape. To achieve improved magnetic properties (Ms = 70 A.m2 /kg), decreased cytotoxicity independent of particle size (Fuentes-García et al. 2020).

4.6 Thermal Decomposition Method Thermal decomposition has become a widely used method for producing magnetic IONPs with controlled shape and size, and magnetic properties. This method involves the thermal decomposition of suitable iron precursors under specific conditions to produce nanoparticles for various applications, including magnetic hyperthermia therapy (Unni et al. 2017; Wierzbinski et al. 2018; Guardia et al. 2011). Essentially, smaller monodisperse and hydrophobic magnetic nanocrystals can be produced by thermally decomposing organometallic compounds in stabilizing surfactant-containing high-boiling organic solvents (Cheah et al. 2021a). Samples produced by the breakdown of precursors in the combination of hot organic solvents and surfactants have significantly improved in terms of size control, narrow size distribution, and crystallinity of individual and dispersible magnetic metal oxide NPs. Because particle size is so important in these processes, MNPs produced by this technology could be used in medical applications such as MRI, protein separation, magnetic cell labelling, and magneto relaxometry. Metallic NPs are also made via the thermal decomposition technique. The benefit of metallic NPs is that they have a higher magnetization than metal oxides (Maity et al. 2009; Sun et al. 2004). Iron precursors, such as iron oleate or iron acetylacetonate, chlorides, are typically dissolved in a stoichiometric ratio in a suitable solvent and stirred to get a

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homogeneous precursor solution. The mixture is then heated at elevated temperatures, usually in the manifestation of a stabilizing agent or surfactant. The thermal decomposition offers the formation of the unstable precursor complex that leads to the nucleation and growth of IONPs. The key advantage of the thermal decomposition method lies in its ability to achieve specific control on the nanoparticle size and morphology. Factors like decomposition temperature, precursor concentration, heating ramp, and reaction time can be manipulated to tailor the desired properties of the nanoparticles. The choice of stabilizing agent or surfactant also influences the size uniformity and stability of the nanoparticles. The resulting MNPs exhibit superparamagnetic or ferrimagnetic behaviour, depending on their composition and size. These nanoparticles can be further functionalized with biocompatible coatings to improve their stability and biocompatibility for biomedical applications (Unni et al. 2017; Lu et al. 2021; Zhang et al. 2017). The intrinsic magnetic properties of these nanoparticles are critical in the domain of MHT. When exposed to an AMF, nanoparticles produce heat via hysteresis losses and rapid friction with solvent while changing magnetic alignment concerning the applied field. This selective heating can effectively induce tumour cell death while minimizing damage to healthy tissues. The thermal decomposition method offers several advantages, including simplicity, scalability, and the ability to produce nanoparticles with tailored properties. Challenges such as controlling particle size distribution and achieving reproducibility need to be addressed. In conclusion, the thermal decomposition method is a versatile and promising approach for synthesizing SPIONs. Its ability to control nanoparticle properties makes it a valuable technique for various applications, including magnetic hyperthermia therapy. Further research and optimization of this method will contribute to advancing nanoparticle-based therapies and their translation into clinical practice.

4.7 Microemulsion Method A Microemulsion can be defined as a thermodynamically stable, isotropic dispersion of two liquids that are normally immiscible. This stability is achieved by coating the micro-domains of one or both liquids with surface-active molecules at the interface. At the interface between the oil and water phases, the surfactant molecules can form a monolayer where the hydrophobic ends dissolve in the oil phase and the hydrophilic groups dissolve in the aqueous phase. In this system, the aqueous phase contains metal salts and/or other components, while the oil phase consists of a complex mixture of hydrocarbons and olefins. Among the different types of microemulsions, water-inoil (w/o) and oil-in-water (o/w) microemulsions are the most commonly used for synthesizing nanoparticles (NPs) (Okoli et al. 2011; Samrot et al. 2021). The morphology and size of IONPs synthesized via the microemulsion process may be influenced by factors such as the molar ratio of water to oil or surfactant, ageing period, alkali concentration, temperature, and initial high concentration of iron ions. Fine micro-droplets of the aqueous phase are contained within surfactant

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molecule assemblies that are disseminated across a continuous oil phase in a waterin-oil microemulsion (Chin and Yaacob 2007; Tartaj and Tartaj 2002). Continuous collisions, coalescence, and re-breaks of these micro-droplets are expected. Conceptually, reactants A and B will combine to generate an AB precipitate when combined in two identical water-in-oil microemulsions. It is possible to conceive the growth of these particles in microemulsions as an inter-droplet exchange and nuclei aggregation process (Zhang et al. 2017; Okoli et al. 2011). The surfactants can be used to separate the finely distributed precipitate that is so formed. The ability to generate nanosized particles using water-in-oil microemulsions has been demonstrated to be effective, adaptable, and straightforward. These characteristics may make this technique useful for both in-vivo and in-vitro applications.

4.8 Electrochemical Method The Electrochemical approach for producing SPIONs relies on the Redox reaction occurring at iron-based electrodes within an electrolyte. During this process, the positive electrode undergoes conversion into metallic ion species within the electrolyte. Subsequently, the metal ions are reduced to their corresponding metals at the cathode, aided by stabilizers. By fine-tuning the applied DC current density, the particle size of the SPIONs synthesized directly on the electrode surface, either as a coating or thin film, can be effectively regulated. The method of using SPIONs in both in-vitro and in-vivo demonstrations restricts access to these pure, unprocessed nanoparticles, making them susceptible to agglomeration and the formation of larger lumps due to strong anisotropic dipolar interaction, particularly in an aqueous phase. Because of this, Fe3 O4 nanoparticles aggregate when given in vivo, resulting in a short blood circulation period, and physical characteristics that reduce SPIONs action. As a result, immobilizing these nanoparticles on a substrate is necessary to stop their aggregation and precipitation in aqueous suspension. To overcome such a problem, Kumar et al. developed GO-based nano platforms for the formation of decorated Fe3 O4 using NaCl solution as an electrolyte in distilled water (DW) (Kumar et al. 2018). The other choices of stabilizers like amine act as supporting electrolytes in the synthesis of SPIONs with proper surface functionalization. The good colloidal stability and low cytotoxicity (70% cell viability) for in-vitro study are major key parameters to carrying out in-vivo study. Due to good colloidal stability and high saturation magnetization (66.6 emug−1 ), a high SAR value of 543 W/g was observed for the nanoparticles with the highest crystallite size of 21.07 nm (Xiao et al. 2018).

4.9 Polyol Method The Polyol method is another simple, fast, and promising method to prepare water-dispersible, uniform, biocompatible single-core nanostructures with excellent

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heating ability that have excellent colloidal stability over time (Cheng and Gu 2011; Hugounenq et al. 2012). Recently, the liquid-phase polyol synthetic strategy paid more attention to the synthesis of SPIONs due to simplicity, reproducibility, and its capability of producing hydrophilic nanoparticles without the need for an inert atmosphere (Cheah et al. 2021b). In multivalent alcohols, this approach generally provides a broad range of selection of parameters like reaction temperature, type of precursors and their relative concentration, reaction time, and capping/stabilizer allowing for the control of the physicochemical properties of the IONPs, such as crystal phase, low polydispersity index, high magnetization, and small particles size (Hachani et al. 2016). E.g. Pohlee Cheah et al. found reaction temperature is a critical factor in the continuous growth of the water-dispersible SPIONs (Hemery et al. 2017; Ling and Hyeon 2013). The apparent advantage of being simple to scale up for industrial manufacturing is no additional reducing agents or surfactants are needed (Fig. 2). In the process of synthesis, polyol not only serves as a high-boiling solvent but also as a reducing agent, which can be used to stabilize (Mi et al. 2017). These diols and glycols have been utilized to effectively control particle development and arrest interparticle aggregation due to the reaction temperature, and solubility of organic and inorganic metal precursors in the solvent provides an advantage too. For slightly reducible metals, the homogeneous solution is heated to a temperature close to the boiling point of the given solvent, resulting in a highly crystalline material with improved yield. The solubility of the inorganic precursor and a slight decrease of

Fig. 2 Polyol route for the synthesis of MNPs

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the dissolved metallic species by the polyol provide good control over nucleation of the metallic monomer phase, and manipulating nuclei growth mechanism using a suitable stabilizer/capping agent is the primary step involved in the polyol process for the production of IONPs. Families of polyols are water-similar and chelating agents including a wide range of ethylene-containing glycols and diols, including with few to high molecular weights. Since polyols instantly coordinate with the nuclei generated and enable good control over particle size to produce water-dispersible, non-aggregated SPIONs with narrow size distribution (Guleria et al. 2019), the shape and size of the SPIONs can be manipulated by altering the solvent nature and amount, reaction time, and the amount of iron precursors (Laurent et al. 2008). Molecules of a long chain in solvent, i.e. polymers produce the smaller and single-core NPs (Porru et al. 2022; Gavilán et al. 2017). Similarly, an increase in the precursor concentration and reaction time produces larger NPs (Storozhuk et al. 2021). Compared with conventional synthesis methods such as thermal decomposition or co-precipitation, the Polyol process yields with highly crystalline nature, and narrow particle size distribution (Walle et al. 2020). The surface of nanoparticles produced through the polyol method provides excellent aqueous stability to the nanoparticles, helps in making them biocompatible as well as found to be enhancing the MRI contrast as well as high saturation magnetization. Nanoparticles grow with increasing initial precursor concentration; hence, depending on the product’s solubility in the polyol, particle size can be kept to a minimum. Both structural and magnetic properties of as-synthesized nanoparticles were extensively studied by Gauvin Hemery et al. based on the influence of the water amount in the reaction. An extremely low amount of water (100 μl) addition results in a larger particle size and lower polydispersity index (0.13), exhibiting the highest SAR (265 w/g). The manufacturing of temperature-dependent highly crystalline oxide nanoparticles using the polyol technique eliminates the need to subject the finished product to post-sintering. Since nanoparticles growing near the critical size have the lowest growth rates and should have stronger crystallinity than in other states of growth, slower growth rates result in crystal growth with fewer defects. A high supersaturation ratio causes a low critical size, which results in more nuclei having positive growth rates exceeding the critical size (Hemery et al. 2017).

4.10 Combustion Synthesis (CS) For the production of inorganic IONPs, simple and rapid Combustion routes become the choice when their applications are specifically in energy storage devices, sensors, water treatment nanocatalysts, and many others (Iano¸s et al. 2018; Buyukhatipoglu 2009). A highly exothermic redox reaction itself acts as the self-estimated powerhouse for ignition and produces crystalline oxide nanoparticles; eliminating procedures of post-annealing treatment of nanoparticles for a prolonged time. This strategy offers a unique mechanism for industry-scale production and continuous synthesis (Rahinov et al. 2021; Tharani et al. 2021; Berry et al. 2019). These characteristics

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make CS an appealing method for producing technologically useful nanomaterials at lower costs than traditional ceramic processes. In the typical synthetic route homogeneous precursor’s solution reacts with the desired quantity of different precursors as oxidizers and reducing agents (fuels). To produce highly pure and pores materials, various operating variables such as the C/ H proportion (fuel type), the ratio between fuel to oxidizer (F/O), the proportion of surfactants to reactants, the moisture content in precursor mixture, and the ignition temperature can all influence the combustion reaction (Ali et al. 2021; Wang et al. 2017; Esmaeilnejad-Ahranjani and Lotfi 2022). Crystallite size, surface area, size distribution, and agglomeration nature of as-synthesized MNPs are mainly controlled by the enthalpy or ignition temperature generated during combustion, which is determined by the characteristic and composition of the fuel and the fuel-to-oxidizer ratio. Particles with desired properties can be synthesized by varying the fuel-to-oxidizer ratio (Salunkhe et al. 2012; Khot et al. 2012).

4.11 Sol–gel Synthesis Sol–gel method is simple and the most suitable non-aqueous synthetic method used to fabricate various nanoparticles due to excellent control over size by optimizing the surfactant to precursor ratio, the viscosity of sol, etc., and low temperature for post-sintering leads to less power consumption, cost-effective, low reaction temperature, and high purity (Kayani et al. 2014; Paulson and Jothibas 2021). This method involving the polycondensation metal precursors to form the inorganic nanostructured framework is performed in liquid/gel form through two different processes. First hydrolysis of metal precursors in acidic/basic reaction media and then the rapid polycondensation of precursor complex called sol at the desired temperature. The aqueous sol–gel approach encounters some serious drawbacks due to highly reactive metal precursors and the dual role of water as a solvent, as well as ligand, which leads to the amorphous and polydisperse nature hence involving an additional post-sintering procedure. A slight change in the experimental conditions seriously affects the reproducibility of nanoparticles (Niederberger 2007). These drawbacks can be eliminated in the non-aqueous route. The non-aqueous method provides several benefits including (i) the ability to achieve materials with a predetermined structure based on experimental conditions, (ii) the potential to obtain pure amorphous phases, uniformity in particle size, and precise control over monodispersity, (iii) control over the microstructure and ensuring homogeneity of the reaction products, and (iv) the option to incorporate molecules within the sol–gel matrix while preserving their stability and properties. These advantages are closely related to the role of solvents and organic or inorganic ligands. The role of solvent and ligands is not only restricted to the source of oxygen rather the formations of intermediate

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complexes highly influence the preferential orientation. Stabilizing agents in combination affect the crystal growth rate leading to the production of crystalline nanoparticles with uniform morphology (Niederberger 2007; Bokov et al. 2021; Mornet et al. 2004; Lu et al. 2007).

5 Conclusion Several methods have been investigated for the production of SPNs; in this chapter, we tried to discuss some of them. During the process, the rate of crystal growth can be tuned by composition, precursor concentration, precursor-stabilizers ratio, capping agents, reaction time, temperature, and heating ramp, such that the iron precursor decomposes, in the suitable temperature window due to the separated nucleation and growth mechanism. The various synthetic parameters optimize to development of monodisperse nanoparticles with desired characteristics. The choice of stabilizing agent or surfactant is also essential for avoiding nanoparticle aggregation and promoting size uniformity. MNPs’ physicochemical properties, such as shape, size, functionalized ligand or moiety, and thus surface charge, as well as magnetic properties such as high magnetic saturation value, magnetic anisotropy energy, and magnetic core size, are critical in their future technology/therapy application. These methods allow precise control over nanoparticle size and morphology, leading to improved magnetic properties and enhanced therapeutic efficacy for future biomedical applications. The versatility of these chemical methods enables the synthesis of nanoparticles with tailored characteristics suitable for specific applications in MRI, drug delivery, and MHT.

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Functionalized Strategies of Superparamagnetic Materials Shubhangi D. Shirsat, Rajaram S. Mane, and Nanasaheb D. Thorat

Abstract Superparamagnetism occurs only in nanocrystals. Superparamagnetic nanomaterials (SPNs) need to be assembled into complex materials, so as to use in particular applications. SPNs must display collective properties such as increased magnetic saturation, biocompatibility, stability, and reactive surface. SPNs are the most considered nanomaterials for subsurface applications. Surfaces of SPNs can be altered by various organic or inorganic materials, like polymers, silica, metals, biological molecules, etc. The main challenges and strategies for the synthesis and properties of surface functionalized SPNs are systematically discussed in this chapter as SPNs are extensively used in numerous medicinal purposes, for example, for early detection of diseases like diabetes and various types of cancers.

1 Introduction Advancements in colloidal sciences have expanded our knowledge to adjust the physicochemical characteristics of nanomaterials. SPNs exhibit magnetization only under the influence of a magnetic field and it gets lost after the removal of the magnetic field (Kashevsky et al. 2008; Zhou et al. 2015). Due to alteration in the magnetization of SPN, it has been used in several fields like microactuators (Perez 2007; Xu et al. 2011; Shao et al. 2012), magnetic separation (Lee et al. 2011; Guzman-Lastra et al. 2016; Chen et al. 2017), and drug delivery (Li et al. 2017; Ulbrich et al. 2016; Peters et al. 2016). Superparamagnetism is seen only in nanocrystal materials. Iron oxide (Fe3 O4 ) displays superparamagnetism when particle size is less than 30 nm S. D. Shirsat Environmental Engineering Program, Guangdong Technion – Israel Institute of Technology, Shantou 515063, China R. S. Mane School of Physical Sciences, S. R. T. M. U. Nanded, Maharashtra, India N. D. Thorat (B) Nuffield Department of Women’s & Reproductive Health, Medical Science Division, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_2

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(Ge et al. 2007; Teja and Koh 2009). For specialized applications of SPNs, they must have assembled in more complex structures, so as to support the variety of functionalities (Ge et al. 2007; Klünker et al. 2018). However, during the formation of complex structure, magnetization, coercitivity of the material could be alter as particles tend to aggregate (Bao and Gupta 2011; Nakata et al. 2008; Lee et al. 2015; Kralj and Makovec 2015; Martinez-Boubeta et al. 2013; Kostopoulou and Lappas 2015) and hence completely changed magnetic response could be detected (Slöetjes et al. 2017). These complex structures are synthesized either by 1D arrays (Sim et al. 2015; Gao et al. 2018) or hybrid clustered beads (Ge et al. 2007; Bannwarth et al. 2015a). Necklace-like chains structure are formed when clustered beads themselves undergo further arrangement (Bannwarth et al. 2013, 2015b). Three-dimensional (3D) structures of magneto-responsive materials show a bigger array of possible applications such as flexible integrated sensors (Zhang et al. 2015; Xu et al. 2015) or biomimetic soft robots (Hu et al. 2018; Diller and Sitti 2014). The efficacy of SPNs for subsurface use has considerably increased due to their exclusive physicochemical characteristics. Among different types of SPNs, magnetite material like Fe3 O4 is most studied due to its increased chemical stability, cheaper, nontoxicity, and effortless synthesis (Daniel-da-Silva et al. 2012). High-performance SPNs are used for industrial uses that basically should avail the following properties: (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x)

Strong magnetic cores with minimum hysteresis at room temperature Aqueous stability in usual situations Increase surface area and mobility Coating for shielding magnetic core due to chemical degradation Functional coating with reactive complexing representatives for specific interaction High density of complexing representatives at the surface Chemical reversibility for replacing complexed metal ions Excellent magnetic recovery for reuse Least toxicity; contributing little environmental damage, and Inexpensive; ease of large-scale production (Condomitti et al. 2018).

For the effective use of SPNs in biological applications, surface functionalization is essential to endow the following properties: i. ii. iii. iv. v. vi.

Improved colloidal stability Increased water solubility Better magnetic controllability Protect and stabilize the surface Biocompatible and nontoxic, and Provide bare functional groups for the attachment of biological substances for nano-bioapplications.

Bare SPNs have a very high surface-to-volume ratio, and consequently, increase surface energies. So, they often aggregate to lower the surface energy. High chemical reactivity and oxidation (especially magnetite) contribute to damage in the magnetic

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and dispersion properties of SPNs. Therefore, for effective applications of SPNs and stable chemical properties, proper surface coating strategies must be used. For coating various organic molecules, polymers, biological molecules, and inorganic molecules, such as metal or nonmetal, metal oxide or metal sulphide and silica are frequently used. Coating gives stability to the SPNs and also make it more suitable for diverse applications (Bohara et al. 2016).

2 Advantages of Surface Functionalization 2.1 Inhibition of Aggregation and Colloidal Stability Aggregation and agglomeration are the serious problems associated with magnetic colloidal nanofluids which are responsible for altering physicochemical properties. Magnetism and thermodynamic factors are mainly responsible for the aggregation of SPN, and surface functionalization can prevent their self-association and increases colloidal stability (Bohara et al. 2014). Colloidal stability is also influenced by interaction with biological medium and magnetic field. Derjaguin–Landau–Verwey– Overbeek (DLVO) model describes that attractive and repulsive forces are responsible for the instability of SPNs in colloidal solution (Bohara et al. 2014; Derjaguin and Landau 1993; Verwey et al. 1999). Short-range isotropic attractions are induced by Van der Waals forces. To achieve good colloidal stability, one must balance electrostatic forces and steric repulsion (Schwertmann and Cornell 2008). Balance among attractive and repulsive forces helps in achieving colloidal stability.

2.2 Steric Stabilization SPNs are used for applications that demonstrate a high salt concentration. Covering of SPNs through ligand shells or surrounding them with an inorganic or polymeric matrix helps to attain steric stabilization. The type of polymers used for the functionalization of superparamagnetic materials decides the steric stabilization, but it is very difficult to predict the quantity of polymer required for the steric stabilization (Napper 1970; Fritz et al. 2002). The molecular weight of the polymer, changeable binding, polymer density, and solvent quality in comparison to the polymers confirm the repulsive potential of SPNs. When two sterically stabilized cores come close to each other, polymer brushes restrict their interaction and increasing the osmotic pressure between SPNs, finally, attaining colloidal stability (Fig. 1a).

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Fig. 1 a Steric stabilization and b electrostatic stabilization of SPNs (Bohara et al. 2016)

2.3 Electrostatic Stabilization The aggregation kinetics of colloidal suspensions are used to assess electrostatic stability. A change in salt concentration changes the aggregation kinetics. Stability factor ‘W’ shows the effectiveness of the potential barrier that prevents aggregation. The ratio of the number of collisions among particles to the total number of collisions ensuing the aggregation represents W. It is calculated by the equation W = kfast/k, where kfast is the rate constant suggestive of rapid aggregation and k is the aggregation rate constant at used salt concentration. The turbidimetric analysis gives an exact measurement of colloidal stability value, this also helps in predicting the critical concentration of salt leading to coagulation (Chen et al. 2012). At critical concentration, the double electrostatic layer is completely repressed and k¼ kfast becomes independent of the used salt concentration. Steric and electrostatic stabilizations are equally important to attain the colloidal stability of SPNs (Fig. 1b) (Chen et al. 2012, 2013; Bychkova et al. 2012; Chandra et al. 2011).

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2.4 Water Compatibilization SPNs are intended for biological applications that must be water compatible, as most of the biological reactions occur in an aqueous environment. Due to many advantages compared to aqueous synthesis, the synthesis of most of the SPNs is carried out in the organic phase at elevated temperatures. However, the resultant SPNs have a polar ligand that shows solubility only in organic solvents like chloroform, toluene, and hexane. Thus, it is essential to make SPNs water compatible, before their use in nanobioapplications. The terms ‘solubilization’ and ‘solution’ when used with respect to SPNs, represent both physical and chemical stability in colloidal suspensions which does not agglomerate, dissociate, or get chemically inert with the solvent or any dissolved gas with time. Water compatibilization of SPNs can be attained by conjugation of unsuspended SPNs with hydrophilic ligands. Water compatibilization can be done at the end or an intermediate stage during the procedure of functionalization. Functionalization with surfactant or exchange by surfactant of SPNs, make it water compatible (Fig. 2). Frequently used surfactant are amphiphilic molecules, which has both hydrophobic and hydrophilic part. The adsorption of the hydrophobic part forms a double-layer with the previous hydrocarbon chain, while the hydrophilic part faces outside of the SNPs, resulting in water solubility. Exchange is done through the exchange of the existing surfactant with a new bifunctional surfactant. It has two functional groups; one binds with the surface of SPNs and the other ends with a surfactant molecule. It is polar, thus SPNs can be dispersed in water or additionally functionalized (Bohara et al. 2016; Hao et al. 2010).

Fig. 2 Water compatibilization of MNP by means of a surfactant (Bohara et al. 2016)

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2.5 Magnetic Controllability Saturation magnetization defines the magnetic response of SPNs in the magnetic field. If saturation magnetization of SPNs remains high, then it can easily separate and transport to desired locations. Functionalization of SPNs helps in achieving high saturation magnetization and allows magnetic controllability through the gathering of particles, which is greater than separate core/shell particles (Nikam et al. 2015).

2.6 Protection and Stability of the Surface Several external factors are responsible for changing the magnetic properties of SPNs; forcing them unsuitable for specific applications. So, the surface of the SPNs must be protected and stabilized from the effect of external factors, this can be accomplished via surface functionalization. Functionalization should prevent SPNs’ reaction with constituents of biological media. Coating materials that give oxygenresistant coatings prove to be the best material and give the best shield to SPNs (Bychkova et al. 2012). SPNs used for biological applications should be stable with respect to their function and coatings. Different coating materials require different methods of preparation for the coating. For example, polymeric coatings can be stabilized through: (i) three-dimensional polymerization of monomeric units on SPNs (Wang et al. 2006), (ii) creation of crosslinks (Du et al. 2011), and (iii) construction of a polymeric layer through chemical bond formation between functional groups and SPNs (Bin Na et al. 2007; Cheng et al. 2009).

2.7 Biocompatibility Increased surface area, high chemical reactivity, production of reactive oxygen species, and penetrating cells and tissues are responsible for the toxicity of nanoparticulate SPNs. Basically, toxicity is present in bound or fixed form as compared to free particles. The hydrophilicity, size, and surface charge of SPNs are key aspects deciding their biocompatibility. The mass and charge of the coating material also help in increasing the biocompatibility followed a subsequent applicability in biological applications. Negatively charged coatings material display increased biocompatibility, while positively charged SPNs show improved cooperation with biosystems. SPNs intended for biological applications greatly vary with respect to their surface charge, so that they can interact with biological molecules (McNeil 2009; Xu et al. 2003). Functionalization SPNs should have one or more functional groups free to bind with biologically active substances for precise bioapplications.

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3 Functionalization Strategies 3.1 In Situ Surface Functionalization In this method, synthesis of SPNs and their surface functionalizations are simultaneously performed. The reaction mixture containing both precursors for the synthesis of SPNs, as well as the coating material, should be instantaneously mixed. Here, the coating procedure proceeds as soon as nucleation starts, thus avoiding overgrowth of the particles. This method helps in achieving lower particle size and a fine particle size distribution. Modifying the group that may react with the particle surface could be a possible limitation of this method. For in situ surface functionalization, carboxylates, thiol, and phosphonates have normally been employed.

3.2 Post Synthesis Surface Functionalization In this method, surface functionalization is carried out after the synthesis of MNPs, thus there are two steps in this method; the first is synthesis and the second one is surface functionalization. In this method, bifunctional groups are used, where the binding of the chelating group is initially fevered, followed by alteration of the group of the coupling site to the final functional group. Several coupling agents are commercially available. This method gives high surface coverage as core–shell structure confirms a strong binding of the functional groups. The functional group should hold a strong affinity for the surfaces of the SPN (Bohara et al. 2016).

4 Types of Surface Functionalized Structures Many surface functionalization strategies and their subsequent applications are dedicated to fabricating four types of surfaces functionalized SPNs, such as core–shell structure, matrix dispersed structure, Janus-type heterostructures, and shell–core–shell structure (Fig. 3).

4.1 Core–shell Structure In this type, the SPN is encapsulated with organic or inorganic coating materials such that the resultant unit exhibit excellent stability and biocompatibility. When SPN is not positioned at the epicentre of the coating; then this type of assembly is called a yolk structure. In the inverse, core–shell type, SPNs will coat the surface of nonmagnetic materials (Fig. 3). In this, SPN not only retains its particular semiconducting

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Fig. 3 Typical morphologies of SPN nanocomposites. Blue spheres represent SNPs, and another colour represents nonmagnetic material and matrix (Wei et al. 2015).

and magnetic properties, but also exhibits some additional properties that are shown by the coating element. Core–shell bifunctional CdSe@Fe2 O3 NPs have claimed applicability in biosensing and biomedical research (Zhan and Zhang 2011).

4.2 Matrix-Dispersed Structure SPNs can be distributed in a matrix to avoid aggregation into a large ferromagnet. Matrix-dispersed SPNs can be prepared in a number of different states, e.g., distributed in an amorphous matrix, mesoscale particles, grafted on threedimensional structures of SPNs (Behrens 2011).

4.3 Janus Structure In this type, one side has SPNs and the other side holds functional materials. The anisotropic surface of different chemical groups reveals exciting applications. For

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example, magnetic γ-Fe2 O3 ||SiO2 Janus particles fabricated by the flame synthesis method have high uniformity and exceptional aqueous dispersibility (Zhao and Gao 2009).

4.4 Shell–Core–Shell Structure This kind of structure in which SPNs lie between the two functional materials, which may be the same or different. Most applications necessitate that SPNs be surrounded by nonmagnetic layers to prevent self-aggregation and subsequent deposition at the bottom. For example, luminescent layers, SPNs, and biocompatible polymer layers are united into nanocomposite, which can be operated via an external magnetic field and real time optical visualization possible simultaneously (Sperling and Parak 2010).

5 Mechanism of Functionalization of SPNs 5.1 Ligand Addition Ligand attachment to the outer surface of SPN without elimination of any previouslyexisting ligands is identified as ligand addition (Fig. 4a). Basically, four methods are used for ligand addition: (1) linking of ligands to SPN (without capping agent), (2) indirect ligand addition; SPN coated with inorganic material first and then adsorption of a ligand species on it, (3) Intercalation of hydrophobic species into the hydrocarbon shell of SPN, and (4) covalent bond formation between the pre-existing and new ligand.

5.2 Ligand Exchange Ligand exchange on SPN typically includes a procedure where the original hydrophobic ligands are substituted by other stronger hydrophilic ligands (Patil et al. 2016) (Fig. 4b). Due to ligand exchange, SPN can form an aqueous phase instead of an organic phase. Different type of hydrophilic ligands has been available, which can replace natural ligand present on SPN surface, subsequent formation of an aqueous solution.

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Fig. 4 Surface functionalization strategies of SPNs a addition, b exchange, and c encapsulation (Bohara et al. 2016)

5.3 Encapsulation It is attained by adding the hydrophobic end of materials with the primary ligand on the surface of the SPN, leaving the hydrophilic portion near the solution (Fig. 4c). The resultant composite becomes water compatible as it contains head groups inside the hydrophilic part of the materials. Additionally, the functional group can be helpful in biomodification of SPNs.

6 Materials Used for the Functionalization of SPNs The specific application requires unique properties of SPN. To impart these special properties, SPN functionalizes with different types of materials, and allow specific targeting, stabilization, and identification of biochemical species. These materials effectively tailor the surface of SPN for specific applications. Different kinds of materials with different strategies can be utilized for functionalization (Fig. 5).

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Fig. 5 Materials intended for the functionalization of SPNs (Bohara et al. 2016)

6.1 Organic Compounds Functionalization with organic material systems can tailor SPN for different mechanical, magnetic, electrical, and optical properties. Organic coating also provides good biocompatibility and biodegradability to SPNs. Organic compounds can be used to functionalize SPN through in situ or post-synthesis coating. They provide reactive groups, e.g., aldehyde, carboxyl, hydroxyl, and amino groups, which link active biomolecules, like an antibody, enzymes, DNA or protein. Magnetic recording, magnetic resonance imaging (MRI), electromagnetic shielding, drug targeting, magnetic separation, etc., are the specific applications where organic molecules are utilized for the coating of SPNs. The following types of organic molecules are of particular attention due to their widespread properties and applicabilities (Chu et al. 2015).

6.1.1

Monomers

(a) Citrates Citrate molecules have carboxylate functionalities which give colloidal stabilization to SPNs. Adsorption of citrate molecules depends upon the shape and size of the SPN that exposes at minimum one carboxylic acid towards the solvent. These exposed

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carboxylic molecules offer hydrophobicity to the SPN as it accumulates sufficient negative charges (Sahoo et al. 2005; Laurent et al. 2008).

(b) Phosphates Surface coatings of SPN such as hexadecyl phosphonic acid, dodecyl phosphonic acid, lauric acid, dihexadecyl phosphate, and oleic acid, have been reported by Sahoo et al. (Bohara et al. 2014) Thermodynamically stable dispersion of SPN is obtained by coating with alkyl phosphonates and phosphates. They form a quasi-bilayer assembly, where the primary layer can be tightly linked to the surface of the particles (Bohara et al. 2014). Alkane phosphonic acids and alkane sulfonic are powerful surfactants, signifying efficient ligands binding on the surface of SPN and stabilization of particle dispersion (Portet et al. 2001; Kreller et al. 2003; Czakler et al. 2014). Phosphonate ions attach to SPN through one or two oxygen atoms. Thus, forming bidentate complexes with SPN (Tejedor-Tejedor and Anderson 1990; Persson et al. 1996; Yee et al. 1999). Biocompatibility phosphonates and phosphate ligands leaves SPNs extremely useful for nano-bioapplications (Roberts et al. 2000).

6.1.2

Small Molecules

Simple preparation, conjugation chemistry, and effective stabilization are major advantageous sides of functionalization with small molecules. Since nanobioapplications generally require a small hydrodynamic radius, functionalization with these is beneficial, as they offer physical barrier protection and also a smaller hydrodynamic radius (Gunay et al. 2012). They also provide functional groups for linking a variety of molecules for biomedical applications (Longmire et al. 2008). (i) Amines and aminosilanes Amines (short chain) and amino silanes are often applied as stabilizers in the synthesis of several types of functionalized SPNs which are extremely water stable and effective for nano-bioapplications. Amine functionalized Fe3 O4 prepared by the thermal decomposition of Fe-chloride precursors in an ethylene glycol medium in the presence of sodium acetate and ethylenediamine was envisaged for the same in the past (Barick et al. 2009a, 2009b). Barick et al. reported several amino acids and peptides containing many amine molecules are the finest stabilizers and can be used in functionalization of SPNs (Lai et al. 2009; Xie et al. 2008). (ii) Thiols Thiol compounds have a high binding affinity to SPNs. The organosulfur compounds like 2,3-meso dimercaptosuccinic acid (DMSA) are commonly used as a stabilizer as they have two thiol and two carboxylic groups in them. The Ligand exchange process is followed for the synthesis of DMSA-stabilized aqueous colloidal SPNs, in this way,

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DMSA molecules are introduced on hydrophobic SPNs (Barick et al. 2009a). DMSAstabilized aqueous SPNs were used for targeted drug delivery. Thiol-functionalized SPNs have extreme stability under physiological pH, which is a necessary condition for nano-bioapplications (Maurizi et al. 2009). The stability of thiols can be enhanced when DMSA protects the polyethylene glycol (PEG) chains on the surface of SPNs.

6.1.3

Macromolecules

(A) Polymers Polymer-functionalized SPNs are receiving more attention from the scientific community as polymer coatings give exceptional colloidal stability. Polymer’s coating increases repulsive forces, balances the magnetic field, and Van der Waals attractive forces acting on SPNs. The properties of SPNs can be tailored by selecting the appropriate polymers and/or reaction conditions. Ligand exchange and ligand adsorption procedures are followed during the functionalization of SPNs with polymers (Patil et al. 2014). Polymers are complex in structure, where exchange or absorption usually requires active terminal groups. So, alkoxysilanes, bisphosphonates, DMSA, etc., can be applied to encourage the polymer coatings on SPNs (Zhang et al. 2007; Amstad et al. 2009). Polymers functionalized SPNs sidestep agglomeration and offer diverse surface properties, which make them highly potential for nano-bioapplications. The working of polymerfunctionalized SNPs depends upon molecular weight, properties of terminal groups, and conformation of polymers (Wu et al. 2008). Both in situ and post-synthesis coating approaches are utilized for SPNs. In an in situ approach, mini/microemulsion and sol–gel process is used (Oh and Park 2011; Zhou et al. 2012a). Here, either core–shell structure or matrix-dispersed structure is formed (Darbandi et al. 2012; El-Sherif et al. 2010). The major disadvantage of in situ approach is that it is ineffective in preserving colloidal stability and the thickness of the shell. In post-synthesis functionalization, firstly, monodispersed SPNs were synthesized with definite shapes, compositions and then functionalized through self-assembly, one-pot route, or heterogeneous polymerization methods (inverse mini/emulsion and dispersion) (Pimpha et al. 2012). The physical adsorption and functional groups attachment on SPNs result in complex SPN such as core–shell structure. Covalent bonding is frequently used in functionalization where the cross-linking is done by using an alkyl chain or carboxylic acid containing thiol and hydrogen bonding (Lu et al. 2010). There are two types of polymer coating materials: natural and synthetic polymers.

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(a) Natural Polymers (i) Dextran Naturally occurring biocompatible polysaccharide polymer. It is made up of R–D glucopyranosyl units having variable chain lengths and branching (Amstad et al. 2009; Gamarra et al. 2005; Lee et al. 2002). Its amination permits facile conjugation between complementary chemical groups (Zhao and Gao 2009). Terminal glucose molecules of dextran polymer are reduced for the coating on SPN and the resultant functionalized material shows changed particle size, magnetic properties, and coating stability. Reduced dextran coating gives more stable particles. The existence of the polymer confines the particle size by preventing aggregation (Lewin et al. 2000). Dextran polymers are attached via chelation or hydrogen bonding with the surface of SPNs. A large number of hydroxyl groups on dextran provide efficient binding (Pardoe et al. 2001) and specific heating or dilution facilitates desorption. Epichlorohydrin prevents the desorption of dextran (Fournier et al. 1995). (ii) Chitosan It is alkaline, biocompatible, hydrophilic, and biodegradable polymer, chitosan encapsulated SPNs has received a great attention for nano-bioapplications (Bhattarai et al. 2007; Kim et al. 2007; Sipos, 2003; Zhi et al. 2006). Chitosan-coated microspheres have shown strong MR image contrast, like ferrofluid in-vitro (Lee et al. 2005). Ferrofluids containing oleic acid-coated SPN can be spread in the chitosan, to coat with chitosan. (Hee Kim et al. 2005). (b) Synthetic Polymers (i) Polyethylene Glycol (PEG) Due to considerable hydrophilicity, flexibility, and biocompatibility of polyethylene glycol (PEG), SPNs coated with PEG have good chemical stability and solubility in aqueous medium and physiological medium. This is the requirement for the biomedical applications of nanomaterials, and by changing molecular weight, variation in the thickness of the coating can be obtained (Sandiford et al. 2013; Xie et al. 2007). PEG having high molecular weight and branching structure can give better dispersion and high stability in biological mediums. Post-synthesis functionalization method is applied for fabricating PEG-coated functionalized SPNs. Several ways of polymer coating have been developed in the past to prepare small and ultra-small particles. Polymerized polyethylene glycosylated bilayers are coated on SPNs to get novel properties (Kang et al. 2002). Extensive investigation showed that stability of SPN in biological environments is useful for vivo use also (Fang et al. 2009). Several approaches are used to prepare PEG-coated SPNs for biomedical applications (El-Sherif et al. 2010; Pimpha et al. 2012; Lu et al. 2010; Cole et al. 2011). Surface functionalization of SPN with PEG has significantly attenuated the

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Fig. 6 Fabrication of PEG/PVP-coated SPN nanomaterial (Jarzyna et al. 2009)

macrophages phagocytosis by reducing cytochrome C (Amara and Margel 2012; Douadi-Masrouki et al. 2010). Fig. 6 represents the fabrication of PEG/PVP-coated SPN, wherein the resultant functionalized material has hydrodynamic size (≤40 nm), neutral or positive zeta potential, and greater stability than PEG alone coating (Jarzyna et al. 2009). (ii) Polyvinyl Alcohol (PVA) A commonly used water-soluble and biocompatible polymer is polyvinyl alcohol (PVA). Functionalization of SPN with PVA avoids the agglomeration, resulting in monodispersed SNPs (Sairam et al. 2006; Sch¨opf et al. 2005). Particle size distribution and stability of PVA-coated SPN varies with fluctuating iron/polymer ratios (Chastellian and Hofman 2004). (iii) Alginate An electrolytic polysaccharide with many carboxyl groups interacting with iron ions of SPN consequences in electrostatic repulsion. Electrostatic repulsion makes a stable assembly of SPN-alginate (Finotelli et al. 2004; Kroll et al. 1996; Llanes et al. 2000; Nishio et al. 2004). Surface coating of alginate on SPN has the following three steps: (a) gelation of alginate and ferrous ions, (b) alkaline treatment of alginate for in situ precipitation of ferrous hydroxide, and (c) oxidation of ferrous hydroxide with an oxidizing molecule. Currently, a new two-step co-precipitation method is being used for manipulating hydrodynamic diameter, size, and magnetic properties (Ma et al. 2008; Morales et al. 2008).

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B. Liposomes Biocompatible and amphipathic forming bilayer with superparamagnetic material. Liposome encapsulation is used for targeted delivery (Kim et al. 2011; Mulder et al. 2006). Liposomes are more advantageous as they can encapsulate several types of biomolecules (proteins, DNA, etc.) and their complexes with SPN as one unit (Kim et al. 2013). While considering organic compounds for functionalization, it is essential that head groups of ligands have a high affinity for the SPNs (Thorat et al. 2013). During the coating of organic compounds with SPN factors like stability, control of the shape, surface structure, biocompatibility, and magnetic properties are still the subject of concern; responsible for the restrictive use of such SPN for nano-bioapplications (Zedan et al. 2013).

6.2 Inorganic Compounds Surface functionalization of SPN via an inorganic layer or shell is of great attention. Inorganic materials hold a number of special properties like strong optical absorption and photoluminescence (e.g., CdSe or CdTe), high electron density (e.g., gold (Au) and silver (Ag)), or phosphorescence (e.g., Y2 O3 ), or magnetic moment (e.g., manganese or cobalt oxide NPs) (Wang et al. 2013; Gowd et al. 2013; Ye et al. 2012). Surface functionalization of SPN with inorganic compounds increases stability and semiconductor efficiency, information storage, catalysis, optoelectronics, optical bioimaging, biological labeling, and many more (Wei et al. 2015). The inorganic materials such as silica (SiO2 ), metals, nonmetals, metal oxides, and sulphides are used for the functionalization of SPN. Silica, Au, and Ag are being widely applied for surface modification of SPNs, resulting in core–shell structures, with various biological molecules and drugs through suitable functional groups (Wu et al. 2008).

6.2.1

Silica

Silica is an imperative composite material for fundamental studies and bioapplication, widely used for SPN functionalization. Silica-coated SPNs result in core–shell structure. Silica coating can enhance the dispersion of SPN in the solution as it lowers the magnetic dipolar movement between them. Additionally, it enhances stability and shield against acidic environments. Presence of abundant silanol groups on the silica layer, silica-coated SPN could be easily activated with many functional molecules. Silica-coated SPNs are colloidally stable, photo-stable, water-soluble, don’t show aggregation, chemically stable, and protect from toxicity (Erathodiyil and Ying 2011). Superparamagnetic silica nanospheres can be synthesized with three different methods; the first is the Stober process, in which silica is designed in situ through the

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hydrolysis and condensation of a sol–gel precursor (Im et al. 2005). Generally, SPN is homogeneously distributed in alcohol, after silane is mixed, and lastly, aqueous solution of water or ammonia is dropped into the mixed solution. Tetraethoxysilane (TEOS), octadecyltrimethoxy silane and vinyltriethoxysilane (VTEOS) are the most frequently used silanes, which bind through OH groups to the surface of SPN (Zhu et al. 2010; Shao et al. 2013; Cao et al. 2013). The final particle size of silica colloids is decided by the concentration of SPNs and the type of solvent used. Lower concertation of SPNs is responsible for the synthesis of larger colloids. Another approach is more efficient and depended on the deposition of silica from the silicic acid solution. This approach occupies a larger portion of the SPN surfaces than the TEOS method. It is the easiest process and particle size can easily manipulate by varying the ratio of SiO2 /SPN or repeating the coating method. The third is an emulsion procedure, in which silica coating is controlled using micelles or inverse micelles. This process needs the separation of the core–shell SPNs from a number of surfactants linked with the emulsion system. Yang et al. synthesized monodispersed silica-coated iron oxide SPNs and successfully entrap the biomacromolecules inside the pores of the nanoparticles (Yang et al. 2004). Ding et al. coated the Fe3 O4 nanoparticles by reverse microemulsion procedure to get silica-coated superparamagnetic core–shell material (Fig. 7). During the formation of core–shell nanoparticles with different shell thicknesses, different sizes of Fe3 O4 nanoparticles are used to avoid the development of core-free silica particles. It is also found that a thicker silica shell avoids the formation of core-free silica particles. Thicker silica shells can be attained by increasing the amount of either TEOS via the equivalently fractionated drops or ammonia with a decreasing one-off TEOS (Ding et al. 2012). It is beneficial as we get uniform silica shells with controlled thickness.

6.2.2

Metal-Coating

The amalgamation of metallic nanoparticles with SPNs has grabbed the attention of materials scientists as their combined physicochemical properties encourage catalysis (Qiao et al. 2011), biotechnology (Thandavan et al. 2011), and biomedicine properties (Barnett et al. 2012; Mahmoudi et al. 2011a). Mostly, monodispersed iron oxide/metal nanostructures in their core–shell, core–satellite, and dumbbell structures display binary/polynary properties. Furthermore, these structures can be improved with dissimilar charges, functional groups to enhance stability and compatibility (Yallapu et al. 2011; Guo et al. 2009). The protective layer can be prepared by using inert metallic elements. The biofunctionalization process is more easily done than the bare SPN surface. Different procedures are available for the functionalization of SPN with metals. One of the most effective and simple procedures of functionalization is the sequential growth of metallic components (e.g., Ag or Au) on a superparamagnetic core. The core–shells, dumbbells, and core–satellites are designed by microemulsion and thermal decomposition methods (Kirui et al. 2010; Chiang and Chen 2009). It is very hard to directly coat the SPN with metal in thermal

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Fig. 7 a La Mer-like diagram; hydrolyzed TEOS (monomers) concentration against time on homogeneous nucleation and heterogeneous nucleation, b the existence of Fe3 O4 @SiO2 core/shell NPs and SiO2 NPs in the reaction production when C > Chomo at some moment, and c only the existence of Fe3 O4 @SiO2 core/shell NPs in the reaction production when C < Chomo at any moment (Ding et al. 2012)

decomposition, because of the unlike nature of the two surfaces and lattice (Gu et al. 2004). Au, one of the most important noble metals, is used for coatings of SPN which give extraordinary advantages if coated along with sulphur. Due to the chemical inertness of Au, Au-coated SPNs are completely stable. Also, the magnetic properties of the SPNs remain stable with the amount of Au addition. Core–shell structure Fe3 O4/ Au fabricated by reducing HAuCl4 on Fe3 O4 nanoparticle was described by Xu et al. (2007). Lin et al. produced core–shell Fe/Au nanoparticles with the reverse-micelle method (Lin et al. 2001). Ramasamy et al. synthesized Au nanorods conjugated with a superparamagnetic nanocomposite (Ramasamy et al. 2014). Silver-coating makes SPN germicidal, as Ag has very good antibacterial properties (Gong et al. 2007). Core–shell structures and heterostructures both can be obtained by a silver coating. Chen et al. prepared Fe3 O4 @C@Ag hybrid nanocomposite (Jiang et al. 2008; Tang et al. 2006). It shows properties like drug release, magnetic resonance imaging, and two-photon fluorescence (TPF) imaging, due to the presence of carbon and Ag on the surface, it acts as a multifunctional system for medical diagnosis and therapy. Some rare-earth elements functionalized SPN forms core–shell structures. For example, a Fe3 O4 @NaLuF4 :Yb,Er/Tm core–shell nanostructure with multifunctional properties (Zhu et al. 2012). Buck et al. constructed hybrid nanoparticle architectures include, M–Pt–Fe3 O4 (M = Au, Ag, Ni, Pd) heterotrimers, Mx S– Au–PtFe3 O4 (M = Pb, Cu) heterotetramers and higher-order oligomers constructed

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Fig. 8 Stepwise construction of M–Pt–Fe3 O4 heterotrimers (M = Ag, Au, Ni, Pd). Multistep synthesis of M–Pt–Fe3 O4 heterotrimers a TEM images of Pt nanoparticle seeds b Pt– Fe3 O4 heterodimers c and Au–Pt–Fe3 O4 d Ag–Pt–Fe3 O4 e Ni–Pt–Fe3 O4 f and Pd–Pt–Fe3 O4 g heterotrimers. All scale bars are 25 nm. h Photographs of a vial that contains Au–Pt–Fe3 O4 heterotrimers in hexane (left), which responds to an external Nd–Fe–B magnet, the same vial with Au–Pt–Fe3 O4 heterotrimers in a larger volume of hexanes (middle) and the same vial after precipitation of the heterotrimers with ethanol (right). The precipitated heterotrimers collect next to the external magnet (Goon et al. 2009)

on the heterotrimeric Au–Pt–Fe3 O4 building block (Fig. 8). This synthetic framework conceptually mimics the total-synthesis approach used by chemists to construct complex organic molecules (Goon et al. 2009).

6.2.3

Metal Oxides/Sulfides

Metal oxides or metal sulphides functionalized SPNs possess exceptional physical or chemical properties. Most frequently used compounds for functionalize SPNs include ZnO (Wu et al. 2012), WO3 (Xi et al. 2011), SnO2 (Wu et al. 2011; Zhang et al. 2013), Cu2 O (Li et al. 2011), CdS (Joseph et al. 2012; Zhou et al. 2012b; Shi et al. 2012), Bi2 S3 (Luo et al. 2012), ZnS (Yu et al. 2009; Liu et al. 2013a), PbS (Zhou et al. 2011), etc. Spindle-like IONP@SnO2 , IONP@TiO2, and IONP@ZnO nanocomposites were functionalized by diverse wet-chemical methods; the resultant

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Fig. 9 Synthesis and modification of Fe3 O4 –TaOx core–shell nanocomposite, and application for simultaneous MRI and CT (Lee et al. 2012)

structure exhibited higher photocatalytic activity for organic dyes because of synergistic effect among narrow and wide bandgap semiconductors and effective electron– hole separation at the interfaces SPN (Zhang et al. 2013). Lee et al. synthesized a sol–gel reaction of tantalum (V) ethoxide in a microemulsion comprising superparamagnetic Fe3 O4 nanoparticles that were used to synthesize multifunctional Fe3 O4 / TaOx core–shell nanoparticles. The resultant nanocomposites were biocompatible with prolonged circulation time in vivo. When these functionalized nanocomposites were intravenously injected, the tumour-associated vessel was visualized by computed tomography (CT), and MRI (Fig. 9) (Lee et al. 2012).

6.2.4

Carbon

Carbon functionalized SPNs have generated massive research activities as they have excellent thermal and chemical stability, and inherent high electrical conductivity. It provides an effective oxidation barrier and stops the corrosion of SPNs. Hydrophilic carbon layering on superparamagnetic iron oxide nanoparticle cores gives improved stability and dispersibility than simple nanoparticles (Bae et al. 2012). Different approaches are being used for producing IONP@C core–shell nanostructures. Most frequently, a three-step process is used; initially, SPNs are synthesized in different

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ways, after that, the polymer is coated by means of the polymerization process, lastly, IONP@C composite formation is concluded through annealing treatment. Scientists worldwide are paying significant attention to the synthesis of Fe3 O4 / graphene hybrid material, due to its inclusive use in ion removal, lithium-ion batteries, catalysts sensors, etc. (Liang et al. 2011). The unusual properties of Fe3 O4 /graphene hybrids are because of their large surface-to-volume ratio, high conductivity, increased magnetism, and environmentally benign nature (Li et al. 2012). Liu et al. manufactured a superparamagnetic graphene oxide–Fe3 O4 hybrid composite (rGO–Fe3 O4 ) by solvothermal reaction, where the reaction mixture contains graphene oxide (GO), Fe(acac)3 in ethylenediamine (EDA), and water (Liu et al. 2013b). Fe3 O4 /graphene hybrid composite has found application in targeted drug delivery and MRI (Fan et al. 2013). Chen et al. functionalized the nanocomposites of amino dextran-coated Fe3 O4 NPs with GO, where the resultant structure was effective for cellular MRI. Figure 10 represents the vivo study of internalization of Fe3 O4 –GO nanocomposites, it did not disturb the normal cellular viability and proliferation. Compared to the bare Fe3 O4 nanoparticles, the Fe3 O4 –GO composites display improved T2 weighted MRI contrast, because Fe3 O4 NPs formed aggregates on GO sheets, caused enhanced T2 relaxivity (Chen et al. 2011).

Fig. 10 Preparation of Fe3 O4 –GO composites for cellular MRI (Chen et al. 2011)

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7 Bioconjugation Biocompatibility and toxicity of SPNs are important factors to take into consideration while using them for a specific biological function. Biocompatibility and toxicity of the material are decided by the nature of the magnetic component and the ultimate size of the composite. Ideal SPNs should have very good magnetization so that they can be controlled with an external magnetic field until they reached near to the targeted diseased tissue through blood (Mahmoudi et al. 2011b). For effective use for biomedical applications, SPNs should have extended blood retention time, low toxicity, and biodegradability. Conjugation with biological molecules of interest (e.g., peptides, proteins, and nucleic acid) is essential for precise biomedical applications. The choice of conjugation protocol is influenced by several factors like shape, size, structure, surface molecules, basic material of SPN, and surface ligands and their functional groups. Different biomedical applications are generally in need of diverse biomolecule to SPN ratios. Bioconjugate can be done with monovalency (Single binding) or higher valencies (multiple binding and avidity). Enzyme, protein, and antibody active sites should have free contact with the outer environment for activity. Nonspecific binding or electrostatic interactions may reveal a mixed attachment and destroy the activity in the final conjugate material. There must be some rigid distance between SPN and biomolecules for some nano-bioapplications, because, sensing arrangements operate on Forster resonance energies. For nearly a few applications, linkage should be permanent, whereas in others, a transient linkage is more necessary, as in drug delivery. In all cases, binding chemistry should not modify the properties of each molecule used (Medintz 2006).

7.1 Overview of Different Bioconjugation Strategies Different chemical methods have been applied for the conjugation of biological molecules with SPNs for diverse biomedical applications. These approaches are grouped into two main types, physical and covalent interaction (Fig. 11). The type of chemical interaction used for the functionalization of SPNs depends upon chemical properties, functional groups found on the surface of SPN, linking biomolecules, and end applications for which it is programmed.

7.1.1

Physical Interactions

Physical interactions are speedy, highly productive, and there is no need for intermediate alteration steps. Physical interaction contains affinity, electrostatic, and hydrophilic/hydrophobic interactions (Fig. 12). Most simple and extensively used

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Fig. 11 Bioconjugation strategies used for SPNs (Bohara et al. 2016)

bioconjugation approach is electrostatic attachment (Fig. 12a), where the attachment is due to the opposite charge on SPN and biomolecule. For example, attachment between negatively charged plasmid DNA and positively charged SPN; but pH and ionic strength need to be wisely adjusted (Sapsford et al. 2013). The electrostatic attraction method was used to prepare lignin-based adsorbents where a protic ionic liquid is applied for the synthesis of polyphenol lignin and acetylation method for lignin nano-microspheres. Self-assembly of lignin microspheres with γ-Fe2 O3 nanoparticles was done under ultrasonic conditions to synthesize polyphenol ligninbased nano-microspheres. This material has superparamagnetic properties which increase the adsorption site for dyes and magnetic properties help in easy recovery (Zhao et al. 2022) (Fig. 13). Hydrophobic/hydrophilic interactions (Fig. 12b) are beneficial when hydrophobic drugs are attached over SPN. Here, firstly SPNs are masked with hydrophobic layers for effective adsorption of hydrophobic drugs and finally triggered to release the drug intracellularly (Liu et al. 2011). The most effective physical interaction is affinity interaction, where the linkage formed is extremely stable and strong of all non-covalent linkages (Fig. 12c). In this case, the surface of SPN is improved with streptavidin, which explicitly binds to biotinylated molecules. Change in environmental conditions like pH, hydrophilicity or salinity does not affect the affinity of binding (Gunn et al. 2008).

7.1.2

Covalent Chemistry

Covalent bonds of covalent chemistry are formed between functional groups, such as amino, thiol, and carboxylic acid on the surface of SPNs. These functional groups cross-linked with diverse biological molecules either directly or by coupling strategies. Some of the coupling strategies are as follows:

(A) Direct SPN Conjugation In this method, the surface of SPNs is functionalized with diverse functional groups, like amine, aldehyde, and sulfhydryl which conjugate with different biological

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Fig. 12 a Electrostatic interaction, b hydrophobic interaction, and c affinity interaction (Bohara et al. 2016)

Fig. 13 Synthetic route showing the preparation of magnetic lignin microspheres (Zhao et al. 2022)

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Fig. 14 Direct conjugating strategies (Bohara et al. 2016)

molecules [Fig. 14(a–c)]. This strategy is more appropriate for small biomolecules (dyes, antibodies, and small peptides) but the efficiency of these chemistries is greatly varied. Direct conjugation methods are likely to intercalate or cross-linking, so restricting their use for biomolecule attachment (Gunn et al. 2008; Sperling et al. 2010).

(B) Carbodiimide Coupling Reaction This is the most beneficial method for bioconjugation as it does not need a lengthy linker molecule and also maintains a low hydrodynamic radius of SPN. The most commonly used coupling agent is 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC). In the ‘carbodiimide coupling’ strategy, carboxylic acids are covalently linked to primary amines through amide bonds. This method is suitable for coupling several proteins (e.g., enzymes and antibodies), amine-terminated nucleic acid, and small molecules with amine groups (Mohapatra et al. 2011; Jordan et al. 2011). Coupling efficacy can be enhanced by adding stabilizing agents like n-hydroxysuccinimide (NHS) or sulfo NHS (Bogdanov et al. 1988) (Fig. 15). This strategy has been useful

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Fig. 15 Reaction outline for EDC–NHS cross-linking with amine functionalized magnetic nanoparticles (Bohara et al. 2016)

for coupling of enzymes to MNPs, with preservation of maximum enzymatic activity (Kumar et al. 2014).

(C) Maleimide Coupling A maleimide is applied to a couple of primary amines to thiol groups in proteins (Erathodiyil and Ying 2011). The most frequently used maleimide-derived coupling reagent is sulfo-SMCC (SSCC-sulfosuccinimidyl-4 (maleimidomethyl) cyclohexane-1-carboxylate). This method is useful to conjugate biomolecules, such as DNA and proteins over the surface of paramagnetic nanomaterial (Fig. 16) (Dubertret et al. 2002).

(D) Click Chemistry This reaction is fast and efficient, carried out in mild reaction conditions, neutral pH, and in an aqueous environment to create biocompatible and water-soluble linkages (electron configuration similar to amide bonds) (White et al. 2007). The reaction mostly includes the coupling of an alkyne to an azide, resulting in 1,2,3-triazole ring formation in the presence of catalysis Cu(I). This technique offers several advantages of which major three are as follows; firstly specificity, azide and alkyne reactive groups are highly compatible with each other and do not react with other functional groups. Second bonds are highly stable, so unaffected by environmental conditions. Thirdly, the rigidity of the bond so preserves the conformation of reacting molecules

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Fig. 16 Reaction schemes for SMCC-mediated conjugation of protein on SPNs (Bohara et al. 2016)

at SPN surfaces and avoids cross-interactions. Despite of a number of advantages, this method has some disadvantages such as often lengthy procedure, low-yield, Cu-based catalysts create problems in vivo due to improper purification before use. Unnecessary Cu consumption has been related to a variety of disorders (Bohara et al. 2016; Li and Binder 2011).

8 Conclusions Studies showed that bare or uncoated superparamagnetic nanomaterials have a tendency to aggregate in suspension which weakens their properties in applications where used. So, for diverse applications of superparamagnetic nanomaterials, properties such as magnetic-electrical, magneto-optical, magnetic-thermal are tailored to acheive biocompatibility, chemical stability, and water solubility. Surface functionalization is necessary to form various complex structures of SPNs like core–shell structure, matrix dispersed structure, Janus-type heterostructure, and shell–core–shell structure. In this milieu, this chapter gives up-to-date information on an array of shell materials with inorganic and organic compositions followed by the most frequently used functionalization procedures to conjugate with biological molecules for their applications in nanomedicine. Coating with silica gives biocompatibility to SPNs, whereas coating with mesoporous silica can intensely increase the surface area. Coating with organic polymer can be beneficial, however, it is toxic due to the presence of organic components. Future work is needed on the toxicity and degradability of naked or

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surface functionalized SPNs. It is synthesized through green chemistry for minimizing environmental pollution. Fruitful growth in this area will result in the development of several scientific technologies or industrial applications and also enhance the quality of life.

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Ferrite- and Non-ferrite-Based Superparamagnetic Materials Ashwini B. Salunkhe, Maithili V. Londhe, and Vishwajeet M. Khot

Abstract Magnetic nanoparticles due to their superparamagnetic behaviour gain a lot of attraction in various fields such as biomedical, industrial and pharmaceutical fields. Superparamagnetic nanoparticles have the potential to generate localized heat when exposed to an alternative magnetic field, resulting in thermal ablation of cancer cells and in combination with chemotherapy. Superparamagnetic iron oxide nanoparticles exhibit a higher specific absorption rate (SAR[W/g]) which is a clinically accepted term for cancer treatments. Magnetic nanoparticles, especially single-domain-based superparamagnetic nanoparticles, are utilized for cancer hyperthermia therapy due to their unique properties such as unique magnetic properties, high biocompatibility and better chemical stability. In this chapter, we discussed superparamagnetic, its multidomain and single-domain systems, synthesis of superparamagnetic materials, biocompatible superparamagnetic iron oxide nanoparticles, ferrites-based superparamagnetic materials (X2+ Fe2 3+ O4 2− , X = Fe2+ , Mg2+ , Ni2+ ) their structure and non-ferrite-based superparamagnetic nanoparticles(Ax A' 1-x BO3 (A = La, Nd, Pr, etc. A’ = Ca or Sr) and their structure are discussed. This chapter explains the properties of superparamagnetic materials for cancer hyperthermia which are applicable in biomedical field such as MRI, targeted drug delivery and magnetic fluid hyperthermia also for environmental applications. Keywords Superparamagnetism · Ferrites and non-ferrites · Cancer · Biomedical applications

A. B. Salunkhe (B) · M. V. Londhe Department of Physics, Rajaram College, Kolhapur, Maharashtra, India e-mail: [email protected] V. M. Khot Department of Medical Physics, Center for Interdisciplinary Research, D. Y. Patil Education Society (Deemed to Be University), Kolhapur, Maharashtra, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_3

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1 Introduction Nanoparticles become an interesting topic for researchers as they exhibit a number of special properties relative to bulk material. Nanoparticles are the bridge between bulk material and atomic or molecular structures. Generally, bulk materials have constant physical properties irrespective of their size, whereas at the nanoscale properties vary with the size. That is why nanoparticles show different and unique properties as compared to the bulk (Bradwell et al. 2001; Ortega-Vinuesa et al. 1998). As the size of the material approaches to nanoscale, the percentage of surface atoms becomes significant; subsequently, there are immense changes occur in various properties like change in color, melting point, hardness, catalytic activity, optical transitions, ionization potential and change in magnetic properties such as coercivity, permeability; saturation magnetization, etc. The drastic change in physicochemical properties at nanoscale of any material motivates their use in a variety of applications. The magnetic nanoparticles (MNPs) are one of the important subclasses of such nanoparticles. The nanoparticles are fundamentally different from the classic magnetic materials with their domain structure. These nanoparticles have giant magnetic moment pseudo-atoms with collective spin which induces gigantic magnetic moment. Because of this, magnetic nanoparticles are mostly accepted for diverse applications including catalyst, gas and humidity sensors, environmental remediation, high-density magnetic storage, multi-tera bit storage, biomedicine and theragnostic biomedical applications, ferrofluid technology, magnetocaloric refrigeration, etc., (Gubin 2009). MNPs can be synthesized by numerous methods that ultimately affect the final properties of the product. Based on the physical state of starting material, the production of MNPs is classified broadly into two strategies; top-down and bottom-up approaches. In the top-down approach, the reduction of starting material to a nanometric scale to one-, two- and three-dimensions, e.g. synthesis through ball milling, whereas, in the bottom-up approach, there is miniaturization of material components (up to atomic scale) with further self-assembly process leading to the formation of nanostructures at atomic and molecular scales (Rodríguez and García 2007). The classification of bulk magnetic material depends upon the magnetic susceptibility (χ ). Magnetic susceptibility is the ratio between induced magnetization (M) and applied magnetic field (H). Diamagnetic materials have small negative susceptibility as magnetic dipoles are oriented antiparallel to applied field and the magnetization is not retained when the externally applied field is removed. Paramagnetic materials possess small positive susceptibility and are dependent on temperature according to Curie law. Magnetic dipoles of paramagnetic material align parallel to applied field H. For ferro-, ferri- and antiferromagnetic materials, susceptibility values are large and positive and largely depend on their atomic structure, temperature and the applied external field H. For these materials, collective magnetism is very critical because

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they contain permanent dipoles which show exchange interactions. This understanding leads to the concept of critical temperature (i.e. Curie temperature for ferromagnetic materials and Neel temperature for antiferromagnetic materials). Below the critical temperature, these materials exhibited spontaneous magnetization. All these materials have large magnetic particles in the bulk and multidomain structure, where domain walls separate the regions of uniform magnetization. As the particle size of such materials is reduced to a critical volume, it will exhibit single-domain state. At single-domain state, particles can exhibit superparamagnetism, where these particles will behave as a giant magnet with zero coercivity and remanence (Salunkhe et al. 2014; Laurent et al. 2011). Fascinating properties of superparamagnetic (SPM) materials can be applicable to a variety of applications from technological to biomedical fields. In this chapter, a deep understanding of superparamagnetism, recent advances and challenges in the development of SPM materials for various applications are comprehensively described. The critical outlines of evolving developments are provided from the view of researchers and nanotechnologists.

2 Superparamagnetism In the early twentieth century, various attempts have been made to produce tiny magnets (Shih 1931; Elmore 1938). In 1946, Kittle put forth a model for nano-metredscale particles (Kittel 1946). According to the early explained models, the magnetic moment of tiny magnets, i.e. magnetic nanoparticles would follow an Arrhenius law with characteristic relaxation time τ. The determination of relaxation time was still an important question at that time, which was further solved by Neel in 1949. Neel assumed that each tiny magnet was formed by rigidly aligned spins which rotate coherently during the reversal of magnetization, when the energy barrier is comparatively much greater than the thermal energy of the system (considering the case of uniaxial anisotropy) (Néel et al. 1949). Furthermore, he derived an expression for τ by equivalencing the system with gyroscopic system and estimated a relaxation time which is to be the order of 10–10 s.   Ke f f V τ = τ0 exp kB T where k B is Boltzmann’s constant and τ 0 ≈ 10–9 s, V is the volume of the particle and K eff is the anisotropy constant. The value was well agreed with the experimental data. It was observed that, as the size of the particles changes, the magnetic properties change as a state varying from multidomain to single domain. When the size of the magnetic particles is small enough (i.e. critical volume), it needs more energy to create domain wall rather than to support the external magnetostatic energy of single-domain state. Depending upon the material, the critical diameter changes which is typically in the range of a few nanometre scales. The critical diameter is

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influenced by the various anisotropy energy terms. Below critical diameter size, the particles composed of magnetic moment behave as a single-domain particle which can show SPM behaviour when the temperature is above blocking temperature (T B ). Such single-domain particles behave like a giant paramagnetic atom having high magnetic moment, negligible remanence and almost zero coercivity. Later, single domain and superparamagnetism were intensively studied under the finite size effect of magnetic nanoparticles on magnetic properties (Laurent et al. 2011).

2.1 Multidomain System All ferromagnetic materials are characterized by multidomain system. In multidomain system, with the application of external field, the domain walls can move in response to the applied field. As a response, there is creation, growth and extinction of domains induced as the externally applied field enforces magnetic moments to align in a preferred direction. As a result, spins present in the domain will change their orientation. To change their orientation, it is necessary to displace the domain walls. This effect is called as Barkhausen effect and is an irreversible process. The trapping and displacement of domain wall depend on structural imperfections of the atomic pre-arrangement. As the effect is irreversible, material exhibits a hysteresis behaviour, i.e. with increasing and decreasing applied magnetic field, the magnetization curve will not coincide. The size of magnetic nanoparticles plays an important role in defining magnetic properties such as coercivity and magnetization. Fig. 1 shows the effect of particle size on magnetic properties. As particle size goes on reducing, the coercivity reaches to maximum and then again tends decreasing towards zero. In the multidomain system magnetization changes in the presence of external magnetic field by domain wall movement. It is found that the coercivity depends on the size of nanoparticles and is experimentally found to be given approximately by Hc = a +

b D

where a and b are the constants and D is the diameter of particle. This relation has no theoretical foundation.

2.2 single-Domain System As the size of the particle is reduced below a certain value known as critical diameter, the proximity of many domain walls in a very small volume is not energetically stable so that single-domain state is adopted. In 1930, Frankel and Dorfmann first time predicted the existence of single-domain particles (Frankel and Dorfman 1930).

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Fig. 1 Effect of particle size on the magnetic coercivity

Further, Kittle estimates the critical size, and relaxation time for single domain. By the definition, the single-domain particle should be always in saturated state in the sense of being spontaneously magnetized in one direction throughout its volume. The single-domain state is reached when magnetostatic energy ‘ΔEMS ’ = Domain wall energy ‘ΔEdw ’ below a critical diameter in the case of spherical particles (Fortin et al. 2007). The critical diameter can be determined as √ Dc ≈ 18

AK e f f μ0 Ms2

where A is exchange constant, μ0 is vacuum permeability and M S is saturation magnetization. Dc vary with materials. The above calculation of Dc is applicable to spherical and non-interacting particles and dependent on anisotropy. Larger the anisotropy, ultimately larger Dc and vice versa. Below Dc , particles will be uniformly magnetized and can change their magnetization by spin rotation as there are no domain walls to move. Shape anisotropy is one of the striking reasons for high anisotropy in single-domain systems. The behaviour of well-isolated single-domain particles helps us to understand superparamagnetism (refer Fig. 1). All magnetic materials have a magnetic anisotropy per particle which can hold the magnetic moments along a certain direction. The magnetic anisotropy energy per particle can be expressed as E(θ ) = K e f f V sin2 θ

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where 8 is the angle between magnetization and easy axis. The quantity K eff V stands for energy barrier which separates the two easy directions of magnetization (energetically equivalent). As the particle size decreases small enough, the thermal energy k B T, exceeds the energy barrier K eff V and magnetization is easily flipped. When k BT > K eff V, the particle will behave like a paramagnet but, instead of atomic magnetic moments, there is the presence of a giant moment inside each particle and such a system is known as superparamagnetism. Such a system has no hysteresis curve and the data of different temperatures superimpose onto the universal curve of M versus H/T. If magnetic moment of the particles reverses in a shorter time than the experimental time scale, the system is said to be in SPM state, if not, then the system is said to be in blocked state. The temperature which splits these two regimes is the so-called blocking temperature T B and expressed as TB =

Ke f f V 30k B

The blocking temperature depends on the effective anisotropy constant, the size of particles, the applied magnetic field and experimental measuring time. Below Dc , because of the thermal effects which are strong to spontaneously demagnetize a previously saturated assembly of particles results in zero coercivity, such particles are known as SPM particles.

2.3 Synthesis of Superparamagnetic Nanoparticles Various physical properties like magnetic, electrical, morphological and structure of the magnetic nanoparticles are strongly influenced by the synthesis method adopted. It has long been a tough task for researchers to develop a synthesis method which helps to achieve anticipated particle size, shape and size distribution. There are two main challenges to develop synthesis method, one is monodispersity and the second is reproducibility. Only care has to be taken while synthesizing to avoid nucleation during the growth period, i.e. nucleation and growth should be separated. However, various approaches had been adopted, mainly classified as physical, chemical and biological. Most of the synthesis methods are summarized in the following Table 1. The physical methods involve planetary ball milling, plasma deposition, lithography, etc., synthesis methods. Various chemical methods are used to synthesize magnetic nanoparticles for a variety of applications. The chemical method involves hydrothermal synthesis, thermal decomposition, sol–gel, combustion, polyol synthesis, etc. Most of the researchers have been following chemical methods to prepare MNPs as the narrow size distribution, homogeneous composition can be achieved easily by chemical route. However, amongst various chemical methods, the most common method used to synthesize is chemical co-precipitation method. Although, with the physicochemical synthesis methods, nanoparticles can be

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synthesized successfully, these are expensive, involve toxic chemicals and are very difficult to achieve monodiameter-sized NPs. Recent advancements in nanotechnology brought a new tool use of MNPs for biomedical application. Keeping in mind the requisites of MNPs for application in biomedical field, clean, non-toxic, monodiameter dispersity and eco-friendly methods were needed to be developed. The biological synthesis method fulfilled the all requirements. Biological methods are broadly divided into two parts intracellular and extracellular syntheses. In intracellular synthesis, magnetotactic bacteria synthesize chemically homogeneous, singledomain nanocrystal, biocompatible magnetosomes intracellularly which are enclosed by protein-embedded lipid bilayer (Salunkhe et al. 2014). Table 1 The table summarizes various ferrite-based and non-ferrite-based SPM nanoparticles SPM material

Compound

Structure

Application

Ferrite-based SPM

1. MnFe2 O4

Spinal

• Magnetic hyperthermia • Magnetic resonance imaging • Lithium-ion batteries • Super capacitors sensors

2. CoFe2 O4

Inverse Spinal

• Recording devices • Magnetic cards • Magnetic drug delivery

3. NiFe2 O4

Inverse spinal

• Microwave absorbers • Catalyst in magnetic solutions • Magnetic refrigeration

4. CdFe2 O4

Spinal

• Electrode materials • Drug-loading materials

1. FePt

Close-packed tetragonal structure

• Targeted drug delivery • MRI • Biological separation

2. LSMO

Perovskite

• Magnetic fluid hyperthermia • Magnetic refrigeration • MRI • Targeted drug delivery

3. NSMO/PSMO

Perovskite

• • • •

4. CoO

Cubic

• Microbatteries • Nanowires • In electronic devices

Non-ferrites-based

Sensors Spintronics Fuel cells Memory devices

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3 Types of SPM Nanoparticles SPM nanoparticles are explored in a variety of applications which include technological as well as biomedical applications. By optimizing the synthesis parameter and appropriate synthesis protocol, SPM nanoparticles can easily be produced. There are a variety of magnetic nanoparticles available in nature for which superparamagnetism can be achieved, e.g. iron oxide-based MNPs (e.g. MFe2 O4 ), bimetallic MNPs (e.g. FePt) and rare earth-based half-metallic ferromagnetic materials (e.g. LSMO). Super paramagnets consist of individual (single) magnetic domains of elements (or compounds) that have ferromagnetic properties in bulk. On the basis of presence of magnetic oxide compound the SPM materials can be classified into two classes.

3.1 Ferrite-Based SPM Materials Ferrites are generally used to describe the class of magnetic oxide compounds that contain iron oxide as a principal compound (Fe3 O4 ). Ferrites are a metal-oxide ceramic made of a mixture of Fe2 O3 and either manganese–zinc or nickel–zinc oxides pressed or extruded into a range of core shapes. Every manufacturer offers a wide variety of shapes and will usually also offer a custom service for large volumes, but for most uses a selection from a relatively small range of standard types is adequate, and offers the benefit of sourcing from different suppliers. Ferrites has many applications because of its very basic properties such as a significant saturation magnetism, a high electrical resistivity, low electrical losses, and a very good chemical stability. Ferrites can be obtained in three different crystal systems by many methods, and the feasibility to prepare a virtually unlimited number of solid solutions opens the means to tailor their properties for many applications. Ferrites are unique class of compounds comprising lanthanides and fast transition metals. The crystal lattice of ferrite is spinel. Spinel ferrites is a special kind, which accommodate cations amongst two possible available interplanar sublattices namely tetrahedral and octahedral. Valence distribution of the cations amongst tetrahedral and octahedral sites decide its structural, electrical, magnetic and chemical properties. In spinal ferrites metal ions are located at octahedral and tetrahedral positions (Wang et al. 2008). Spinel ferrites are homogeneous materials with a general chemical formula AB2 O4 . Where A and B are metal cations whereas O is oxygen anion. A and B are the metallic cations positioned at two different crystallographic sites. The general formula for spinel ferrite is, X2+ Fe2+ O2− 4 where X2+ is a divalent metal ion such as Fe2+ , Mg2+ , Ni2+ , etc.

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Fig. 2 a Normal spinel and b Inverse spinel Structure

Depending upon the divalent atom and trivalent atom position and direction, we have two types of atomic distribution such as A-site and B-site distributions. A-site is called tetrahedral site, where each metal ion is surrounded by four O2− ions. Bsite is called octahedral site, where each metal ion is surrounded by six O2− ions as shown in Fig. (2a). Fe2+ , Ni2+ and Mn2+ ions prefer to occupy the octahedral site (Rondinone et al. 1999). The spinel ferrites are the special kinds that consist of cubic close-packed or face-centred cubic oxide with M' occupying 1/8th position of the tetrahedral holes and M'' cations occupying ½ of the octahedral holes. The nickel ferrites, i.e. NiFe2 O4 crystallize in an inverse spinel structure and can be represented as (Fe1-γ 3+ ) A [Ni1-γ 2+ Fe1+γ ]B O4 , where ‘γ’ is an inversion parameter and depend upon the preparation condition and heat treatment. In inverse spinel structure, B-site is occupied by 50% of divalent atoms and 50% by trivalent atoms, and the A-site is occupied by trivalent atoms, i.e. each divalent metal ion (Fe2+ ) is in octahedral form and 50% of trivalent metal ion (Fe3+ ) is in tetrahedral form and remaining 50% trivalent metal ions is in the octahedral form (Limaye et al. 2009) (Fig. 2). The spinel ferrites, e.g. ZnFe2 O4 , MnFe2 O4 and (ZnMn)Fe2 O4 are the most focused ferrite materials in recent studies and wildly used in technical fields. The bulk Zn ferrite is a regular spinel with Zn2+ at tetrahedral (8a) and Fe3+ at octahedral sites and shows antiferromagnetism. And, bulk Mn ferrites have mixed cation distribution with Mn2+ and Fe3+ at both crystallographic sites. Ferrites are classified into two classes according to their crystal structure: soft ferrite and hard ferrite. Soft ferrites are materials with low coercivity and are easily demagnetized. These are used in electronic industries to make efficient magnetic core called ferrite core for high-frequency inductors, transformers, antennas and various microwave components. Hard ferrites are the materials with high coercivity and these are the materials which are very difficult to demagnetize. Hard ferrites are used for the preparation of permanent magnet used in refrigerators, loudspeakers and electric motor (Veverka et al. 2007).

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3.2 Non-Ferrite-Based SPM Materials Non-ferrites-based material in which Fe2+ or Fe3+ group is not present but these materials also exhibit the magnetism. This type contains the doped rare earth manganites with a general formula Ax A' 1-x BO3 (A = La, Nd, Pr, etc., A’ = Ca or Sr) such as a series of LSMO, NSMO, PCMO, etc., and bimetallic ferromagnetic compounds, e.g. CoPt, FePt, etc. Many authors have demonstrated the possible application of such non-ferrite-based SPM nanoparticles in biomedical field. LSMO compounds have large magnetic moments at room temperature and can show a wide range of Curie temperatures from 283 to 370 K, whereas nanosized manganites show very high magnetoresistance compared to bulk. Magnetoresistive effects are very interesting and important for technological applications. Magnetoresistive effect is mostly related to structural, electrical and magnetic properties of the material. This effect has very much importance in technological applications: magnetic data storage, magnetic recording, etc. (Thorat et al. 2013).

3.3 Structure of LSMO/NSMO Manganites have a perovskite structure. Perovskite denotes a general group of crystal systems having the basic chemical formula ABO3 , where A and B are cations of different sizes and O is an anion which bonds them together. The following figure shows a typical perovskite structure (Thombare et al. 2019). From Fig. 3, at the corner of the cube, A-site cations are present, and at the centre, B-Site cations with oxygen ions at the face-centred position. Here, A-site cations are usually rare earth elements and B-site cations are transition metals. The most important feature of perovskite system is structural flexibility due to some interesting facts (i) relative size mismatch created by A and B site cations (ii) breaking of local symmetry emerged through a delicate interplay within long-range columbic forces and short-range repulsive forces. As a result of structural flexibility, various perovskites undergo a series of phase transitions and exhibit a structure including orthorhombic, rhombohedral and tetragonal symmetries at low temperatures. In the perovskite, A-site ions are larger than B-site ions. The structure is stabilized by sixfold coordination of B-cation and sixfold coordination of A-cation. Even though the perovskite structure is cubic the compounds in this family may possess some distortion. These compounds are generally used as sensors, as a catalyst electrode in certain types of fuel cells, in memory devices as well as in spintronics (Kulkarni and Bodasa 2015).

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Fig. 3 Perovskite structure with B-type metal ions surrounded by six oxygen ions

3.4 Bimetallic Nanoparticles Metal alloy nanoparticles are called bimetallic nanoparticles. These nanoparticles also can show SPM behaviour which makes them potential Magnetic Resonance Imaging (MRI) contrast agents and magnetic carriers for drug delivery. Compared to other metallic nanoparticles interaction between the two metals offers greater chemical stability. Besides, their surface chemistry helps to bind carboxylate- and amine-based surfactants on the surface. Surfactants improve their water solubility and make them a possible candidate for biomedical applications. The best example of such bimetallic nanoparticles are FePt, CoPt, etc. (Shubayev et al. 2009).

3.5 Metallic with a Shell This class includes the surface of a magnetic metallic core passivated by gentle oxidation, surfactant, polymers and any valuable metals. The most important feature of this type is the exchange bias effect. When two magnetic phases come in close contact with other, the exchange coupling across the interface between metallic core and shell can induce the exchange bias effect. This effect offers stabilization in magnetization by providing an extra source of anisotropy. This effect first time was observed for cobalt nanoparticles coated with a CoO layer. Exchange bias between core Co NPs and shell antiferromagnetic CoO will allow easy tailoring of the magnetic properties and controls the anisotropy by tunning the magnitudes of core and shell. These types of nanoparticles are usually used in microbatteries, nanowires, in electronic devices, etc. (Zhou et al. 2011).

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4 Important Properties of SPM Nanoparticles to Be Used in Biomedical Applications Recent advances in nanotechnology brought us new tools for research in material science, environmental and biomedical engineering. Particularly, nanobiotechnology has evolved as a challenging area which can provide commercial biomedical products using nanoparticles for mankind. The global market for various nanoparticles for drug formulation, drug delivery has reached $ 79.8 billion in 2019, with a compound annual growth rate of 22%. SPM nanoparticles are one of such new tools emerging as a potential candidate for various applications like targeted drug delivery, magnetic fluid hyperthermia, stem cell labelling, nanobiosensors, etc. (Ha et al. 2018). SPM nanoparticles possess superparamagnetism, which makes them an attractive candidate for biomedical applications. SPM materials have received intense focus because of their controllable nanoscale dimensions and superior magnetic properties: hysteresis-free reversible magnetization, i.e. zero coercivity and remanence and high saturation magnetization, quick magnetophoretic response, by modifying their surface by various organic and inorganic materials the biocompatibility can be improved. When SPM nanoparticles come in contact with a controlled external magnetic field, they are acting as giant-nanomagnets with numerous striking properties: i. They can lose their magnetism instantly and completely once the external field is removed. ii. They can be actuated by a magnetic field gradient: action at a distance. iii. They can resonantly respond to a time-varying field—energy transfer. iv. Their presence can be detected remotely. v. Due to magnetic losses of SPM nanometric particles in colloids, they can generate intense localized heat. Furthermore, some other properties of SPM materials highlighted them as a potential agent for various applications (Basic Fundamentals of Drug Delivery 2019): i. The surface of SPM nanoparticles can be passivated by reactive functional groups without hampering their magnetic properties. ii. Surface can easily decorate with antibodies, which can allow them to target biological or chemical entity. iii. SPM nanoparticles can be manipulated for sensing, fractionation, filtration and collection and other purposes. iv. The large surface area-to-volume ratio of SPM nanoparticles make them potential to detect a very dilute concentration of analyte and subsequently can improve detection sensitivity.

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5 Role of SPM Nanoparticles for Various Applications The above-described competences make SPM materials one of the important aspects of research to explore them in medical, technological and other applications. Maximum of these applications required very sophisticated instruments and highend research laboratories. However, these SPM materials can be incorporated into kits for Rapid Diagnosis Test (RDT) for malaria detection which is very cheap, easy to use and portable. Keeping in mind their applications in various fields, intensive research has been carried out on the development of SPM nanoparticles for different applications (Mahmoudi et al. 2011). Some of them are discussed in detail here.

5.1 MRI Early-stage diagnosis makes it easier to treat any disease. Nowadays, so many modalities are available for diagnosis of many diseases, but still, some types of cancer, like pancreatic cancer, unfortunately cannot be detected until their later stage. In this case, accurate diagnosis can enhance the chances of accuracy of treatment. SPM nanoparticles have high imaging contrast effect due to which they can enhance the difference between pathogenic targets and normal tissue through the MRI (Salunkhe et al. 2020). MRI is working on the basis of directional magnetic field or magnetic moment associated with charged particles in motion. Nuclei containing an odd number of protons and neutrons have a precession (characteristic motion), as the nuclei are charged particles this precession produces a small magnetic moment. MRI uses high magnetic fields to align the nuclear magnetization of the body’s hydrogen atoms because when the human body is placed in large magnetic fields many of the free hydrogen nuclei align themselves with the direction of magnetic field and these nuclei precis about the magnetic field direction like gyroscope called Lamour precession. When the radiofrequency pulse is removed from the applied magnetic field then the nuclei ‘relax’ back to their original state. This process can be measured by either its longitudinal relaxation (T 1 ) or transverse relaxation (T 2 ), each of which can be used to generate an MR image (as shown in Fig. 4) (Barrow et al. 2017).

5.2 Targeted Drug Delivery Drug delivery is the process of administering a pharmaceutical compound to achieve therapeutic effect in human or animal body. In drug delivery, drug targeting plays a vital role and can be defined as the optimization of drug’s therapeutic index by strictly focusing its pharmacological activity at the site or organ of action. For targeted drug delivery, SPM nanocarriers are attached with a cytotoxic drug. The prepared

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Fig. 4 a alignment and realignment of nuclei by applying and removing RF signal for contrast image capturing b Drug release mechanisms with SPM nanoparticles c Magnetic fluid hyperthermia with SPM nanoparticles

drug/carrier complex is generally prepared in the form of injectable biocompatible ferrofluid, and is inserted into the patient’s body through a circulatory system. When such nanocarriers enters the bloodstream, an external high-gradient applied magnetic field is used to concentrate the carrier complex at a particular tumor site within a body (Palanisamya and Wang 2019). Once the drug/carrier is concentrated at the target, drug can be released either via enzymatic activity or changes in physiological conditions such as pH, osmolality or temperature, and be taken up by the cancerous or tumour cell. Drug-released mechanisms are shown in Fig. 4.

5.3 Magnetic Fluid Hyperthermia SPION is one of the important subclasses in cancer therapy. The MNPs used in magnetic hyperthermia therapy. The hyperthermia therapy with MNPs in AC magnetic field started in late 1950. In magnetic thermotherapy, the affected cancer cell or tissue are heated to the temperature range of 41–46 °C by exposing to highfrequency external AC magnetic field. Heat generated by the magnetic nanoparticles due to hysteresis loss, Neel and Brownian relaxation. SPM nanoparticles show only Neel and Brownian losses. Heat is generated due to oscillation in magnetic moment, and due to this, temperature of cancer cells is elevated up to 41–43 °C; generally, at

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that temperature, cancerous cells die. The magnetic heating through the mediators depends on the size and magnetic property of the medium (Kandasamy et al. 2018).

5.4 For Environmental Application Environmental pollution is a serious problem worldwide due to the influence of various industries such as petroleum refining, leather tanning, paper, textile, cosmetics, electronics, mining and metallurgy. The contamination caused due to this industrialism is removed by using absorption technique. The absorption technique is a very convenient technique which offers selective accumulation and separation of toxic contaminants through facile. It is efficient, environmentally friendly and cost-effective process. Selecting the right absorbent is very important for the early stages of the contamination removal process. Commonly used absorbents include clay, biodegradable polymers, activated carbons, synthetic polymers, etc. The application of adsorbents has been limited by several problems such as low adsorption capacities, separation inconveniences, e.g. tedious filtration and centrifugation and turbidity in effluent (Sezer et al. 2021). The SPM nanoparticles as absorbents are superior to the traditional absorbents because of their inherent properties. Different SPM nanoparticles can be prepared by attaching the specific functional groups on the particle surface which can effectively remove the organic and inorganic contamination including dyes, heavy metal ions and wastes. Magnetic separation of SPMNPs allows for easier and faster contaminant removal as compared to centrifugation and filtration processes. Under the application of the external magnetic field, the SPM nanoparticle absorbent and absorbed contaminants are easily separated from the solution which enables for safe disposal of the wastes. Also, there is no power source is required when the external magnetic field is applied from the permanent magnet.

6 Conclusion SPM materials are emerging as probable nanocarriers for various biomedical applications. The SPM materials exhibit striking properties when come in contact with externally applied magnetic field, which make them more efficient and potential candidates than conventional carriers. Superparamgnetism is a size-dependent phenomenon of MNPs which can be controlled by choice of synthesis method and also depend upon the application intended. SPM materials are mainly classified into two types on the basis of containing magnetic oxide compound. Both ferrite-based and non-ferritebased SPM materials have been studied intensively for a variety of applications from technology to biomedical. SPM materials offer more attractive and added advantages to biomedicine and can help the clinician to detect and treat cancer simultaneously.

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References Barrow M, Taylor A et al (2017) Biomater Sci 6:1. https://doi.org/10.1039/C7BM00515F Basic Fundamentals of Drug Delivery (2019) Advances in pharmaceutical product development and research. pp 685–731, https://doi.org/10.1016/B978-0-12-817909-3.00017-0 Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R (2001) Clin Chem 47:2069 Elmore C (1938) Phys Rev 54:309 Fortin JP, Wilhelm C, Servais J, Me´nager C, Bacri JC, Gazeau F (2007) J Am Chem Soc 129: 2628–2635 Frankel J, Dorfman J (1930) Nature: 126–274 Gubin SP (2009) Magnetic nanoparticles. Wiley-VchVerlag GmbH & Co. KGaA, Weinheim Ha Y, Ko S, Kim I, Huang Y, Mohanty K, Huh C, Maynard JA, Appl ACS (2018) Nano Mater 1:512–521 Kandasamy G, Sudame A, Luthra T, Saini K, Maity D (2018) ACS Omega 3(4): 3991–4005 Kittel C (1946) Phys Rev 70:965 Kulkarni VM, Bodasa D, Paknikar KM (2015) RSC Adv 5:60254–60263 Laurent S, Dutz S, Häfeli UO, Mahmoudi M (2011) Advan Colloid Inter Sci 166:8–23 Limaye MV, Singh SB, Date SK, Kothari D, Reddy VR, Gupta A, Sathe V, Choudhary RJ, Kulkarni SK (2009) J Phys Chem B 113:9070–9076 Mahmoudi M, Sant S, Wang B, Laurente S, Senf T (2011) Adv Drug Deli Rev 63(1–2):24–46 Néel L (1949) CR Acad Sci Paris 228. Ann Geophys 5(99): 664 Ortega-Vinuesa JL, Hidalgo-Álvarez R, de las Nieves FJ, Davey CL, Newman DJ, Price CP (1998) J Colloid Interface Sci: 204–300 Palanisamya S, Wang YM (2019) Dalton Trans 48:9490–9515 Rodríguez JA, García MF (2007) Synthesis, properties, and applications of oxide nanomaterials. Wiley Interscience Rondinone AJ, Samia ACS, Zhang ZJ (1999) J Phys Chem B 103:6876–6880 Salunkhe AB, Khot VM, Pawar SH (2014) Curr Top Med Chem 14(5):572–594 Salunkhe A, Khot V, Patil SI, Tofail SAM, Bauer J, Thorat ND (2020) ACS Appl Bio Mater 3(4):2305–2313 Sezer N, Arı ˙I, Biçer Y, Koç M (2021) J Magn Magn Mater 538(15):168300 Shih W (1931) Phys Rev 38:2051 Shubayev VI, Pisanic TR, Jin S (2009) Adv Drug Deliv Rev 61:467–477 Thombare B, Dusane P, Kekade S, Salunkhe A, Choudhary RJ, Phase DM, Devan RS, Patil SI (2019) J Alloys Comp 770:257–266 Thorat ND, Khot VM, Salunkhe AB, Prasad AI, Ningthoujam RS, Pawar SH (2013) J Phy D: Appl Phy 46(10):105003 Veverka M, Veverka P, Kaman O, Lancok A, Zaveta K, Pollert E, Knzek K, Bohacek J, Benes M, Kaspar P, Duguet E, Vasseur S (2007) Nanotechnology 18(345704): 7 Wang Z, Liu X, Lv M, Chai P, Liu Y, Zhou X, Meng J (2008) J Phys Chem C 112:15171–15175 Zhou L, Yua J, Wei Y (2011) J Mater Chem 21:2823–2840

In Vitro and In Vivo Assessment of Superparamagnetic Materials Ahmaduddin Khan , Chandunika R. Kalaiselvan, Shalmali Sudhindra, Mohammad Suhaan Dar, and Niroj Kumar Sahu

Abstract Magnetic nanoparticles, particularly, superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for biomedical applications for so long. Their biocompatibility, drug loading efficiency, cytotoxic potential, and diagnostic abilities are well researched. SPIONs can be surface functionalized to enhance their biocompatibility and to provide possibility for further conjugation with targeting moieties thus minimizing the side effects by effectively delivering the anticancerous drugs to the specific tumor location. SPIONs can be utilized effectively for diagnostic imaging and magnetic hyperthermia for combinatorial therapeutic purpose. The biomedical applications of SPIONs are promising because of non-invasiveness. In this chapter, the synthesis and functionalization of SPIONs, applications in magnetic imaging, hyperthermia and as drug cargo, in vitro and in vivo assessment, and toxicity have been described thoroughly. Keywords Iron oxide · Nanoparticles · Chemotherapy · Hyperthermia · Magnetic resonance imaging · Drug delivery

1 Introduction Cancer is an ailment in which cells grow uncontrollably and quickly and can metastasize to other parts of the body. Human cells grow and multiply for the formation of new cells through cell division. When this sequence breaks, it can form lumps of tissue called tumor. It can be benign or cancerous. According to the report by the A. Khan · C. R. Kalaiselvan · S. Sudhindra · M. S. Dar · N. K. Sahu (B) Centre for Nanotechnology Research, Vellore Institute of Technology, Vellore, TN 632014, India e-mail: [email protected] S. Sudhindra School of Advanced Sciences, Vellore Institute of Technology, Vellore, TN 632014, India M. S. Dar School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, TN 632014, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_4

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American Cancer Society, nearly 609,360 deaths are expected in the US from cancer in the year 2022. It is one of the most common causes of death in the US only after heart ailments (American Cancer Society 2022). Therefore, early detection of cancer before the metastasis of cancer cells is necessary for the diagnosis of cancer for the effective treatment of early-stage cancers. Still, early diagnosis is difficult in most of the cases due to the later-stage appearance of clinical symptoms. Thus, minimally invasive or non-invasive methods for the early detections are required urgently for the efficient treatment of cancer (Bakhtiary et al. 2016). Several types of cancer therapies are available such as chemotherapy, radiotherapy, hyperthermia, surgery, etc., depend on the cancer and its stage. Patients have to undergo either single therapy or a combination of treatments. Chemotherapeutic treatment utilizes drugs to kill the cancer cells. Radiation therapy uses high radiation doses for the killing of cancer cells and to shrink the tumor. In hyperthermia, cancerous tissues are heated to an elevated temperature to damage the cancer cells. In surgery, the cancer cells or the infected organs are removed surgically from the body. Chemotherapy is the most widely used treatment procedure for the therapy of cancer. However, it suffers from various drawbacks such as non-specific biodistribution, toxicity to vital organs, low therapeutic efficiency, etc. Therefore, nanotechnology-mediated cancer therapy and diagnosis are in trend. The nanoscale engineering of materials is fascinating due to their astonishing optical (Tari et al. 1979), electronic (Poizot et al. 2000), and magnetic properties (Mahmoudi et al. 2010). The dimensional tuning of these nanoparticles leads to surface modification and the fabrication of nanoparticles suitable for cancer therapy (Mahmoudi et al. 2011). Among several nanoparticles, SPIONs, mainly magnetite and maghemite nanoparticles are mostly used in cancer theranostics such as magnetic hyperthermia, MRI, and drug delivery because of their substantial magnetic attributes and biodegradable nature (Kandasamy and Maity 2015). SPIONs show superparamagnetism at room temperature below a critical size of approximately 30 nm (Kandasamy and Maity 2015). In SPIONs, iron oxide core imparts them the exclusive property of superparamagnetism, high field reversibility, and anisotropy thus making them a valuable agent for targeting specific cells (Mahmoudi et al. 2011; Kodama, et al. 1996; Ansari et al. 2018). SPIONs are found in various forms such as maghemite, hematite, and magnetite with a broad range of applications (Ansari et al. 2017, 2018). SPIONs require proper surface functionalization with a biocompatible polymer to enhance the residence time in blood and to avert the aggregation. Non-functionalized SPIONs can affect cell viability by causing toxic effects. SPIONs size can be tuned for their buildup in tumors by using the enhanced permeability and retention (EPR) effect. The shape, size, and surface chemistry of SPIONs have a very important role in the specificity and theranostic efficiency. This chapter includes an overview of the synthesis of SPIONs, their biomedical applications, in vitro and in vivo assessment and toxicity.

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2 Synthesis of SPIONs SPIONs are produced in a variety of ways, including chemical, physical, and biological methods (Ali et al. 2021; Samrot et al. 2021). The method of synthesis used is crucial in achieving the desired shapes, sizes, morphology, stability, and magnetic property (Samrot et al. 2021). Chemical co-precipitation synthesis is the most widely employed method for making iron oxide magnetic nanoparticles. In this process, iron precursors are turned into iron oxides with the help of a mild reducing agent like sodium hydroxide, ammonia, tetramethylammonium hydroxide (TMAOH), etc. (Samrot et al. 2021). Synthesis of magnetic nanoparticles through co-precipitation technique is facile and suitable when the nanocrystals are required in higher quantities. This method is very popular for the synthesis of nanoparticles with good magnetic attributes and controlled size (Ali et al. 2021). Physical methods include several approaches such as ball milling, laser evaporation, laser pyrolysis, electron beam lithography, etc., (Majidi et al. 2016; Samrot et al. 2021). Ball milling is a top-down method for the synthesis of magnetic nanoparticles from the bulk. It is a simple technique which includes grinding mechanically the coarse textured particles to fine textured particles (El-Eskandarany 2001; Ali et al. 2021). Laser evaporation is a bottom-up method in which nanoparticles are generated from condensation of the liquid or gas phase (Ali et al. 2021; Biehl et al. 2018). It is also called laser ablation, it’s an easy method in which laser of high energy is used for the formation of magnetic nanoparticles (Ali et al. 2021; Shin et al. 2004). In laser pyrolysis, flowing gases’ mixture is heated with a constant CO2 laser wave which starts and withstands a chemical reaction (Majidi et al. 2016; Veintemillas-Verdaguer et al. 2002). In electron beam lithography, electron beams are emitted in a pattern on a substrate coated with resin/film and then lifted off selectively the non-exposed/ exposed regions of the resin (Corbierre et al. 2005; Samrot et al. 2021). Green synthesis of magnetic nanoparticles has gathered much attention due to environmental safety. It includes several techniques which can minimize or eradicate toxic materials and substances for the restoration of the environment (Majidi et al. 2016). In a report by Sathiskumar et al., magnetic iron oxide Fe3 O4 nanoparticles were fabricated by fruit extract of C. guianensis also called cannonball tree. Various extracts from the fruit and leaf of this tree have several medicinal values. The nanoparticles formed were spherical and polydispersed having a size in the range of 7–80 nm (Sathishkumar et al. 2018). Yusefi et al. reported a facile method for the formation of Fe3 O4 nanoparticles using an extract of Garcinia mangostana fruit peel. As evident from the scanning electron micrographs, the synthesized nanoparticles were surrounded by the extract. The synthesized nanoparticles possess a maximum saturation magnetization of 69.42 emu/g and an acceptable SAR value (Yusefi et al. 2021). Additionally, magnetic nanoparticles having superparamagnetic properties can also be synthesized through microbes by using several species of fungi, bacteria, and yeast (Nadeem et al. 2021).

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3 Surface Functionalization of SPIONs Magnetic iron oxide nanoparticles suffer from problems such as quick aggregation and oxidation in the tumor environment because of large surface area, high surface energy, and chemical activity thus compromising the magnetization (Mohammadi et al. 2013). Hence, proper surface modification of the iron oxide nanoparticles is necessary for their biocompatibility toward normal cells. Coating of the biocompatible polymers is the most convenient method to functionalize the surface of the nanoparticles by ligands or other targeting materials. This prevents the oxidation and agglomeration of the nanoparticles as well as provide the scope of further functionalization (Sun et al. 2014; Zhu et al. 2018). A number of coating agents such as albumin, chitosan, polyethylene glycol, etc., are used to functionalize the surface of the SPIONs. These agents avert the aggregation of the SPIONs either by steric stabilization or electrostatic repulsion (Sodipo and Aziz 2016). Albumin is a natural protein which transports minerals, vitamins, and other several hydrophobic compounds like steroids to several tissues. One of the advantages of using albumin is that the therapeutic drug can be conjugated effortlessly by covalent or non-covalent interaction (Moku et al. 2019). In a report by Vidawati et al., human serum albuminSPIONs-loaded PLGA nanoparticles are effectively used for the delivery of protein (Vidawati et al. 2018). Another biocompatible polymer is chitosan which is a natural cationic polysaccharide produced by the deacetylation of chitin chemically (Janes et al. 2001; Moku et al. 2019). In the polymer backbone of chitosan, there are primary amino groups which provides a positive charge to the polymer. It is also useful for oral drug delivery because of its mucoadhesivity (Moku et al. 2019). As reported by Shete et al., magnetite nanocrystals were prepared by alkaline precipitation. The nanoparticles coated with chitosan showed superparamagnetic behavior and were biocompatible on L929 cells even after 48 h of incubation (Shete et al. 2014). Another conventional polymer polyethylene glycol (PEG) is widely used as a biocompatible coating for magnetic nanoparticles. It is convenient for biomedical applications because of its biocompatibility, high aqueous solubility, and well tolerance (Hoang Thi et al. 2020). As reported by Dai et al., PEG-coated SPIONs were synthesized by one-pot approach. Its toxicity was assessed both in vivo and in vitro. PEG-coated SPIONs are biocompatible to the organs as per the results of H & E staining. At 192 μM concentration, 86% of the NIH/3T3 cells were viable after 24 h confirming its biocompatibility (Dai et al. 2014). Khan et al. reported magnetic nanoclusters (MNCs) coated with a functionalized polymer PEG-diacid where the free carboxyl end of the polymer was utilized for the further formation of hydrazone bond with DOX for pH-sensitive delivery (Khan and Sahu 2021).

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4 Applications of SPIONs in Cancer Therapy and Diagnostics 4.1 SPIONs in Magnetic Hyperthermia Frans Westermark, in 1898, was the first to utilize heat as a treatment for cancer in which a hot water tube was used to treat cervical cancer (Westermark 1985). Later, Gilchrist et al. in 1957, first time administered magnetic nanoparticles for selective killing of cancer by induction heating (Gilchrist et al. 1957). They administered 5 mg of iron oxide nanoparticles to the lymph nodes and acquired a 14 °C rise in temperature with an applied alternating magnetic field (AMF) of 15.9–19.1 kAm−1 at a frequency of 1.2 MHz. The experiment was a success with a substantial cancer cell death rate. Since then, the development of magnetic nanoparticles has been on focus as a heating system to deliver heat. The effect of overheating the body over the normal temperature (normal body temperature is 37 °C) is known as hyperthermia. In medical terms, hyperthermia is the body’s elevated temperature in the range of 40–45 °C. Since cancer cells are more vulnerable to this temperature range than normal cells, hyperthermia has been preferred as a non-invasive anticancer therapy. Several methods such as laser, high-frequency current, microwave, water bath, etc., can induce this temperature. However, these techniques heat the whole body or broad area rather than selective heating which affects healthy cells. Therefore, specific heating using magnetic nanoparticles known as magnetic hyperthermia (MHT) is preferred for enhanced cancer treatment, a schematic is shown in Fig. 1. Here, the heat is generated by applying an appropriate alternate magnetic field (AMF) to the magnetic nanoparticles. The interaction between the magnetic nanoparticles produces heat and is classified into Neel relaxation related to the direction flip of magnetic moment inside the single nanoparticle and Brownian relaxation related to the flip in the direction of the entire nanoparticle. The heating capacity of the magnetic nanoparticle can be regulated by tuning their physicochemical properties like size, shape, composition, polydispersity, surface coating, etc. (Périgo et al. 2015). MHT overcomes several challenges faced by conventional methods and importantly targets specific heating. The other notable advantages are tunable magnetic/heating properties, easy surface functionalization with antibodies, drugs, or ligands, and can be utilized as contrast agents for diagnostic purposes like MRI imaging (Korchinski et al. 2015; Shen et al. 2017; Wei et al. 2017). The SPIONs are the most promising material because of the ease of synthesis and superparamagnetic behavior. It has been given approval by the Food and Drug Administration for use in nanomedicine (Thakor et al. 2016). Compared to normal magnetic material, SPIONs have large magnetic susceptibility, zero coercivity, and zero remanent magnetization. As a result, upon the application of an external alternating magnetic field, the magnetic moments in the material get aligned in the direction of the applied field resulting in saturation. Moreover, when the external field is removed, it gets demagnetized without remanent magnetization. Thus, with an alternate magnetic field, the magnetization of SPION goes back to its initial zero magnetization state by Neel relaxation mechanisms

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Fig. 1 Schematic illustration of magnetic hyperthermia therapy (MHT). Adapted and reprinted from Rajan and Sahu (2020)

resulting in heat generation. The most exploited iron oxide particles at the nanoscale for MHT are magnetite and maghemite. Rego et al. examined aminosilane-coated SPION for MHT therapy in a glioblastoma tumor model (Rego et al. 2019). They used C6 cells for in vitro and rats implanted with C6 cells for in vivo assessment. 52% and 32.8% of cancer cell death are observed in in vitro and in vivo, respectively, upon the application of AMF to the SPIONs at the field strength of 200 Gauss (20 mT) and frequency of 874 kHz. The heat generation directly depends on the applied field strength. However, an allowed upper limit for electromagnetic field that can be used on living organs according to the Brezovich criterion is H.f = 4.5 × 108 Am−1 s−1 (Brezovich 1988). In the past decades, the main focus of MHT research has been to understand the mechanisms of cell death activated by SPIONs under the influence of AMF. Several studies suggested that cell death is mainly due to the increased intracellular temperature. The studies show permanent molecular impairment occurs at an elevated temperature like DNA double-strand breakage, protein aggregation and unfolding, and plasma membrane permeability (Pucci et al. 2022). Marino et al. proposed an effective magnetic hybrid lipid nanoparticle for the synergic magnetothermal hyperthermia and chemotherapy treatment. They observed the SPIONbased hybrid nanoparticles increase the permeability of the plasma membrane of glioblastoma multiforme cells upon AMF (Marino et al. 2019).

4.2 SPIONs in Magnetic Resonance Imaging Among the various diagnostic approaches for imaging cancer cells, MRI is the most preferred tool for its non-invasiveness. The principle of MRI depends on the hydrogen

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atoms which are abundant in the living organism. The individual hydrogen atoms with a single proton act as a magnetic moment upon the external field. On applying an external alternating magnetic field, these hydrogen protons align parallel or against the applied magnetic field. However, a large number of protons will align parallel to the field, occupying a lower energy state, and some will align in an antiparallel direction to the field by occupying the higher energy state. This phenomenon of two possible energy states in the presence of an external magnetic field is called the Zeeman effect (Grover et al. 2015). The energy difference between the two states is given by ΔE = γhB0 /2⊓ (where γ is the gyromagnetic ratio, h is Planck’s constant, and B0 is the external magnetic field). The aligned proton tends to rotate with an angular momentum along the axis of an applied field known as Larmor frequency (ω0 = γB), where ω0 is the angular frequency of the protons and γ is the gyromagnetic ratio (a constant fixed for a specific nucleus). On the application of short pulses of radiofrequency (RF) equal to the Larmor frequency, perpendicular to the applied external magnetic field, the protons absorb this excess energy causing a transition of the proton from higher to lower energy levels and vice versa. The protons relax back to their equilibrium state through two relaxations: longitudinal relaxation (T1 relaxation) and transverse relaxation (T2 relaxation) after removing the RF field. The image based on T1 relaxation produces brighter images (positive image) and T2 relaxation produces darker images (negative image). In the past decade, many attempts have been made to improve the sensitivity of contrast agents for MRI. Among other contrast agents, SPIONs have been widely investigated for MRI application for their higher MRI signal contrast (Wu and Huang 2017). SPIONs have several advantages like high saturation magnetization, biocompatibility, long shelf life, etc. Additionally, the main component of SPIONs is “Fe” which, by nature, is an abundant biomineral found in the living system and thus results in low toxicity and is easily biodegradable compared to other metals. In general, for positive T1 images, “Mn” and “Gd” based compounds are used as contrast agents (Banerjee et al. 2019; Cai et al. 2020; Kim et al. 2011; Yang et al. 2016), and for T2 images, Fe-based compounds like SPIONs are utilized as contrast agents (Moradi Khaniabadi et al. 2017; Yang et al. 2022). Wang et al. reported the synthesis of monodispersed with high relaxivity ultrasmall-sized SPION for MRI application by facile synthesis method using albumin (Wang et al. 2017). The resulting particles are in the size of 4.78 ± 0.55 nm and with a high MR relaxivity of 444.56 ± 8.82 mM−1 s−1 . In another study by Marashdeh et al., different sizes of SPION particles were investigated for MRI effect where 22 nm particle exhibits shorter dephasing compared to the 30-nm-sized particle (Marashdeh et al. 2019). They also study the effect of concentration of the contrast agent and the result proves that with the concentration, the relaxivity of the material increases. Thus, the results of this study indicated that SPIONs are the promising agent for MRI. According to several researches, the shape of the SPIONs also highly influenced the relaxivity property of the material. For example, Wei et al. investigated the influence of shape of the SPION in MRI relaxivity and reported that nanoplates (25 nm edge-to-edge length and 3 nm thickness) exhibited T2 relaxivity of 571.21 mM−1 s−1 at 0.5 T which around six folds higher than spherical particles of size 34 nm as shown in Fig. 2 (Wei et al. 2018).

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Fig. 2 In vivo liver tumor T2-weighted MR images before and after intravenous injection of iron oxide nanoplates or spherical nanoparticles for 0.5 h, 1 h, 2 h, and 4 h at a dose of 2 mg Fe per kg body weight. Adapted and reprinted with permission from Wei et al. (2018)

This is attributed to the superparamagnetic behavior and higher saturation magnetization of nanoplates (Ms = 84.4 emu/g) than the spherical nanoparticles (Ms = 69.7 emu/g). The main attractive feature of SPIONs is, at a certain level by decreasing their particle size into the ultrasmall range leads to a small r2 /r1 ratio resulting in a pronounced T1 effect means the SPIONs can also be utilized as T1 contrast agents in the ultrasmall range. For example, Zang et al. (2017) studied ultra-small MnFe2 O4 NPs of size 3 nm synthesized by the thermal decomposition method as a T1 MRI nanoprobe. The MRI measurements were taken at 3 T for different concentrations of [Fe + Mn], and it measured a r1 relaxivity of 8.43 mM−1 s−1 .

4.3 SPIONs in Anticancerous Drug Delivery Chemotherapy is the most widely used procedure for the treatment of cancer. The chemotherapeutic drugs can be used in combination or single drug itself is able to avert the quickly dividing cancer cells. Though it is used very often, it has several drawbacks such as toxicity to the normal cells due to non-specific distribution and harmful effects towards the vital organs. Therefore, nanoparticle-mediated delivery of anticancerous drugs has paved the way to deliver the drugs specifically towards the cancer cells thus the side effects can be minimized. Magnetic iron oxide nanoparticles have arisen as one of the dynamic materials for the treatment and diagnosis of cancer-like dreadful disease. Several researches are being conducted for cancer therapy using magnetic nanoparticles, especially SPIONs. In a report by Singh et al., iron oxide was coated by casein by emulsion crosslinking and the nanoparticles were prepared in situ. Iron oxide nanoparticles coated with casein and loaded with cytarabine show inhibition of the formation of the cell colony of HepG2 liver cells. It also suppresses the invasion and relocation abilities of HepG2 cells (Singh et al. 2020).

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The nanoparticles also assist in increasing the solubility of various anticancerous drugs having poor water solubility. Telmisartan, an anticancerous drug, was loaded on chitosan-coated magnetic nanoparticles and tested on human prostate cancer PC3 cells. The nanoparticles are superparamagnetic in nature with a large surface area of 134 m2 /g. At 100 μg/ml, drug-loaded chitosan-coated magnetic nanoparticles exhibit 34.2% viability of cells (Dhavale et al. 2021). In another report, the bioavailability of camptothecin, a drug with poor water solubility was enhanced by its attachment to the magnetic nanoparticles. The nanoparticles were surface modified with 3aminopropyltroethoxysilane and then the drug was attached to enhance its solubility. This formulation is superparamagnetic in nature (Patil et al. 2020).

5 In Vitro Assessment of SPIONs When the studies are done with cells, and microorganisms outside the normal biological environment, it is called in vitro. SPIONs with suitable functionalization have the least toxic effect on the cells and are recognized as potential candidate for the applications of drug delivery, MRI, and hyperthermia. SPIONs are categorized as inert materials which reduce the toxicity (Bustamante-Torres et al. 2022). These nanoparticles interact with the cells through different mechanisms. Some of the possible ways of uptake are receptor-mediated endocytosis, passive diffusion, caveolin-mediated internalization, clathrin-mediated endocytosis, and some other caveolin- and clathrinindependent endocytosis (Hillaireau and Couvreur 2009; Singh et al. 2010). SPIONs will apparently degrade into ions of iron in the lysosomes. Free iron has the ability to cross the nuclear or mitochondrial membrane. In mitochondria, this free iron may react with H2 O2 and O2 generated by mitochondria and forms hydroxyl radicals through Fenton’s reaction which are highly reactive. These radicals can damage proteins, DNA, lipids, etc. (Singh et al. 2010). In one of our reports using magnetic nanoclusters (MNCs), it was observed that nearly 37% of MCF-7 cells were killed by ROS-dependent mitochondrial pathway (Khan and Sahu 2021). Surface charge also plays an important role in the colloidal stability at a particular pH. Nanoparticles having positively charged groups can increase the cellular uptake of SPIONs in comparison to the cationic surface which can cause aggregation of platelets and hemolysis (Lei et al. 2013). The nanoparticles should be stable in the physiological medium so that their residence time should increase to perform all its functions. In this direction, Shang et al. fabricated biocompatible, biostable magnetic nanoparticles coated with reducible polydopamine (SPIONs@PDA) which can perform both drug delivery and MRI diagnoses. The nanoparticles showed good stability and enhanced drug loading efficiency and efficient T2 relaxivity with good negative contrast for MRI. The experimental analysis showed nearly 21% cell viability at the DOX concentration of 14.4 μg/ml after treatment with DOX-loaded SPIONs@PDA, whereas the bare SPIONs@PDA showed nearly no cytotoxicity on the HeLa cells which demonstrates the efficient internalization of the nanoparticles by the cells (Shang et al. 2017).

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Functionalization of the nanoparticles surface by targeting ligands can enhance the in vitro cellular uptake of the drug-loaded nanoparticles. In a report by Mosafer et al., DOX and SPIONs were entrapped in PLGA-based nanoparticles which are modified by multiple emulsion solvent evaporation techniques. AS1411 aptamer was attached to the surface of the nanoparticles by EDC/NHS mechanism. The conjugation of aptamer to the nanoparticles (Apt-NP) increases the cellular uptake of DOX in murine colon carcinoma C26 cells. Apt-NPs increase the cytotoxic effect of DOX and further inhibit the tumor effectively prolonging the survival of tumor-bearing mice (Mosafer et al. 2017). In a report by Sagir et al., magnetic nanoparticles coated with mesoporous silica (M-MSN) and functionalized with polyamidoamine (PAMAM) dendrimer were fabricated for the delivery of drug for photodynamic therapy. It was conjugated with folic acid for targeting (Sagir et al. 2022). Indocyanine green was loaded onto the fabricated nanoparticles for photodynamic therapy on the MCF-7 breast cancer cells. The PAMAM dendrimer was chosen to coat the M-MSN to make the nanocargo stimuli-sensitive. PAMAM dendrimer acts as imaging probes and gatekeepers for intelligent and fluorescent MSNs. Decrement in the viability of cells when treated with the nanoparticles in the generation manner was checked on MCF-7 cells by WST-1 cell proliferation assay. Viability of both the unirradiated and irradiated cells decreased depending upon the dose for all the generations. Concentrations of 50 and 100 μg/ml were substantially toxic in both light and dark conditions, respectively, for all the generations. G3-FA decreased the viability of cells even at low doses in comparison to the other generations in PDT. The highest cell apoptosis was attained at the concentration of 100 μg/ml of G3-FA in irradiated conditions (Fig. 3a). Morphological assessment of the cells treated with 10, 25, 50, and 100 μg/ ml of G3-FA was done after 24 h in both unirradiated and irradiated conditions. Control cells irradiated with light only didn’t show any difference from the features of MCF-7 cells showing light irradiation alone didn’t cause any cytotoxicity. Cells which are tested with the modified nanoparticles (G3-FA) were also similar to the control showing the biocompatibility of the nanoparticles. However, cells tested with the similar concentrations of G3-FA in light conditions were blebbed and shrinked (Fig. 3b) (Sagir et al. 2022). Sahoo et al. reported a pH dual-responsive core–shell nanoparticles containing polymer shell functionalized magnetic nanoparticles for drug cargo and targeting agent for cells (Sahoo et al. 2013). Magnetite nanoparticles having superparamagnetic nature were prepared by co-precipitation method. Further, amine groups were introduced on the surface by using APTES. Poly(N-isopropylacrylamide)-blockpoly-(acrylic acid) copolymer having dual-responsive nature were then conjugated to the amine groups by EDC/NHS technique. For targeting abilities, folic acid was attached to the abundant surface amine groups on the MNPs by EDC/NHS mechanism. FA-MNPs showed no toxicity on HeLa cells as well as L929 cells. However, DOX-MNPs cause substantial toxicity to both the HeLa and L929 cells. When incubated with 100 μg/ml of DOX-MNPs, 70% of the L929 cells were viable. Similar drug-loaded nanoparticles caused 84% apoptosis of HeLa cells. At the concentration of 10 μg/ml DOX-MNPs, IC50 values for the HeLa cells were observed. The

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Fig. 3 Cell proliferation percentage of MCF-7 cells treated with 10, 25, 50, and 100 μg/ml of G0FA, G1-FA, G2-FA, and G3-FA under light and dark conditions, respectively (a), Morphological alterations in MCF-7 cells treated with G3-FA under light and dark conditions (b) Reprinted with permission from Sagir et al. (2022)

noteworthy death of HeLa cells in comparison to L929 cells was because of folate facilitated receptor-mediated endocytosis (Sahoo et al. 2013).

6 In Vivo Assessment of SPIONs In vivo studies are those in which the biological objects are tested in living organisms. In vivo testing in clinical trials is an important aspect of medical research. It provides important information concerning the effect of a specific substance or progression of a disease in a living organism. For the last two decades, magnetic nanoparticles have been utilized as an imaging agent for detection based on MRI and for specific delivery of anticancerous drugs in vivo (Sun et al. 2008; Jiang et al. 2021). Magnetic

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nanoparticles produce MRI contrast by varying the relaxation of water protons after excitation by the radio frequency and then return to the equilibrium (Jiang et al. 2021). Because of the high surface-to-volume ratio, magnetic iron oxide nanoparticles have the capability to be utilized in several diseases (Khan and Sahu 2021; Quinto et al. 2015). For example, magnetic nanoparticles coated with PEG-DOXindocyanine green can deliver the DOX in the tumor, while indocyanine green can perform imaging of the drug-loaded nanoparticles (Jiang et al. 2021; Shen et al. 2019). In a report by Huang et al., photosensitizer-conjugated magnetic nanoparticles were fabricated for gastric cancer therapy and imaging. Chlorin e6 (Ce6) was anchored covalently on the magnetic nanoparticles surface (Ce6-MNP) and showed brilliant targeting and imaging abilities for near-infrared fluorescence imaging and MRI-monitored photodynamic therapy. Ce6-MNPs are biocompatible on MGC803 cells in the range of 0–84 μM concentration. Xenografts tumor tissues in the mice, when treated with Ce6 MNP and irradiated with a 632.8 nm laser for 10 min with a light dose of 5.88 J/cm2 , there was substantial tumor regression which specifies that Ce6-MNPs have exceptional imaging and targeting capability (Huang et al. 2011). In a report by Wang et al., PEG/PEI-SPIONs and PEG-SPIONs were prepared by a simple process of thermal decomposition in the mixture of PEI and PEG using iron acetylacetonate. The nanoparticles showed a high r2 /r1 ratio and after the intravenous injection of SPIONs, the in vivo MRI of the mouse brains showed a better contrast effect (Wang et al. 2015). For the improvement of the pharmacokinetics and therapeutic efficiency, construction of protein corona on the nanoparticles is an efficacious approach. In this direction, Zhang et al. demonstrated the in vivo magnetothermal regulation of protein corona on magnetic nanoparticles. This magnetothermal regulation uses the localized induction heat produced by the magnetic nanoparticles in the presence of AMF for the modulation of the composition of protein corona during the construction process (Fig. 4). Both the in vivo and in vitro results demonstrate that magnetothermal regulation causes the modulated protein corona with up-regulated dysopsonins and down-regulated opsonins. Magnetic nanoparticles with superparamagnetic nature, when exposed to AMF exhibited better pharmacokinetic behavior which includes lesser liver and spleen accumulation and enhanced blood circulation (Zhang et al. 2021). In a report by Hałupka-Bryl et al., PEGylated iron oxide nanoparticles were loaded with DOX (PEG-PIONs/DOX). Poly(ethylene glycol)-block-poly(4vinylbenzylphosphonate) block copolymer (PEG-b-PVBP) was used for surface modification. It was loaded with DOX and utilized as a potential drug cargo for targeted delivery of drug assisted by AMF. PEG-PIONs/DOX was easily gathered in the tumor area of 5–6 weeks old BALB/c mice thus making it highly efficacious for cancer therapies (Hałupka-Bryl et al. 2014). In the report by Pham et al., superparamagnetic iron oxide nanoparticles (sSPIONs) with two different diameters, 10 and 25 nm, were synthesized for in vivo assessment (Pham et al. 2018). They fabricated customized Fe2 O3 s-SPIONs with reversible addition–fragmentation chain-transfer diblock copolymer. Nude mice (BALB/c-Foxn1nu /Arc) were administered with PBS with or without s-SPIONs intraperitoneally at 90 mg iron/kg body weight dose which is equal to 7.3 mg/kg

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Fig. 4 In vivo magnetothermal regulation of protein corona on the iron oxide nanoparticles. Reprinted with permission from Zhang et al. (2021)

in humans. The 10 nm s-SPIONs were scattered in all the tissues with no buildup in kidney or brain. After 1 h of injection, iron level peaked in heart and colon, and after 4 h of post-injection, iron level peaked in spleen, liver, and ovary. After 24 h of post-injection, iron level peaked in mesentery, omentum, peritoneal lining, lungs, bladder, uterus, and stomach (Fig. 5). In between 48 h and 1 week, most of the 10 nm s-SPIONs got cleared from nearly all of the tissues. Omentum was the tissue with the highest concentration of nearly 1000 μg Fe/g tissue, 1 week post-injection. Nearly the same biodistribution profile was visible for the 25 nm s-SPIONs (Fig. 5). s-SPIONs of 25 nm were quickly cleared from the peritoneal cavity as the level of iron dropped back after 4 h. Iron level peaked after 1 h in blood and return to the control level in 48 h. There was no indication of uptake of 25 nm s-SPIONs in heart and brain and a slight increase was observed in bladder, uterus, and kidney. Iron level peaked in liver, lung, stomach, and spleen after 1-h post-injection; in omentum, colon, peritoneum, and ovary after 4 h; and in uterus and mesentry after 24 h. Within 1 week, iron level comes back to control for all the tissues. s-SPIONs of 25 nm tend to depart from the peritoneal cavity quicker and gather in several tissues earlier. They also got cleared from the tissues in the same time frame as the 10 nm s-SPIONs (Pham et al. 2018).

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Fig. 5 Biodistribution of s-SPIONs in the tissues after IP injection of 10 and 25 nm s-SPIONs at a concentration of 90 mg of Fe/kg in PBS. Adapted and reprinted from Pham et al. (2018) (Open access)

7 Toxicity of SPIONs The biocompatibility of a nanodrug cargo is associated with both the immune system response after its administration and the inherent toxicity of the carrier. From the toxicological stance, the degradation products and the colloids should be removed from the body in the smallest time after the drug release (Reddy et al. 2012). Bearing in mind the utilization of magnetic nanoparticles in the healthcare area, toxicity is the key area of concern since not much investigation has taken place in this area (Markides et al. 2012). Basically, toxicity is defined as the destructive effects in terms of chemical, biological, and physical agents(Malhotra et al. 2020; O’Keefe

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et al. 2014). Toxicity of these particles can also reduce the therapeutic efficacy (Malhotra et al. 2020; Huang et al. 2008). In a report by Gupta et al., SPIONs were synthesized and modified with pullulan to reduce the cytotoxic effects of the naked SPIONs and to enhance the cellular uptake (Gupta and Gupta 2005). Cytotoxicity studies on human dermal fibroblast cells showed that the bare particles are toxic which induced a substantial reduction in the viability of cell at a low concentration of 0.05 mg/ml and nearly 60% loss of cell viability at 2 mg/ml. Pullulan-coated SPIONs were slightly toxic as nearly 92% of cells were viable at 2 mg/ml concentration (Gupta and Gupta 2005). When dextran-coated iron oxide nanoparticles were incubated with primary human fibroblasts hTERT-BJ1, it induces apoptosis and cytotoxicity same as the uncoated nanoparticles. Though cellular uptake of both the uncoated and dextran-coated nanoparticles was same, still the dextran-coated nanoparticles induced reduction in the motility of cells as well as cell membrane disruption (Berry et al. 2004; Reddy et al. 2012). An ultra-small SPIONs coated with dextran, Ferumoxtran-10 didn’t show any cytotoxic effect on human monocyte–macrophage interaction at 1 mg/ml concentration for 72 h (Malhotra et al. 2020; Müller et al. 2007). When n-octyltriethoxysilane-coated MNP and uncoated MNP were checked on rat pheochromocytoma (PC12) cells and human neural stem cells (ReNcell Vm) for 24 h at a concentration of 0 to 64 μl, the results showed lesser cytotoxic effects from the coated MNPs (Malhotra et al. 2020; Ma et al. 2019). Stroh et al. reported citrate coated SPIONs cause a significant enhancement in the protein oxidation and oxidative stress. This study also showed that behind the generation of ROS, iron was the source as iron chelator administration causes reduction in the ROS levels (Stroh et al. 2004; Patil et al. 2018). In a report by van den Bos et al., they showed that dextran-coated SPIONs induce lipid peroxidation with an increase in the dose (Patil et al. 2018; Bos et al. 2003).

8 Conclusion SPIONs have been developed for multifunctional purpose. They offer possibilities for further functionalization of the nanoparticles. They can be utilized for magnetic hyperthermia, anticancerous drug delivery, magnetic resonance imaging, etc. They can be tuned according to the need for effective and specific delivery of the therapeutics. Still, toxicity is a major concern for the SPIONs for its utilization in clinical therapies. Therefore, proper modification of the nanoparticles’ surface is of prime importance for its utilization in cancer therapy and diagnostics. They should be nontoxic to normal cells along with enhanced retention time in blood. Thus, in vitro and in vivo assessment of the SPIONs is of much importance for its utilization in the area of biomedicine.

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Applications of Superparamagnetic Materials in Drug Delivery Toward Cancer Therapy Jagriti Gupta, P. A. Hassan, and K. C. Barick

Abstract Superparamagnetic nanomaterials are very important and extensively studied for various biomedical applications, especially in therapy and diagnosis. More specifically, they have received great attention as nanocarriers for drug delivery applications for cancer therapy due to their unique physio-chemical properties and excellent biocompatibility as well as biodegradability. Besides, other types of nanoparticles, superparamagnetic nanoparticles (SPMNPs) with suitable surface ligands have engrossed a great deal of attention in cancer treatment. Owing to high magnetization and surface-to-volume ratio, SPMNPs can be employed at the desired site of tumors by applying an external magnetic field, and consequently enhance the release of drug molecules with high efficiency. This chapter aims to provide a detailed study on SPMNPs, focusing on their fundamental properties and surface chemistry required for biomedical applications. This chapter also covers the recent advances in the development of SPMNPs together with their possibilities and limitations in drug delivery. Furthermore, we have discussed the effects of the sizes, shapes, and surface modification with the principle of passive and active drug targeting and stimuli-responsive drug release systems, i.e., pH-responsive, temperature/thermal-responsive, enzymatic-responsive, and redox-responsive drug release systems. Keywords Superparamagnetic · Nanoparticle · Targeted drug delivery · Functionalization · Toxicity

J. Gupta (B) · P. A. Hassan · K. C. Barick Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India e-mail: [email protected] P. A. Hassan · K. C. Barick Homi Bhabha National Institute, Anushaktinagar, Mumbai 400 094, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_5

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1 Introduction Cancer is one of the most incurable life-threatening diseases universally. In 2020, approximately 19.3 million new cases and almost 10.0 million deaths were reported globally (Sung et al. 2020). Various types of therapeutic approaches were proposed and developed for cancer treatment including chemotherapy, radiation therapy, photodynamic therapy, immunotherapy, and surgical. Besides them, chemotherapy (delivery of therapeutic agents) has been considered one of the most effective and economical ways of cancer treatment. However, non-selective nature and non-specific distribution, poor aqueous solubility, development of multidrug resistance, and serious side effects on healthy tissues are the biggest challenges for chemotherapy. Recent advances in nanoscience and nanotechnology have provided a new technique by bringing various branches of science together such as chemistry, materials engineering, and biology, and designed various types of novel nanomaterials for therapeutic and diagnostic applications. Specifically, magnetic materials in the nanoscale regime have received great attention owing to their unusual physiochemical, electronic, optical, and magnetic properties. Therefore, the exceptional properties and nano dimensions make them ideal candidates for various biomedical applications, especially drug delivery applications. Among numerous nanomaterials, superparamagnetic nanoparticles (SPMNPs) have gained significant attention and broadly demonstrated for various biomedical applications in the past few decades, for example, targeted drug delivery, magnetic hyperthermia, and magnetic resonance imaging (MRI) (Singh et al. 2010; Shabestari Khiabani et al. 2017). Currently, the main associated problems in the drug delivery system are the systemic drug administration and their distribution, the lack of selectivity towards a targeted tumor site, required higher dose, non-specific toxicity, and other adverse side effects. SPMNPs are one of the potential solutions to resolve the entire prevailing problem in the drug delivery system. Foremost, SPMNPs-based drug delivery systems can be straightforwardly manipulated towards a specific or targeted site with the assistance of a magnetic field that enhances the indigenous concentration and reduces the side effects of therapeutic agents. Consequently, the therapeutic agents can be effortlessly transported to the specific or targeted tumor site without damaging healthy tissues. Iron oxide mainly exists in γ-Fe2 O3 (maghemite), Fe3 O4 (magnetite), or α-Fe2 O3 (hematite) cores that exhibit superparamagnetic properties in nanosize (size smaller than 20 nm in diameter) (Akbarzadeh et al. 2012). Similar to iron oxide, mixed oxides of iron with transitional metals (copper, cobalt, nickel, and manganese) also exhibit superparamagnetic properties and are considered the family members of SPMNPs. Amongst the other members, SPMNPs are the most auspicious and received tremendous interest and outstanding accomplishments for various biological applications due to their unique properties, excellent biocompatibility, and biodegradability. Moreover, strong magnetic response selectively targeting tissues, and appropriate surface functionalization modifications accompanied by the diagnostic capability make them more attractive aspirants for drug delivery applications. Their magnetic

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Fig. 1 Applications of superparamagnetic nanoparticles (SPMNPs)

properties are not only useful for diagnostic purposes but also important for magnetically targeted/guided drug release and hyperthermia effect at the desired location by applying a magnetic field (Chauhan et al. 2021; Dennis et al. 2008; Wang et al. 2021). Consequently, SPMNPs can be accomplished as integrated next-generation therapeutic agents for cancer diagnosis and treatment. Figure 1 shows the various applications of superparamagnetic nanoparticle in the biomedical field. Furthermore, the shape, size, and surface characteristics of SPMNPs have significant importance for the development of successful drug delivery systems for both in-vitro and in-vivo (Wang et al. 2022a,b; Karaagac and Köçkar 2022; Angelopoulou et al. 2019). Functionalization of SPMNPs with suitable molecules including organic compounds like citrate, glycine, polymers, lipids, various surfactants, biomolecules or inorganic compounds like silica, metal oxides, sulfides, metal nanoparticles is usually applied to achieve better physical and chemical properties (Karaagac and Köçkar 2022). Figure 2 shows the different surface functionalization of magnetic nanoparticles. Moreover, the surface functionalization of SPMNPs not only improves water dispersion, and stability against oxidation but also enhances the payload amount of drugs. The presence of various functional groups on the surface of SPMNPs could be used for further functionalization with specific or targeting ligands (Liu et al. 2020; Zhu et al. 2018; Dutta et al. 2021). For the effective drug delivery for cancer, it is preferred that the size of SPMNPs after surface functionalization should

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Fig. 2 Different surface functionalizations of SPMNPs

not exceed 100 nm to escape rapid clearance by the reticuloendothelial system (RES) (Nie 2010; Tang et al. 2019). It was observed that the surface functionalization of SPMNPs not only improves various properties but also helps to enhance biocompatibility. Recently, magnetic vortex Fe3 O4 @PVP@DOX nanostructures were reported for magneto-triggered on-demand magnetic responsive controllable drug release, magnetic hyperthermia, and MRI as a synergetic theranostic agent for cancers (Wang et al. 2022b). It was observed that the surfaces of the magnetic nanoparticles with the poly (ethylene glycol) (PEG) improved the biocompatibility of the nanoparticles by resisting protein adsorption and increasing their intracellular uptake. To date, substantial progress was made in the controlled shape and size of the magnetic nanoparticles by developing various synthesis approaches such as coprecipitation, hydrothermal, solvothermal, thermal decomposition, microemulsion, sonochemical, microwave-assisted, and physical methods, etc. (Gupta et al. 2016; Kumar et al. 2020; Mohapatra et al. 2015; Salvador et al. 2021; Majidi et al. 2016; Ansari et al. 2019). Among other methods, the co-precipitation method is the most popular and widely used method for magnetic nanoparticle synthesis due to the non-toxic nature of the chemicals employed during synthesis. However, the particles obtained by co-precipitation, unfortunately, tended to agglomerate and polydisperse. Conversely, high temperature and organic precursors are widely used in other methods for controlling the shape and well-defined monodispersed size having narrow size distribution. However, the obtained particles are hydrophobic and hence, surface modification was usually applied to make them hydrophilic for biomedical

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applications (Liu et al. 2014). Additionally, the prepared nanoparticles with dimensions on the order of 10 nm possess low magnetization per particle and hence separation of the particles is difficult from the solution and their movement in blood by applying magnetic fields. The chapter aims to consolidate the recent advances in superparamagnetic nanoparticles (SPMNPs) for drug delivery in cancer treatment. Various important aspects of SPMNPs for cancer therapy were discussed in detail. Furthermore, future research prospects, the potential of SPMNPs, and associated challenges in cancer theranostics were deliberated. However, there are still many challenging issues associated with the clinical precision medicine of cancers such as targeting and precisely controlling the drug release in vivo.

2 Important Characteristics of SPMNPs Required for Drug Delivery The required characteristics for drug delivery, SPMNPs should be superparamagnetic with high magnetic saturation, narrow in size, large surface area, and good chemical and colloidal stability in water along with excellent biocompatibility. The basic requirements that are important for superparamagnetic-based nanoparticles to be used in drug delivery applications are presented in Fig. 3.

Fig. 3 Physicochemical parameters of SPMNPs required for effective drug delivery

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2.1 Superparamagnetic Nature The magnetic nanoparticles possess distinct physical and chemical properties from the bulk material. The magnetic nanoparticles (Fe3 O4 ) show superparamagnetic behavior when composed of a single magnetic domain. The superparamagnetic nanoparticles exhibit high magnetization under the influence of a magnetic field and they are easily demagnetized after the removal of the magnetic field (Xiao and Du 2020; Singamaneni et al. 2011). In SPMNPs, thermal fluctuations are strong enough to spontaneously demagnetize a previously saturated assembly; therefore these particles have zero coercivity and have no hysteresis. Superparamagnetic behavior is crucial for various biological applications such as magnetically targeted drug delivery, MRI, cell separation, etc., (Ma et al. 2021). Moreover, the SPMNPs with higher magnetization is not only advantageous to facilitating the release kinetics of drugs but also avoid agglomeration of NPs after removal of the magnetic field. Another important characteristic shown by magnetic nanoparticles is Neel and Brownian relaxation formed as a result of energy absorption. Upon exposure to alternating magnetic fields, the dipole moments of the SPMNPs are easily rearranged, depending on the particle size, magnetic field strength, frequency, and temperature. The generated energy due to Neel relaxation and Brownian relaxation disintegrates in heat and raises the temperature of the solution. The increase in the temperature of the solution of SPMNPs is utilized in cancer cells as magnetic hyperthermia. Moreover, magnetic nanoparticles show shape and size-dependent magnetic properties. Therefore, they possess different heating behaviors under various magnetic fields (Nemati et al. 2018; Mohapatra et al. 2018; Ali et al. 2019).

2.2 Effect of Shape and Size on Cellular Uptake Besides magnetic property, another important feature that needs to be understood for biomedical applications is the shape, size, and surface properties of superparamagnetic nanoparticles. Figure 3 discribes the basic requirement of drug delivery using SPMNPs. A great effort has been made by designing the different shapes of magnetic nanoparticles to prolong them at the targeted site and increase their blood circulation time, cellular uptake and biodistribution as well as improve the cellular toxicity. Various shapes of the SPMNPs like spherical, cube, rods, etc. were reported for biomedical applications including drug delivery, magnetic hyperthermia, and MRI (Dutta et al. 2018; Nizamov et al. 2018). However, the study on the shape of superparamagnetic nanoparticles was not extensively demonstrated as it concerns its effect on biodistribution. Nevertheless, few shape-dependent studies with other nanomaterials reported that non-spherical and rod-shaped nanoparticles possess a longer blood circulation time compared with the spherical nanoparticles (Huang et al. 2011; Shao et al. 2017; Salatin et al. 2015). Huang et al. designed two different shaped fluorescent MSNs and investigated the effects of particle shape on biodistribution,

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clearance, and biocompatibility in vivo (Huang et al. 2011). Short-rod MSNs were presented in the liver, whereas long-rod MSNs were presented in the spleen showing the shape-dependent in-vivo behavior and the clearance rate of MSNs are primarily dependent on the particle shape, where short-rod MSNs have a more rapid clearance rate than long-rod MSNs in both excretion routes. In another study, Shao et al. reported the shape influence on the endocytosis, biocompatibility, and biodistribution of magnetic mesoporous silica nanoparticles (M-MSNPs), three FITC-labelled M-MSNPs with a different aspect ratio (AR = 1, 2, and 4). Long-rod M-MSNP2 possessed higher intracellular internalization than the short-rod M-MSNP1 and the sphere-like M-MSNP0 in both cancer cells and normal cells due to the difference in the endocytosis pathways without any significant shape effects on biocompatibility. Moreover, the biodistribution study showed that M-MSNPs were mainly presented in the reticuloendothelial system (RES) organs including the liver, spleen, and kidney while sphere-like M-MSNP0s were easily trapped in the liver and long-rod M-MSP2 exhibited more retention in the spleen. Rod-like M-MSNPs are preferentially accumulated in tumor sites than sphere-like M-MSNPs, indicating an improved drug delivery efficacy in cancer therapy. Over the other nanostructures, spherical nanoparticles show more significant advantages for instance controlled the size of the sphere, and even surface functionalization with suitable ligands as compared to other shapes. These factors significantly affect the loading content of the drug molecules on the surface of nanoparticles for better drug release at the targeted site and hence illustrate better cellular toxicity. Based on the existing literature, the reported nanoparticles are spherical and extensively used in both in-vitro for various cancerous cells and invivo studies. The numerous studies on SPMNPs show substantial biocompatibility and high payload of therapeutic agents for effectiveness in inhibiting cancer cell growth, which reveals the auspicious perspective of nanoparticles for drug delivery application. Besides the shapes, the size of the nanomaterials also possesses significant importance in cellular uptake and determination of their half-life in the blood circulation (Chouly et al. 1996; Di et al. 2021; Feng et al. 2018). For illustration, particle sizes smaller than 10 nm are easily removed by renal clearance while larger particles (larger than 200 nm) accumulate in the spleen or are harvested by phagocytic cells of the body. Henceforth, the size of the nanoparticles should be optimized to maximize the cellular uptake rate of the nanocarrier. Generally, a particle size in the range of 10–100 nm diameter is reported an optimal size for their effective delivery to target sites for various cancer cells and showed longer circulation times due to easy escape from the reticuloendothelial system in the body. Moreover, larger particles of about 100 nm possess a high affinity to bind with a large number of receptors and might restrict the binding of other molecules or additional particles. Moreover, various biological applications of SPMNPs such as magnetic resonance imaging (MRI), magnetic hyperthermia, magnetic separation, and targeting depend on the magnetic properties which largely depend on the size of the particle (Mohapatra et al. 2018; Unterweger et al. 2018; Chen et al. 2018; Caro et al. 2019). The small size of SPMNPs also improves the permeability and retention effect which causes the maximal accumulation of nanoparticles at the targeted tumor site. Conversely,

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nanoparticles size less than 2 nm are not acceptable for biological applications due to the potential diffusion through the cell membrane. Consequently, small size nanoparticles damage intracellular organelles and reveal potential toxic effects. Besides these, nanoparticles of different shapes and sizes internalize through narrower capillaries. Their agglomeration might cause blockage in the blood system. Therefore, controlled and well-defined shape and size of particles should be an important consideration for an effective drug carrier.

2.3 Surface Functionalization and Colloidal Stability of SPMNPs A surface property of SPMNPs strongly affects the colloidal stability and efficacy of drug loading content and their performance in drug delivery. Mostly, nanoparticles used in delivery in-vivo must have a good antifouling property to avoid the nonspecific adsorption of proteins, macrophage cellular uptake, and pharmacokinetics. The adequate surface functionalization of magnetic nanoparticles can significantly prolong blood circulation time (Ma et al. 2017). The surface functionalization of nanoparticles has a substantial effect on their physicochemical characteristics that significantly affect circulation, biodistribution, cellular Internalization, and trafficking (Duan and Li 2013). The surface functionalization of SPMNPs with silica, small organic molecules (citric acid, glutamic acid, L-cysteine, etc.), dendrimers, protein, polymers, peptides, lipids, and biological molecule (folic acid, RGD) like are an effective approach to improve the physicochemical properties of nanoparticles (Gupta et al. 2016; Ma et al. 2017; Dutta et al. 2020; Villanueva et al. 2009; Singh et al. 2021; Gawali et al. 2021; Ahmad et al. 2020; Veloso et al. 2021; Deng et al. 2015). The surface functionalization not only increases the colloidal stability but also improves the dispersibility of nanoparticles and bioavailability as well as the biocompatibility of the SPMNPs. Moreover, various surface groups are available on the surface of the functionalized magnetic nanoparticles that help to conjugate various targeting and therapeutic agents on them. Moreover, they also increase blood circulation time by preventing the clearance through the reticuloendothelial system (RES). Besides them, magnetic nanoparticles are not only very susceptible to oxidation under ambient conditions but also have a tendency to agglomerate owing to their high specific surface area to volume ratio and strong magnetic dipole–dipole interactions (Liu et al. 2020). These properties significantly affect the rapid total clearance of magnetic nanoparticles by the reticuloendothelial system (RES) (Hu et al. 2018). Henceforth, suitable surface functionalization needs to be done to sustain the desirable properties of magnetic nanoparticles in drug delivery applications. Furthermore, SPMNPs possess both positive and negative surface charges depending on the surface groups, indicating good dispersion stability, and consequently do not agglomerate in different dispersion media on storage. The surface charge on the surface of SPMNPs greatly affects the internalization behavior in their

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target cells and their distribution in the body (Villanueva et al. 2009; Maurizi et al. 2015). Furthermore, positively charged nanoparticles possess better cellular internalization but show poor distribution and short circulation time due to the opsonization. Whereas, negatively charged nanoparticles have poor cellular internalization but they are very well distributed in the tumor and have a longer circulation time (He et al. 2010). However, the cellular internalization of nanoparticles is also governed by the type of cells. The cell membrane is slight negatively charged as it consists of a phospholipid bilayer, glycolipids, and glycoproteins, and the electrostatic attractions play an important role in the uptake behavior of the nanoparticles. Moreover, the surface functionalization of SPMNPs provides a specific mechanism to allow the endorsement of the nanoparticles either through endocytosis, macropinocytosis or phagocytosis, or diffusion (Guggenheim et al. 2020). Furthermore, the surface functionalization SPMNPs with hydrophilic polymers (PEG, dextran) and surfactants (polysorbate 80 and 20 (PS-80 and PS-20)) inhibit the formation of the protein layer at the surface of the particle by providing hydrophilic and neutral ligands. It was found that PEG and polysorbate 80 functionalized nanoparticles are not only effective to improve the pharmacokinetic profile but also assist to enhance the accumulation of nanoparticles at the tumor site as well as show an improvement in the transport of nanoparticles to the blood–brain barrier (BBB) (Huang et al. 2016).

2.4 Biocompatibility and Toxicity of SPMNPs Biocompatibility and associated toxicity of SPMNPs are important aspects before applying in in-vivo (Malhotra et al. 2020a; Kim et al. 2006). Fe is present in ferritin as a naturally occurring metal in the human body. Consequently, SPMNPs are reported as safe and biocompatible materials for the body (Markides et al. 2012). Moreover, the location of SPMNPs in the cells is an extremely important consideration. For example, a cytotoxic response of SPMNPs could be associated either with their internalization by potentially interfering with the biological function or interfering with the cell surface interaction when nanoparticles are attached to the cell surface (Reddy et al. 2012). Besides, specific characteristics such as structure, size, shape, chemical composition, surface modification, dosage, biodistribution, bioavailability, solubility, immunogenicity, and pharmacokinetics could further induce a toxic response due to aggregation and coagulation (Malhotra et al. 2020b). With the use of SPMNPs in vivo application, it is correspondingly imperative to study the degradation, accumulation, and excretion of the nanoparticles or by-products in various tissues and organs. The reaction of the degradation products with various components of the body or cells needs to be understood and therefore appropriate investigations should always be performed. SPMNPs-based products such as Ferumoxtran-10, Feridex, Endorem, Combidex, and Sinerem are approved by FDA and are currently used for MRI. Among them, Ferumoxtran-10 is a dextran-coated USPIO (ultra-small SPIONs) NPs, which is accepted for humans. However, this product induces transient effects including

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urticaria, diarrhea, and nausea (Anzai et al. 2003). SPMNPs demonstrate tolerable safe and non-cytotoxic concentration ≤ 100 μg/ml (Caro et al. 2019). Pradhan et al. demonstrated a non-toxic effect of a thin lipid layer with mesoporous magnetite nanoassemblies (LMMNA) up to 1 mg/mL against sensitive (A2780S) and cisplatinresistant (A2780-CisR) ovarian cancer cells (Pradhan et al. 2016). It was suggested that magnetic nanoparticles biodegraded and cleared from the body through the endogenous iron metabolic pathway (Nosrati et al. 2019; Marín-Barba et al. 2018; Rojas et al. 2017). The first released iron is metabolized in the liver or either used in the production of red blood cells or eradicated from the body through the kidneys (Jiang et al. 2019). Specific pathways administrate the iron contents released from the magnetic nanoparticles in the body. The biotransformation of MNPs was also reported in atherosclerotic LDLR−/− mice. The uptake of SPMNPs was revealed by macrophages and endothelial cells in the liver, spleen, and atherosclerotic lesions. The magnetic nanoparticles are accumulated in the lysosomal compartment for degradation (Poller et al. 2018). The biotransformation of the magnetic nanoparticles takes place in the lysosomal environment, resulting in a transfer of iron from magnetic particles to ferritin in macrophages and endothelial cells (Levy et al. 2011). Therefore, the lyzosome-mediated degradation mechanism for SPMNPs is generally considered the main intracellular metabolic-degradation process. Besides, it was reported that the presence of ferritins was found to delay the degradation process of magnetic particles by forming a complex colloidal mixture in an acidic medium (Volatron et al. 2017). The magnetic nanoparticles are stored as maghemite oxide or ferritin which makes them useful for MRI detection of atherosclerotic plaques in clinics (Maraloiu et al. 2016).

3 Tumor Targeting Pathways The non-specific or non-target nature of therapeutic agents is one of the biggest challenges in drug delivery in cancer, causing severe adverse effects to the healthy cells/tissues. Therefore, substantial efforts were made for designing proficient strategies for drug delivery applications. The introduction of various types of nanomaterials (organic and inorganic carriers) in drug delivery is a promising approach to reducing the adverse effects of therapeutic agents by delivering them at the targeted site. The therapeutic agents are attached to the nanocarriers either by electrostatic interaction or covalent conjugation. For the effective drug delivery, the nanocarriers must possess a higher payload capacity of therapeutic agents and their site-specific targeting ability having maximized blood half-life (minimal loss in blood circulation) as well as deliver the effective concentration of the therapeutic agent to the targeted site without any undesirable harmful effects and interactions with blood, cells, and proteins. Once the nanoparticles enter the body, opsonin proteins in the blood attach to the particles (opsonization) and form a large aggregate of the nanoparticles that are easily recognized by the reticuloendothelial system (RES) or the mononuclear phagocyte system (MPS). As a result, it reduces the phagocytosis of MPS, and the

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Fig. 4 Different targeting mechanism of nanoparticles (Dutta et al. 2021)

circulation half-life of the nanoparticles in the body. The opsonization greatly affects and governs the fate of the nanoparticles in the biological system (Yallapu et al. 2015). The cellular internalization of nanoparticles is predominantly governed by two types of targeting pathways i.e. passive targeting and ligand-receptor mediated active targeting. Figure 4 shows the different targeting mechanisms of nanoparticles.

3.1 Passive Targeting In the passive targeting, SPMNPs enter the tumor due to their small size and ability to accomplish longer retention time and enhanced cell permeability (EPR) and the hyperpermeable vasculature and defects in lymphatic drainage systems in tumors as compared to normal tissues (Dutta et al. 2021). Besides them, surface functionalization with suitable ligands or molecules with appropriate size (86% of cells were viable

>90% of cells were viable

>90% of cells were viable

Sulphorhodamine B

MTT

∼60% of cells were viable >90% of cells were viable

MTT

Assay

78% of cells were viable

Effect on cell viability

(continued)

Calmon et al. (2012)

Patil et al. (2016)

Patil et al. (2016)

Thorat et al. (2014b)

Thorat et al. (2014b)

Thorat et al. (2014b)

Bhardwaj et al. (2020)

Pradhan et al. (2007)

Pradhan et al. (2007)

Pradhan et al. (2007)

Lotfi et al. (2017)

Lotfi et al. (2017)

References

202 K. Parekh and N. Jain

TEG

TEG

Silica

Polyvinylpyrrolidone

Polyvinylpyrrolidone

Alginate coated and functionalized with galactosamine

Poly amidoamine (G4) dendrimers

Co.Zn ferrites

Co.Zn ferrites

Mn ferrite nanoassembly

Fe3 O4

Sulfur doped iron oxide

Fe3 O4

Iron oxides

MNPs prepared with KOH (74%)

None

Magnetite a MNP-KOH

a

HeLa

None

Magnetite a MNP-KOH

MC4L2

HepG2

HeLa

BT-474

HeLa

L929

MCF7

HeLa

Cells

MNP composition Coating

Table 1 (continued)

10, 100, 500, 1000, 1500 μg/mL

24

24

1,000 μg/ml

24/48/72

800 μg/ml 24/72

24/48

600 μg/ml

0-400 ppm

24

24

24/48

24/48

Hours of treatment

2 mg/mL

2 mg/mL

0.1 mg/ml

0.05 mg/ml

Concentration of MNP

MTT

∼40% of cells were viable

MTT

MTT

MTT

No significant cytotoxicity up to 500 ug/ml (91.3% cell viability) cell viability reduced at 1000 ug/ml-66% 1500 ug/ml-31%

No significant decrease in cell viability

MTT

MTT

>80% of cells were viable CCK8 over the concentration range from 0 to 400 ppm

79/68/61% of cells were viable respectively

90% of cells were viable

Up to 78% of cells were viable

MTT

MTT

∼50% of cells were viable

Up to 82% of cells were viable

Assay

Effect on cell viability

Salimi et al. (2020)

Liao et al. (2015)

Guan et al. (2020)

Aliakbari et al. (2019)

Kumar et al. (2015)

Bohara et al. (2015)

Bohara et al. (2015)

Calmon et al. (2012)

Calmon et al. (2012)

References

Toxicology of Superparamagnetic Materials 203

Surfactant coating

Lauric acid

Lauric acid

Lauric acid

Folic acid and polyethylene glycol

MNP composition

Fe3 O4

MnFe2 O4

CoFe2 O4

Allyl-SPION

Mice

Mice

Mice

Mice

Model

Table 2 Effect of MNPs on mouse models

Intravenous/ 48 μmol Fe/ kg

Intravenous/ 50 mg, 200 mg, and 400 mg/kg body weight

Intravenous/ 50 mg, 200 mg, and 400 mg/kg body weight

Intravenous/ 50 mg, 200 mg, and 400 mg/kg body weight

Mode/ Dosage

24 h

24 h

24 h

24 h

Treatment time

Not performed

No significant change except an increase in SGPT levels at a dose of 400 mg/kg body weight

No significant change

No significant change

Hematology

Very little difference compared to control

No significant change

No significant change

No significant change

Biochemistry

No pathological changes

No pathological changes

No pathological changes

Histology

(continued)

Hayashi et al. (2013)

Pradhan et al. (2007)

Pradhan et al. (2007)

Pradhan et al. (2007)

References

204 K. Parekh and N. Jain

Surfactant coating

PEG

MNP composition

Fe3 O4

Table 2 (continued)

Mice

Model

Intravenous/ 1.5, 2.5, 5 mg/kg

Mode/ Dosage 4h

Treatment time On day 7 post-injection, Blood counts were in normal range

Hematology Four indexes: AST, total bilirubin, BUN, and creatinine were within the normal range ALT enzyme slightly increased compared to PBS control. On day 14 post-injection, the increased ALT level returned to normal

Biochemistry

References

Slight cell infiltration in Feng et al. the portal area of the (2018) liver was identified. Splenic plasmacytosis was also noted, which may represent a response to antigenic stimulation, presumably due to the uptake of exogenous IONPs

Histology

Toxicology of Superparamagnetic Materials 205

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K. Parekh and N. Jain

Further, the heating efficiency of magnetic fluid, a suspension of MNPs, depends on the specific absorption rate (SAR) (W/g) which is a function of material composition and its structural properties such as material type, particle size, size distribution, shape, dipolar interaction, surface functionalization, etc. To determine SAR, MF of known concentration is subjected to an AMF of known amplitude and frequency. SAR is equal to the dissipated power divided by the magnetic material density, showing that the SAR varies linearly with the product of frequency ( f ) and the square of the magnetic field (H2 ) which is calculated using the following equation: SAR = C p ·

1 T · t ϕmagnetic

where C p denotes the specific heat capacity of the magnetic fluids given by C p = mparticles ∗ C p -particles + mcarrier ∗ C p -carrier where T denotes the slope of the graph between temperature rise and induction t heating time. ϕ magnetic is the weight fraction of the magnetic content of the particles. A major requirement for MFH, in addition to a biocompatible MF with a higher SAR value so that a smaller amount would be required to inject into a tumor, is an induction heating device that consists of coils to generate alternating magnetic field within the safety limits. Iron oxide nanoparticles magnetite (Fe3 O4 ) and maghemite (γ-Fe2 O3 ) are mostly used for different biomedical applications due to their low toxicity index on normal cells and apoptotic effects on cancer through hyperthermia. However, mixed ferrite such Ni, Zn, Mn ferrite as well Cobalt ferrite are in development due to their high SAR for hyperthermia, but their cytotoxicity is being evaluated.

4 In Vitro Magnetic Hyperthermia Toxicity For any non-surgical-based cancer treatment approach, it is important that the therapeutic strategy should lead to the killing of cancer cells with minimal or no effects on normal cells. Moreover, the death of cancer cells should occur through the mechanism of apoptosis and not necrosis to avoid any type of undesirable inflammatory responses. In vitro approach, therefore, becomes an important tool to obtain a preliminary insight into the effectiveness of MFH contemplating the concentration of MF, time of treatment, and applied AMF within safety limits. Magnetic Hyperthermiabased cell toxicity studies have focused to develop MF with higher SAR values so that a hyperthermic temperature window of 42–45 °C can be reached at the earliest and kill a maximum number of cancer cells. The said temperature is reported to kill cells through apoptosis and temperature beyond 45 °C leads to cell necrosis. Further, it has also been observed that a single session of magnetic hyperthermia may not be enough to kill a large number of cancer cells, therefore multiple sessions

Toxicology of Superparamagnetic Materials

207

of magnetic hyperthermia have also been attempted for the maximal killing of cancer cells in vitro. After analyzing the in vitro cytotoxicity of MF, as mentioned in Table 1, most authors proceeded to validate the cytotoxic effect of the same MF under AMF with a frequency between 100 and 500 kHz maintaining the upper safety limit of applied AC magnetic field either less than or close 5 × 109 Am−1 s−1 . Furthermore, the killing of cancer cells was achieved by two approaches, one using single session and other using multiple session hyperthermia (Table 3).

5 In Vivo Magnetic Hyperthermia Toxicity Taking into account the obvious potential of MHF for cancer treatment based on the promising clinical trial results on late-stage, recurrent cancer cases as discussed later in this chapter, and encouraging results on cell lines using the different combinations of MF, concurrent attempts have also been made to investigate the effect of MFH on animal models to systematically develop MFH to treat early-stage cancer patients as well. With regard to in vivo magnetic hyperthermia toxicity studies on mouse models, the animals were mostly xenografted subcutaneously to develop solid tumors of the breast, lung, colon, pancreas, brain, etc. The hyperthermic studies revealed either regression of tumor growth or complete elimination of tumors after MFH as mentioned in the Table 4, with the exposure time of induction heating ranging from a minimum of 2 to a maximum of 20 min. Although intravenous, tracheal instillation and inhalation-based MFH treatment have also been reported on animal models, the present chapter has focused on intratumoral injection of MF and the subsequent effect under AMF. Direct injection of MF into the tumor is the most preferred way of tumor ablation using MFH as it supplies MF straight into the tumor and the tumor site can be placed precisely under the magnetic field. However, in most of the presently reported animal studies, the magnetic field crossed the biological safely limit of the magnetic field of 5 * 109 Am−1 s−1 . Moreover, it is well known that the temperature of the tumor tissue under treatment should be maintained between 42 and 45 °C to kill the cells through apoptosis, however, many studies reported the ablation of tumor after crossing 45 °C temperature. A temperature above 45 °C is known to cause cell death through necrosis that could be detrimental to the patient during MFH treatment since necrosis leads to adverse inflammatory responses. Nevertheless, in vivo results available to date are propitious and have paved the way to further investigate various MF types under the different fields for the development of MFH for cancer treatment.

Coating

Hydroxyapatite

SiO2

PEG

Terephthalic acid (TA) and aminoterephthalic acid (ATA)

Terephthalic acid (TA) and aminoterephthalic acid (ATA)

MNP composition

Fe3 O4

Fe3 O4

La.Sr.MnO3

Iron oxide

Iron oxide

MCF7

MCF7

MCF7

HeLa

MG-63

Cell line

Table 3 In vitro cytotoxic effect of MFH

1 mg/ml

0.5 mg/ml

1 mg/ml

2 mg/ml

0.1 mg/ml

Concentration of MF

Not performed

Not performed

Not performed

Not performed

∼80% of cells died, 60 min HT after 24 h MF treatment

751.5 kHz/10.9 ∼90% cells died, kA/m 60 min of HT after 15 min MF treatment 751.5 kHz/10.9 ∼100% cells kA/m died, 60 min HT after 15 min MF treatment

265 kHz 23.87 kA/m

250 kHz 26.66 kA/m

∼60% cells died, 20 min HT after 2 h of MF treatment

Effect on cell viability due to two or more MFH sessions Not performed

Effect on cell viability due to single session MFH Almost all cells died, 30 min HT after 12 h of MF treatment

307 kHz 628 Oe

AMF strength kHz or SAR

TB

TB

MTT

SRB

TB

Assay

(continued)

Kandasamy et al. (2018)

Kandasamy et al. (2018)

Thorat et al. (2016)

Majeed et al. (2014)

Mondal et al. (2017)

References

208 K. Parekh and N. Jain

Coating

Lauric acid

Lauric acid

Triethylene glycol

None

MNP composition

Mn.Zn.Fe2 O4

Mn.Zn.Fe2 O4

γ- Fe2 O3

Mn.Fe2 O4

Table 3 (continued)

Saos-2

MCF7

MCF7

HeLa

Cell line

0.25 mg/mL

2 mg/mL

0.35 mg/ml

0.35 mg/ml

Concentration of MF

765 kHz 23.87 kA/m

765 kHz 24.67 kA/m

330 kHz 15.3 kA/m

330 kHz 15.3 kA/m

AMF strength kHz or SAR

1st session ∼25% cells died, ∼5 min HT after 48 h of MF treatment

Assay

Not performed

2nd session performed after 48 h ∼75% cells died. 18–23 of HT

TB

MTT

MTT

2nd TB session performed after 24 h ∼80% of cells died. 60 min of HT 3rd session performed after 24 h of 2nd session Almost all of the cells died. 60 min of HT

Effect on cell viability due to two or more MFH sessions

Up to 99% cell Not performed death, 90% of cells died, 60 min HT after 24 h of MF treatment

1st session Up to 60% of cells died, 60 min HT after 24 h of MF treatment

Effect on cell viability due to single session MFH

(continued)

Makridis et al. (2014)

Gkanas (2013)

Bhardwaj et al. (2020)

Bhardwaj et al. (2020)

References

Toxicology of Superparamagnetic Materials 209

Coating

None

MNP composition

Mn.Fe2 O4

Table 3 (continued)

Saos-2

Cell line

0.5 mg/mL

Concentration of MF

Effect on cell viability due to single session MFH

765 kHz, 23.87 1st session kA/m ∼25% cells died, ∼5 min HT after 48 h of MF treatment

AMF strength kHz or SAR

2nd session performed after 48 h ∼90% cells died 18–23 of HT

Effect on cell viability due to two or more MFH sessions TB

Assay

Makridis et al. (2014)

References

210 K. Parekh and N. Jain

PEG

Mg.γFe2 O3

Nude mice/ Hep3B hepatocellular carcinoma cells xenografted

Nude mice/MB231 breast cancer cells xenografted

PEG 600

Fe3 O4 calcium phosphate cement

Model

Nude Mice/MCF7 breast cancer cells xenografted

Surfactant coating

Fe3 O4 nanorings mPEG

MNP composition

Table 4 In vivo effect of MFH on tumor growth Treatment Temperature Modality

74.73 ± 9.12 °C ∼2 min (Very high temperature)

≈50.2 °C ∼10 min (High temperature)

A 100 μL of Intratumoral 99 kHz 12.33 kA/ 1.15 mg mL−1 conc. injection MF for ≈1000 mm3 m tumor volume 5 min

400 kHz 44 ± 0.5 °C Intratumoral 31.83 kA/ 10 min injection m (Above biosafety limit) 10 min

Mode

0.36 g/0.2 ml MCPC Intratumoral 626 kHz 28.6 A injected in tumor of injection 3 min ∼2 cm3 volume

0.3 mg/ml MNPs in 0.5 cm3 tumor volume

Dosage

References

Tumor killed in 2 days

After two weeks, black necrotic tissue separated from the body and fell off

(continued)

Jang et al. (2018)

Xu et al. (2015)

No tumor Liu et al. was (2015) detected after 6th day. No recurrence observed up to 40 days. Untreated mice showed 25 fold tumor growth by the 40th day

Effect on Tumor

Toxicology of Superparamagnetic Materials 211

Hydroxypropyl methyl cellulose, polyvinyl alcohol

Fe3 O4

Nude mice/MB231 breast cancer cells xenografted

Phosphorylated-PEG Nude mice/MCF7 breast cancer cells xenografted

Fe.Mn.O

Model

Surfactant coating

MNP composition

Table 4 (continued)

Intratumoral 366 kHz 31.8 kA/ injection m Above biosafety limit 10 min Intratumoral 626 kHz 28.6 A injection

≈0.1 m MNPs in 200 mm3 tumor volume

60 μl of HPMC/ 60%Fe3 O4 >60 °C Very high temperature

≈45 °C

Treatment Temperature Modality

Mode

Dosage

References

(continued)

Tumor Wang et al. disappeared (2017) after 3 min

Tumor Liu et al. eliminated (2016) completely on 10th day

Effect on Tumor

212 K. Parekh and N. Jain

Surfactant coating

Citric acid

Poly amidoamine (G4)

MNP composition

CoFe2 O4

Iron oxide

Table 4 (continued)

BALB/c mice/ MC4L2 breast cancer cells injected into right inguinal flank

BALB/c mice/4T1 breast and CT26 colorectal cancer cells injected into right hind flank

Model Intratumoral 261 kHz 42–43 °C and 19.89 kA/ 46–48 °C injection m (Near to upper biosafety limit) For CT26 tumors, single 30 min exposure to AMF For 4T1 tumors 3 cycle of MHT with interval of 24 h b/w each cycle Intratumoral 300 kHz 45 °C 12 kA/m injection 20 min Treatment repeated 3 times with a gap 1 day

≈100 μl 60 mg/ml iron for ≈30 mm2 tumor volume

5 mg/ml for ≥50 mm3 tumor volume

Treatment Temperature Modality

Mode

Dosage

References

Significant Salimi reduction in et al. tumor (2020 ) volume

Complete Garanina regression et al. of CT26 (2020) and 4T1 tumors at 42–43 °C (single cycle) and 46–48 °C (3 cycles) respectively

Effect on Tumor

Toxicology of Superparamagnetic Materials 213

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K. Parekh and N. Jain

6 Magnetic Hyperthermia in Clinical Trials The very first and the only nanotechnology device (NanoActivator) which is approved for the treatment of brain cancer by the EU is installed in Germany and Poland for treatment of brain tumors/glioblastoma (https://www.magforce.com/en/home/ ). MagForce Nanotechnologies AG, Berlin, Germany has the technical capability to establish NanoTherm therapy (treatment of cancer using magnetic fluid hyperthermia) as a valued combined or monotherapy for the treatment of brain and prostate cancers with the medical community. The increase in numbers of commercial treatments in Germany and Poland for Glioblastoma, has shown a potential of this therapy and consequently the proof-of-principle for other solid tumors are also being carried out. The applicator MFH300F at MagForce Nanotechnologies AG, Berlin, Germany generates a variable magnetic field strength of 0–18 kA/m at 100 kHz frequency. The applicator complies with the safety criteria for medical use imposed by the respective European authorities. Due to its universal design, it can be used for the treatment of malignancies at almost every location of the human body. Fiber-optic thermometry probes are integrated for minimally invasive measurements of the treatment temperatures. The magnetic fluid used for the treatment contains 15 nm size iron oxide nanoparticles and manufactured according to European medical device regulations. The first clinical feasibility study on magnetic fluid hyperthermia was carried out with 14 patients (2 with primary tumors and 12 with recurrences) suffering from glioblastoma multiforme (GBM) in March 2003. All patients received a neuronavigationally guided injection of the magnetic fluid into the tumor. The study showed that the thermotherapy was well tolerated at magnetic field strengths of 3.8–13.5 kA/ m with minimally observed side effects (Maier-Hauff et al. 2007). In phase I trial, a total of six thermal therapies at weekly intervals or sometimes two treatments per week with 60 min duration of each therapy was conducted using an AMF applicator. The intratumoral temperature was kept around 44.6 °C. This results showed that MFH technique is feasible and patients exhibited tolerability towards MFH. The success of the phase I study was followed by a phase II study. The treatment efficacy in 65 patients suffering from recurrences of GBM and its evaluation is in progress. In 2013, the post-marketing clinical study in recurrent glioblastoma with NanoTherm® Therapy was started by MagForce AG. The trial is an openlabel, randomized, and controlled study to determine the efficacy and safety of NanoTherm® monotherapy alone and also when used in combination with radiotherapy versus radiotherapy alone in up to 280 glioblastoma patients. In 2019, MagForce USA, Inc. successfully completed stage 1 of its clinical study on the focal ablation of intermediate-risk prostate cancer, and the next stage is under preparation. The clinical trials of MFH therapy is more effective when it is combined with the radiotherapy, chemotherapy, immunotherapy and photothermal therapy. The combined therapy will not only reduce the toxicity and side effect of the individual treatments but also improve the prognosis of patients. The advantage is ascribed to the

Toxicology of Superparamagnetic Materials

215

fact that MFH increases the blood circulation due to the expansion of tumor blood vessels. Also, the thermal enhancement improves the drug cytotoxicity, increases intracellular uptake of drugs by changing the permeability of the cell membrane, and controls drug release for an on-demand need basis. Combining MFH with radiation therapy reduces the required dose of radiation, effectively kills resistant cells and reduces the toxicity to normal tissue. The heat generated during MFH therapy is not only use to kill the cancer cells but also helps in triggering an anti-tumor immune response by releasing tumor antigens and endogenous adjuvants (Lin et al. 2018; Sato et al. 2010; Kobayashi et al. 2014). Acknowledgements The authors wish to acknowledge the support of research scholar Ms. Dharti Bhadla in the preparation of the chapter. The authors’ study have been funded by Department of Science and Technology-Science and Engineering Research Board DST/SERB/CRG/2021/001587 and Indian Council of Medical Research, ICMR-NCD/AD-HOC/64/2020-21, New Delhi, India.

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In Vivo Mechanistic Study of Superparamagnetic Materials Rakesh M. Patil, Prajakta B. Shete, Prabhanjan S. Giram, Sandeep B. Somvanshi, and Nanasaheb D. Thorat

Abstract The recent, fast track advancements in the field of nanotechnology have encouraged the use of nanoparticles as nanomedicine in the biomedical field. Especially, the special class of magnetic materials such as superparamagnetic nanomaterials are highly recommended in cancer theranostics (diagnosis and therapeutics). Mechanistic studies of these superparamagnetic nanomaterials mean the biological processes associated with the nanomaterials coming in contact with the biological environment. The biological environment quickly interacts with the nanomaterials, remodels their structure i.e. formation of protein corona results in changes in their physicochemical properties which is responsible for their biodistribution, biotransformation, potential toxicity and fate. The biodistribution and fate of superparamagnetic nanomaterials determines the effectiveness and safety of the cancer nanomedicine. In order to use the cancer nanomedicine very efficiently it is important to understand the mechanistic studies very well. The chapter involves the understanding of mechanistic studies of superparamagnetic nanomaterials. The main focus is on the biodistribution, biotransformation, toxicity and fate or elimination pathways R. M. Patil Regional Forensic Science Laboratory, Home Department, Nashik 422003, MS, India P. B. Shete Krishna Institute of Allied Sciences, Krishna Vishwa Vidtapeeth (Deemed to Be University), Karad 415539, MS, India P. S. Giram Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA S. B. Somvanshi School of Materials Engineering, Purdue University, West Lafayette 47907, USA e-mail: [email protected] N. D. Thorat (B) Nuffield Department of Women’s and Reproductive Health, Medical Sciences Division, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK e-mail: [email protected] Department of Physics, Bernal Institute and Limerick Digital Cancer Research Centre (LDCRC), University of Limerick, Limerick, Ireland © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_11

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followed by nanomaterials after their short term and long term exposure. Although, the use of nanomedicine is highly advanced, the mechanistic studies from exposure to long term fate of newly synthesized nanomaterials must be cautiously evaluated prior actual clinical use. Current understanding of the adverse effects and the exact fate of the nanomaterials inside the human body is very inadequate. Keywords Mechanistic studies · Nanomedicine · Biodistribution · Fate

1 Introduction The advancement of nanomedicine and the subsequent exposure of humans to them require thorough exploration of biodegradation, toxicity and fate of nanomaterials in vivo. Nontoxic application of nanomedicine needs thorough understanding of nanoparticles (NPs) pharmacokinetics in the organism. The physiological parameters of NPs along with their potential interactions, retention time and elimination routes must be addressed when administered in an organism. Though, information about the different pathways of NPs biodegradation is still very limited because of the difficulty in long-term measurements. Nanotechnology has developed very quickly and we are now successfully approaching towards solutions to various day to day challenges. Few of the current challenges of nanomedicines are making of drug nano-carriers which are biologically inert, are easy internalization in cells and reaching their desired target with a great specificity. The unique properties of NPs (optical, electrical, magnetic etc.) point out them as favorable candidates for various biomedical applications, such as in vitro investigative tests and in vivo drug delivery, therapy and imaging. Gold NPs and quantum dots are used for high resolution imaging due to their optical properties (Dung et al. 2021), while magnetic NPs (MNPs) are used for diagnosis and therapy of cancer, magnetic resonance imaging (MRI) technique for diagnosis and magnetic fluid hyperthermia as therapy. Superparamagnetic iron oxide NPs (SPIONPs) with diverse dimensions and morphologies when surface modified, not only serve as contrast probes for MRI, but also act as therapeutic agents or delivery platforms while they are laden with other therapeutic agents (Patil et al. 2019). For the use of NPs as nanomedicine comprehensive understanding of interactions between NPs and cells in the biological environment is necessary. In major studies acute effects of NPs are assessed through response or exposure assays and on the other hand the biotransformations of NPs originated by the biological environment are very less explored. According to National Institute of Health, USA, mechanistic studies help to understand a biological or behavioral progression, the action mechanism of an intervention or the pathophysiology of a disease. The mechanistic studies of superparamagnetic nanomaterials in any organism involves biodistribution, biotransformation, biodegradation, bio-assimilation and elimination processes. These processes are governed by energetic and complex interactions with various biological components during

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MNPs’ journey throughout the organism. Figure 1 shows the schematic view of MNPs’ lifecycle after intravenous administration. After intravenous administration of NPs, they interact with the components of the biofluids. Present investigations showed that biological interactions constantly modify the physical appearance and properties of NPs. Biomolecules continuously modify the surface of NPs which may results in NPs’ aggregation, opsonization and degradation. Such alteration may control the cellular internalization, biodistribution, potential toxicity and fate (Kolosnjaj-Tabi et al. 2016). NPs whenever encountering a cell they will be quickly internalized into the cells. The NPs are first internalized in the cell and then transferred to different cell organelles (Patil and Shete 2019). MNPs are getting growing attention in recent years. MNPs have a range of superior properties such as superparamagnetic behavior at room temperature, high saturation magnetization, biocompatibility and very low toxicity. Due to such superior properties these NPs are well suitable for in vivo applications as MRI contrast enhancement agents, magnetic fluid hyperthermia therapy for cancer (MFH), targeted drug delivery and cancer diagnosis. In all the available MNPs, SPIONs are the most investigated

Fig. 1 Schematic representation of NPs’ fate after intravenous administration (Reproduced with permission from Kolosnjaj-Tabi et al. 2016)

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nanomaterial for in vivo applications (Patil 2020). Hence in this chapter mechanistic studies of SPIONPs are mostly discussed. SPIONPs are extensively used in medical science (Salunkhe et al. 2016; Phadatare et al. 2012; Shete et al. 2013; Patil et al. 2015, 2014). SPIONPs are now routinely used to target cancerous tumor by applying an external magnetic field due to their excellent magnetic properties. Ease of synthesis, unique magnetic properties, multifunctionality with various chemical agents, and biocompatibility make SPIONs promising candidate for biomedical application. Multifunctional NPs based technologies for in vivo applications are continuously developed. Therefore, it is important to know their biodistribution, biodegradation and elimination pathways in vivo and also their short-term and long-term fate in the organism. Many different types of MNPs were studied and are being studied for various biomedical applications but the most extensively studied is SPIONPs. SPIONPs passed most of the barriers to enter into clinical trials. Hence this chapter mainly focuses on the lifecycle of SPIONPs, a versatile and biocompatible class of NPs.

2 Factors Controlling the Mechanistic Studies The size, shape, surface properties and in vivo characteristics (like biocompatibility, stability and permeability) strongly influence the use of MNPs for therapeutic applications. As we know, NPs possess high surface to volume ratio. The consequently increased surface area can be used to anchor a range of imaging modalities and/or targeting ligands making them multifunctional in use. For example, IONPs based liposomes can be used as drug carriers. Such multifunctional NPs are suitable to minimize toxicity, preserve drug integrity and reduce side effects to normal tissues during drug delivery. On other hand IONPs can be used as powerful MRI contrast agents and also used to destroy cancer cells by MFH therapy (Gupta and Gupta 2005; Thorat et al. 2014). MNPs’ blood circulation time is controlled by the reticulo-endothelial system (RES), which in turn influences the biodistribution, biodegradation and potential clearance of MNPs. The biodistribution and fate of MNPs affect their effectiveness and safety, which determines if a technology which uses MNPs, may be efficiently applied in the clinic or not. For example, it is important to understand the concentrate of NPs accumulated at the targeted site to evaluate how effectively the NPs are targeted, passively or actively. Supervising the accumulation in normal tissues and long-term fate of MNPs are necessary for their safe use as nanomedicine (Almeida et al. 2011). This part tells us about the major factors influencing on the biodistribution, biotransformation/biodegradation and fate of MNPs. Size, shape, surface charge, surface functionality, mode of administration and amount of dose injected play a significant role in biodistribution, biodegradation and subsequent clearance of MNPs.

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2.1 Surface Charge and Functionalization Surface nature of the NPs plays an important role for its therapeutic application and subsequent elimination from the body. The biodistribution as well as biotransformation of MNPs depend on the functional groups and charge present on the surface. The biodistribution is also determined by the surface charge, polarity and type of tissue and/or cell they are targeted in. The surface properties comprise surface charge, hydrohilicity/hydrophobicity and functional groups (–OH− , –COO− , –NH3+ , etc.) of polymers and surfactants attached onto the surface. A lot of efforts have been taken to manufacture different types of MNPs with different surface charges and polarities. The MNPs were functionalized with different organic materials such as surfactants, polymers and biomolecules (Patil and Shete 2019). A neutral surface charge of the MNPs results in reduced uptake by RES organs with longer circulation time. The biodistribution of IONPs with respect to the surface charge was studied by Papisov et al. (1993). They immobilized poly-l-lysine (PLL) and succinate followed to PLL on dextran-coated IONPs. The positive and negative surface charges were developed on the IONPs, respectively, as a result. A faster clearance in 1–2 min was observed for positively charged PLL-dextran-coated IONPs while 40–50 min time was required for the negatively charged succinatePLL-dextran-coated IONPs. The biodistribution was observed chiefly in the liver and spleen for PLL-dextran-coated IONPs and in lymph nodes for succinate-PLLdextran-coated IONPs. In a parallel study Chouly et al. (1996) observed increased liver uptake of both positive and negative charge IONPs and lesser circulation time related to neutral IONPs. The surface charge has major influence on the mechanistic studies of MNPs which in turn influences the potential applications of the nanomedicine. The surface charge on NPs is measured in terms of zeta potential. Zeta potential of NPs with high positive or negative values designates greater colloidal stability. It plays a vital role in physiological and colloidal stability of NPs. The functional groups on the NPs at a specific pH determine their surface charge. The cellular interactions of NPs mostly depend on the surface charge (generally in endocytosis and phagocytosis). For example, if the NPs are hydrophobic in nature it causes their agglomeration and subsequent rapid elimination through RES. Thus to reduce the opsonization followed by elimination and to lengthen the circulation time, surface modification of NPs with hydrophilic groups is crucially important (Chandra et al. 2011). Their surface modification is extremely vital when designed to use as nanomedicine. Amphiphilic polymers like polyethylene oxide (PEO) and polyethylene glycol (PEG) are used for surface functionalization of NPs to achieve water solubility and biocompatibility. Chen et al. (2009) used an amphiphilic PEO-block-poly (γmethacryloxypropyl trimethoxysilane) (PEO-b-PγMPS) copolymer to functionalize IONPs and compared the biodistribution of these IONPs with an amphiphilic triblock polymer (ATBP) or dextran functionalized IONPs. PEO-b-PγMPS functionalized IONPs demonstrate lesser biodistribution in liver and spleen in comparison with

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dextran or ATBP- functionalized IONPs on intravenous injection to mice. In addition, it is found that pluronic or PEG-coated IONPs can efficiently disperse in the tumor. A negative MR imaging contrast was observed by Cole et al. for PEG-starchcoated IONPs in a rat model (Cole et al. 2011) at the tumor site. The starch-PEG coating offered increased circulation time in blood which assists better localization of the NPs at the tumor site. Pluronic-Oleic acid (OA) IONPs were administered at the tumor site by Foy et al. (2010) and increase in signal on 2nd day and decrease in signal through 11th day was found. The decrease in signal may be due to the elimination of NPs.

2.2 Size and Shape The morphological characteristics of NPs such as size, shape and surface functionality are key factors to understand their biodistribution, biodegradation and fate. NPs below 100 nm size have higher active surface area which enables efficient ligand binding, have improved tissue diffusion and have reduced sedimentation rates. To escape from the RES, the NPs’ size should be sufficiently small with proper surface coating. The NPs should remain for a prolonged period in the blood circulation. Size of the NPs highly influences on their concentration and clearance from the blood circulation (Patil and Shete 2019; Chandra et al. 2011). Shape (geometry) of NPs is a crucial factor which governs NPs behavior in blood circulation. NPs geometry has considerable influence on phagocytosis by macrophages. Macrophages readily phagocytize spherical NPs while elliptical disk shaped NPs reveal diverse interactions with macrophages subject to their local geometry. Macrophages are incapable to complete phagocytosis when come in contact with these NPs on the smooth side. Conversely, when contacted on the pointed end, normal phagocytosis is performed. The inhibition of phagocytosis is observed in highly elongated worm-like NPs. Longer circulation times were observed for elliptical disk-shaped NPs than spherical NPs (Yoo et al. 2010). The small sized MNPs have easy entry into the lymph nodes while the large sized MNPs have been rapidly taken up by the liver and spleen. Bourrinet et al. (2006) studied biodistribution of two different sized dextran-coated IONPs, 30 nm ferumoxtran and 80 nm ferumoxide in rats. 37–46% of ferumoxtran NPs primarily biodistributed in the spleen, 25% in the liver and 5–11% in the lymph nodes, 24 h post injection. On the other hand the larger ferumoxide NPs were predominantly taken up by the liver (83% and >50%, 1 h and 24 h post injection) (Bourrinet et al. 2006; Weissleder et al. 1989). Moreover, the circulation time in the blood of ferumoxtran NPs was higher than the ferumoxide NPs. A rapid and enhanced uptake for dextrancoated IONPs in liver was observed with a range of 33–90 nm in size (Chouly et al. 1996). Larsen and colleagues (Larsen et al. 2009) observed size dependent biodistribution of 40 and 20 nm PEG-silane coated IONPs, the larger ones biodistributed at the tumor site and the smaller in the normal tissues.

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2.3 Mode of Administration and Dose Injected The mechanistic studies of MNPs are also influenced by the mode of administration. Different routes of administration of MNPs for theranostics applications are oral, intravenous, dermal, pulmonary and peritoneal administration. Out of these various routes of administration, the most preferred and studied route for MNPs into the body is intravenous injection to reach the targeted organs effectively through blood circulation. The intravenously injected MNPs expose to an environment which is rich in cells, proteins and other biomolecules. The size of the MNPs and morphology of endothelium in blood vessels limit their transport into surrounding tissues (Patil and Shete 2019; Almeida et al. 2011). In oral administration of MNPs, some factors like their shape, size, surface functionalization and their absorption through the gastrointestinal cells govern their transport into the blood stream. The acidity of the stomach may alter their physicochemical properties. Therefore the mode of administration is vital for the optimal use of MNPs for biomedical applications. MNPs may accumulate in the normal tissues rather than the therapeutic location if the mode of administration is altered which may be harmful to the normal tissues. As investigated by Weissleder et al., 1 h after intravenous injection in rats, the biodistribution of dextran-coated IONPs (ferumoxide) showed 82.6% of the ferumoxide NPs biodistributed in liver and 6.2% in spleen (Weissleder et al. 1989). The biodistribution and fate of MNPs are also influenced by the amount of dose injected. A higher dose of a nanoformulation induces agglomeration of MNPs followed by earlier clearance from the RES. It might rapidly trigger the immune response. Similarly, a low dose may also be instantaneously cleared by RES. In both the cases, MNPs will not accomplish their objectives (Patil and Shete 2019). So the mechanistic studies are highly influenced by the amount of MNPs dose injected and their mode of administration.

2.4 Biopersistance and Protein Remodelling The elongated occurrence of a substance in a biological system is called biopersistance. In nanomedicine, the biological half time of the NPs is studied for in vivo applications (Patil and Shete 2019). It is significant to determine the biopersistance of NPs as inadequate data is available on the biopersistance study. In case of ferumoxtran and ferumoxide NPs, ferumoxtran NPs were found to have 97–222 min blood clearance half-life while ferumoxide NPs were found at approximately 6 min plasma clearance half-life (Bourrinet et al. 2006). IONPs are generally taken up by the macrophages present in the spleen, liver, bone marrow and lymphatic system. The blood half-lives of SPIONs are dependent on their size and surface properties and may vary from 1 to 24–36 h. NPs with smaller

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size with same surface coating have longer half-lives compared with larger size NPs with the same surface coating (Bohara and Thorat 2018; Schrand et al. 2009). Protein remodeling or formation of protein corona is also responsible for NPs’ biodistribution and fate. It is a vital parameter which has profound impacts on the fate of MNPs. The formation of the protein corona can modify surface properties of NPs like hydrodynamic diameter and surface charge. As soon as NPs come in contact with body fluids like plasma, blood or interstitial fluid, proteins in the fluids are adsorbed on their surface leading to the changes in the conformation, resulting in the formation of new epitopes and altered functions. It also increases the hydrodynamic diameter and alters the surface charge of NPs. For example, immune response like complement cascade can be initiated if complement proteins or antibodies adsorb onto the NPs resulting in phagocytosis by macrophages (Patil and Shete 2019).

3 Biodistribution of MNPs The study of biodistribution of NPs is a key topic of the present nanomedicine research. Nanomedicine based theranostics technologies for therapeutic and diagnostic functions are being developed for in vivo biomedical applications. So it is very much essential to study the biodistribution of the nanomedicines in the body. Primary studies conducted on IONPs to investigate their biodistribution show their accumulation in the RES organs. Hence IONPs are used as MR contrast agents in the liver and spleen. Biodegradation of the IONPs along with their surface coating and subsequent metabolism of the iron, also affects biodistribution of IONPs. The biodegradation of IONPs core and its surface coating is a well-known phenomenon (Chouly et al. 1996). Small molecules or polymers like citrate, OA, chitosan, dextran, PEG, pullunan or starch are usually used to coat IONPs. Subtle variations in the coating of IONPs have major effects on their biodistribution (Duli´nska-Litewka et al. 2019). It is important that IONPs should have a high blood circulation time and reduced uptake in the RES organs, as required for their use in various biomedical applications. In the last decade, several researchers synthesized IONPs coated with PEG or PEO holding different chemical groups to stimulate longer circulation time and improved uptake at the desired target (Soenen and Cuyper 2010). For instance, Chen et al. (2010) synthesized IONPs coated with OA and an amphiphilic Pluronic® block copolymer. OA was used to form the hydrophobic surface which can effectively hold hydrophobic drug molecules. They investigated a total of 55% of the dose of 193 nm Pluronic-OAIONPs biodistributed to liver, 6 h after injection. That means the Pluronic-OA-IONPs were taken up by the RES after 6 h. The bare IONPs are constantly modified by interactions with biomolecules present in the biological fluids. These dynamic interactions are responsible for change in the overall shape, hydrodynamic diameter, and surface charge of IONPs initiating aggregation and opsonization, followed by biodegradation. According to Jain et al. (2008) biodistribution of magnetite (average 20 nm size) NPs was traced mostly in the liver

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and spleen. In some extent these NPs were biodistributed in other tissues (heart, kidneys, brain, lungs, stomach and small intestine). In a similar way, the effect of surface modification of bare IONPs with PEG, dextran and polyvinyl pyrrolidoneon on circulation time was observed by Wang et al. (2010). An increased blood circulation time of 3 h was observed after surface modification of IONPs while bare IONPs were trapped by RES cells in 1 h.

4 Biotransformation/Biodegradation The basic need for long term use of NPs for biomedical applications is their safety. So it is significant to identify the pathways or processes by which they will degrade after achieving their therapeutic goal. It is not known if the biodegradation products formed are bio-assimilated or eliminated through body. For biodegradable NPs their biodegradation products after biodegradation may cause unforeseen biological reactions. On the other hand, non-biodegradable NPs might accumulate in the lysosomal compartments and saturate them which in turn disturbs the autophagic and degradative pathways that are vital for cells to degrade proteins (Tomasovicova et al. 2017). In pharmacokinetics, metabolism and excretion are vital mechanisms by which drugs or xenobiotics are removed through the body. In the cellular environment, MNPs start rapidly degrading which results in biotransformation of NPs into released ions. Biodegradation of IONPs’ core results in the release of the iron ions. In macrophages the released iron ions subsequently integrate into the physiological iron metabolism. IONPs’ biodegradation mechanism inside macrophages is shown in Fig. 2. IONPs injected intravenously are generally phagocytized by macrophages present in the liver and spleen. These NPs then transferred to the lysosome. Their degradation takes place in the acidic environment of the lysosome (Stern et al. 2012; Baboci et al. 2020; Yu et al. 2018). After degradation the released iron ions joined the normal iron metabolism as shown in Fig. 2. In oral administration low pH of the stomach dissolves IONPs releasing iron ions inside the gastrointestinal tract. The released iron ions are readily absorbed into the blood and integrated into normal iron metabolism. Metabolism of intravenously injected IONPs is much faster than orally administered IONPs (Mao et al. 2013). In inhalation, IONPs are taken up and degraded by alveolar macrophages. The released iron ions in all these mechanisms are used for the synthesis of myoglobin and hemoglobin or stored with the help of iron-regulating proteins ferritin and hemosiderin in the body (Mao et al. 2013; Arami et al. 2015). The iron is stored inside ferritin molecules in the form of non-magnetic ferrihydrite (Martinkova et al. 2018). Bourrinet et al. (2006) examined the biodegradation of bare and dextran coated IONPs by using radio-labeled iron oxide (59 Fe) and dextran (14 C). They found that dextran gets rapidly degraded and eliminated earlier than the iron of IONPs. It is thought that iron that is released from IONPs may bind to apoferritin and forms

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Fig. 2 Overview of IONPs’ biodegradation inside macrophages and released iron ions’ fate (Reproduced with permission from Jain et al. 2008)

ferritin, the iron–protein storage complex, in liver (Walle et al. 2020). In a study by Levy et al. (2011), it is found that IONPs are digested within the lysosomes of Kupffer cells of liver. After degradation the released iron is transported in the form of ferritin or hemosiderin to the spleen. Better understanding of IONPs biodegradation and absorption will help to understand their biodistribution. As stated earlier, the nature and quality of the coating material, dose injected and the type of phagocytizing cells they enter limits the rate of biodegradation of IONPs. In mice, Lartigue et al. (2013) observed long-term biotransformation of nanocubes (PEG coated IONPs) and their intracellular biodistribution. PEG coated nanocubes were intravenously injected to mice and ex vivo transmission electron microscopy (TEM) analysis is carried out at different time points as shown in Fig. 3. One day after injection, the nanocubes’ maximum accumulation was observed in endosomes or more contrasted lysosomes. At day 7 and day 14 post-injection more isolated and rarer nanocubes were observed mainly on the periphery of lysosomes. They also found the existence of smaller monodisperse electron-dense circular NPs coexist with the residual cubes. The intracellular entities observed were representative of ironfilled ferritin proteins. Faster biodegradation of IONPs is observed in liver Kupffer cells than spleen macrophages. The main problem obstructing biodegradation studies of MNPs for months and years is the limitation of the techniques available for their detection. Invasive and non-invasive techniques are used to quantify the MNPs (Tseng et al. 2012). The techniques like TEM, inductively coupled plasma mass spectrometry (ICP-MS), Mossbauer spectroscopy and ICP-OES (optical emission spectroscopy) are invasive in nature (Kolosnjaj-Tabi et al. 2015; Ruiz et al. 2015; Gabbasov et al. 2013). These techniques involve time-consuming sample preparation procedures and sacrificing a

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Fig. 3 PEG coated nanocubes in murine liver and spleen are degraded intracellularly after intravenous injection. Red arrows show the presence of monodispersed iron-rich ferritin proteins (Reproduced with permission from Lartigue et al. 2013)

number of animals. Furthermore, the total number of macrophages and their activity involved in the assays may be subject to the inherent features of the animals (Jones et al. 2013; MacParland et al. 2017). Accordingly, in these methods the MNPs’ mass is averaged over various animals. Ferromagnetic resonance (FMR), Electron paramagnetic resonance (EPR) and superconducting quantum interference device (SQUID) can be used as non-invasive techniques. A magnetic spectral approach was described by Zelepukin et al. (2021) for in vivo monitoring of biodegradation of MNPs. They performed comprehensive one year fate study of 17 different types of IONPs. The factors such as dose, surface coating, hydrodynamic size, zeta potential, internal architecture and degradation half-life of MNPs influences on their fate. They studied the biodegradation of six types of MNPs (100 nm) surface modified with different hydrophilic polymers. Polyglucuronic acid coated MNPs showed faster (t 1/2 = 15 ± 4 days) biodegradations; in contrast PEG coated MNPs showed slower (t 1/2 = 38 ± 6 days) biodegradations. The internal architecture of MNPs has the most significant influence on the biodegradation. To understand the in vivo biodegradation of the MNPs, an approach was developed which is a method based on the MNPs quantification (MPQ). The biological and magnetic properties of the spleen and liver were altered by the biodegradation of 1 mg of citrate-coated MNPs. Firstly the mice were injected with MNPs intravenously. Later, samples of liver, bile, spleen, urine and feces were collected for examination. ICP-MS was used to detect the iron concentration in tissues. MPQ and SQUID were used to analyze the magnetic properties of organs. Eosin-Perls staining was used to study the biodistribution of iron in the tissues as well as to determine the number of iron-positive cells. All the observations were shown in Fig. 4. Figure 4a and b show the MPQ and ICP-MS measurements of degradation of MNPs in liver and spleen respectively. The obtained data showed that there was 27% and 54% decrease in MNPs’ integral amount in the liver and spleen after a month respectively. Figure 4c– f show the MPQ and ICP-MS measurements of urine and feces respectively. The data obtained from MPQ and ICP-MS measurements show that iron was not excreted into the urine. At day 1 and 7, an enhanced MPQ signal was observed in feces. The increase in MPQ signal might be due to the decrease of the intestinal iron absorption from diet, as already iron is overloaded in the organism. Figure 4h–i show the histological

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study of liver and spleen tissues stained with Perls Prussian blue and eosin. Blue dots were iron localization points obtained by the Perls reaction (Zelepukin et al. 2021). The data obtained by Zelepukin et al. (2021) showed that complex mechanisms are involved in the biodegradation of MNPs, which involves the biotransformation of MNPs into the MPQ-undetectable forms of iron. The iron present in the soluble form is toxic to the cell hence it is stored in the form of a protein, called ferritin. Ferritin enters the lysosomes when cells are overloaded with iron. Hemosiderin compounds were formed and detected in the histological studies when ferritin was destroyed in the lysosome.

Fig. 4 Magnetic and biological assessment of biodegradation of 1 mg of citrate-coated magnetite NPs in mice (Reproduced with permission from Zelepukin et al. 2021)

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5 Fate of MNPs The main limiting factor for theranostics applications of MNPs is their long-term fate after completing the therapeutic goal. The preliminary steps to determine their fate is to study their transformation in vivo. In recent years, many studies were conducted to understand the biodegradation of NPs along with the surface coating. Overall, after entry of MNPs into the blood stream they are biodistributed by the RES. The RES includes macrophages, phagocytic monocytes mainly present in the bone marrow, liver, spleen and lymph nodes (Patil and Shete 2019). MNPs are phagocytized by macrophages after recognition and biodistributed to these organs. Liver is the major organ of RES with non-RES cells and Kupffer cells. MNPs accumulated in the nonRES cells get eliminated through bile while those phagocytized by Kupffer cells are usually present for a long time within the cells (Longmire et al. 2008; Poon et al. 2019). Moreover, MNPs are also eliminated through the kidneys. Inhalation, ingestion, dermal penetration and intravenous injection are four different exposure and or entry routes of MNPs (Matteis 2017). For most of the therapeutic applications, MNPs are generally administered orally or intravenously (Wu et al. 2015; Akbarzadeh et al. 2012). MNPs are directly injected into the blood by the intravenous mode. On the other hand, they can diffuse across the gastrointestinal mucosa and enter the blood circulation in case of oral ingestion. MNPs circulating in the body may have different fates like renal clearance (Croissant et al. 2017; Du et al. 2018), phagocytosis (Liu et al. 2017; Feng et al. 2018) or self-degradation (Feng et al. 2018). NPs with >10 nm diameter were excreted by the body as this size of MNPs can cross the kidney’s glomerular filtration membrane. But NPs with 5.5 nm diameters are found to be non-renal clearable. Though, some NPs with diameters 5.5–10 nm are actually excreted via urine (Senthilkumar et al. 2021). The IONPs and its surface coating materials degrade with time. The bare IONPs were dominantly accumulated in the liver and spleen. The coating of the surface of MNPs is responsible for stability, aggregation and cellular interactions of NPs (Cho et al. 2009). As stated earlier, the liver of rats show 55% accumulation of the intravenously injected pluronic-OA-IONPS. On the other hand, around 25% of injected dextran-coated SPIONs were eliminated through urine and feces (Chen et al. 2010). Bourrinet et al. (2006) radio-labelled 59 Fe and 14 C of dextran-coated SPIONs and reported that the liver showed 24% and the spleen showed 40% accumulation of the injected dose while 9% MNPs were circulating in the lymphatic system. In different animal models, urine and feces show around 20% dextran-coated SPIONs. Iron and dextran were mainly eliminated through feces and urine respectively. Similarly in other studies biodistribution of intravenously injected MNPs were detected in different organs (Bourrinet et al. 2006; Jain et al. 2008; Mejías et al. 2013). MNPs were mainly biodistributed to the liver and spleen where these MNPs undergo an acid induced degradation. MNPs radio-labelled with 59 Fe or 57 Fe were used to determine

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how the exogenous iron incorporated into endogenous iron metabolism. The radiolabelled isotopes are assimilated within the hemoglobin (Rojas et al. 2017). Such studies helped to understand the fate of IONPs and notably prove the degradation of IONPs. In a recent study, Stepien et al. (2018) used a biomagnetic detection system (alternating current biosusceptometry) to detect and quantify MNPs. They studied the biodistribution MnFe2 O4 MNPs, clearance of MnFe2 O4 MNPs and determine the elimination time of MnFe2 O4 MNPs. They collected liver, spleen, kidneys, lung, heart and blood sample along with feces for biodistribution and elimination analysis for a period of 60 days. Greater uptake of MNPs in the liver and spleen was found. After 60 days, a significant decay of MNPs in the liver and an absence of MNPs in the spleen were recorded. Similarly, Masthoff et al. (2019) studied biodistribution and elimination of curcumin coated IONPs (Cur-IONPs) in mice. A long term of 3 weeks of biodistribution of Cur-IONPs was assessed for a single-dose of 5 mg/kg. It was found that the MNPs were mostly taken up by liver and spleen than kidney and brain. The considerable accumulation of the MNPs was seen in the brain between 1 h and 1 day after injection, proving the successful transfer of Cur-IONPs through the blood–brain barrier. MNPs are intended for various biomedical applications especially for brain MRI or targeting tumors within the brain. To reach their target cells, these MNPs are supposed to cross the blood–brain barrier (Soares et al. 2022). After achieving their desired goal these MNPs should be cleared out from the brain as their buildup will result in neurotoxicity. Elbialy et al. (2019) examined the elimination of PEG-b-poly(lactic acid) copolymer NPs (PEG-PLA) and reconstituted high density lipoprotein (rHDL) NPs from the brain. After injection of both the NPs within the intraparenchymal route, it was found that both the NPs were eliminated through the paravascular glymphatic pathway. The clearance of PEG-PLA and rHDL NPs from brain through paravascular glymphatic pathway is schematically shown in Fig. 5. The IONPs with two different surface coatings, viz. glucose or PEG, were investigated over 4 months to study in vivo biotransformation and in vitro protein adsorption, biodegradation. Interestingly, faster degradation of IONPs functionalized with glucose was observed in vitro. In contrast, in vivo results of the IONPs coated with PEG were opposite with faster biodegradation and clearance (Mejías et al. 2013). All these studies show that the biodistribution, biotransformation and clearance of MNPs are complex mechanisms. Extensive investigation along with expertise is needed to know the behavior of MNPs in the body. Successful clinical translation of MNPs requires thorough understanding of their elimination from the body.

6 Assessment of Toxicity of Magnetic Material Assessment of toxicity of the magnetic material is important for drug delivery, biomedical, healthcare, imaging, diagnostic applications, etc. Toxicity of the magnetic material was assessed by In-vivo, In-vitro, Ex-vivo, and In-silico method

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Fig. 5 The schematical representation of paravascular glymphatic pathway for clearance of PEGPLA and rHDL NPs from brain

for approval from US FDA (Food Drug Administration). Nanotoxicology includes the study of nanomaterials’ toxicity for human application. According to regulatory guidelines, the safety and non-toxicity levels of superparamagnetic iron oxide nanomaterials (SPIO) are important for the translation of the nanomaterial for clinics. Once this nanomaterial is administered or implanted inside the human body, the immunological system responds to it quickly based on the ability to produce reactive species. In-vitro, Ex-vivo, and In-silico methods are cost-effective, performed without regulatory stringency. In case of In-vivo studies regulatory approval of institutional animal ethical permission is mandatory. Physicochemical and morphological properties of the nanomaterial such as size, shape, surface charge, preparation method, and catalyst/crosslinkers used affect toxicological properties. In case of In vitro toxicity tests if no significant results or no effect was observed, then we need to perform In vivo and further human studies for clinical investigation after registering the products with FDA.

6.1 In-Vitro Toxicity Study In case of In-vitro toxicity of nanomaterial cell viability performed with fibroblast NIH3T3 , human umbilical vein endothelial cells with MTT(3-[4,5-dimethylthiazol2-yl]-2,5 diphenyl tetrazolium bromide), XTT (2,3-Bis-(2-Methoxy-4-Nitro5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide), lactate dehydrogenase (LDH), Trypan blue, Neutral red and other assays were used for measurement of cell viability. The cell viability is indirectly a measure of cytotoxicity potential of the nanocarriers for cytotoxicity, oxidative stress, inflammatory reactions, and genotoxicity. In contact with mitochondria, SPION produces damage. These assays are based on the presence

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of reductase enzymes in mitochondria (Stoddart 2011). Cytokine assays were used to assay superparamagnetic nanocarriers tested in the human monocyte macrophage culture and the production of various cytokines such as IL-12, IL-6, IL-1β and TNFα was measured by the Immunoassay kit. Production of cytokines correlated with the toxic potential of nanocarriers. Oxidative stress potential of nanomaterials tested with the nitro blue tetrazolium (NBT) assay involved conversion of NBT to blue formazan (Müller et al. 2007). Phagocytosis assay of the nanocarriers was performed with RAW 264.7 cells and fluorescent beads and phagocytosis potential was measured with flow cytometry and fluorescence microscopy. The phagocytosis potential of the nanomaterial determined as % of fluorescent beads engulfed by cells (Bancos et al. 2014; Semerad and Pacheco 2020). Similarly, cellular uptake pathway of SPION was performed to understand the fate of the nanocarriers inside the cells by endocytosis (Weissleder et al. 1997). Cellular oxidative stress production ability of the magnetic nanomaterial exposed to cells was measured by the dichlorofluorescein assay. In the cells non-fluorescent derivatives were oxidized to DCF fluorescent derivative by various oxidants and the emitted fluorescence was quantified by various methods used for oxidative stress measurement (Wang and Joseph 1999; Schreck et al. 1992). Comet assay and cytokinesis-block micronucleus assay were used for genetic toxicity study of the nanocarriers. In the literature dextran coated SPION nanocarriers produce reactive oxygen in HepG2. This result indicates that these nanocarriers have the potential of genetic toxicity (Seo et al. 2017). Figure 6 showed various in-vitro cell-based assays.

Fig. 6 In-vitro cell-based assay for toxicity assessment

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Hemolysis assay of magnetic nanoparticles performed with blood of rabbit, balb/ c mice, and humans according to the American society of testing material (ASTM) standards. The value of % Hemolysis below 10% indicates safety of the nanocarriers and its hemocompatiblity. For implantable devices it would be less than 5%. In the literature the applicability of heparin-based SPION anticoagulants for blood purification was tested by the hemolysis test. These SPION loaded heparins showed concentration dependent hemolysis (Gertz et al. 2012; Zhao et al. 2018). In-vitro cellular assay used before In-vivo study of magnetic nanomaterials to get insights of the toxicity prediction based on lethal dose, safety profile, IC50 and other parameters etc.

6.2 In-Vivo Toxicity Study In-vivo toxicity method measures hematological parameters, serum biochemistry, bio-distribution, histopathology, clearance, and organ-specific toxicity. In-vivo test is performed in rat, mouse and guinea pig-based animal model after approval of the institutional animal ethical committee permission protocol. This was followed by the administration of SPION based magnetic nanomaterials to the animal by oral (most commonly used), Intravenous administration (for maximum concentration in blood and immediate effect), transdermal, Subcutaneous, Inhalation (for pulmonary toxicity) and Intraperitoneal modes. Animals were regularly observed for physiological observation. After a specific time point, based on the types of toxicity study like acute, subacute or chronic, blood was collected from the animals and evaluated for hematological parameters such as % Hemoglobin, Red blood cells, granulocyte, agranulocytes and immunological cells. For serum biomarker study liver (SGPT, SGOT, APL, and AST) and kidney (urea, creatinine) quantification tests are important as elevated level of the biomarker indicated the toxicity potential of the nanocarriers. At the end of the study the animals were culled and different organs like heart, brain, liver, kidney, spleen, uterus and lungs stained with H & E sating and observed under microscope for any abnormal changes induced in the tissue by the toxicity potential of the magnetic nanocarriers (Giram and Garnaik 2021). Figure 7 showed general outline for In-vivo toxicity study of the magnetic nanocarriers. Various types of toxicity Iron oxide based magnetic nanomaterial are considered biocompatible at concentration, showing no severe toxic effects in vivo (Jain et al. 2008). Iron based nanocarriers reported to be safe and non-toxic below 1–10 μg/ml but toxicity was observed at 100 μg/ml. In the in-vivo study ultra-small superparamagnetic iron oxide, a dextran-coated nanocarrier, showed diarrhea, urticaria and nausea as short periods. In the body Iron based nanocarriers metabolized in the liver, subsequently used for iron formation, and are excreted by the kidney (Ankamwar et al. 2010). Excess amount of Iron results in DNA damage, oxidative stress, and cytotoxicity (Anzai et al. 2003). OECD guidelines are very important for toxicity of the iron oxide nanocarriers (Veranth et al. 2007).

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Fig. 7 In-vivo toxicity study

Figure 8 shows In-vitro toxicity study for SPIONs of uncoated, SPION modified NH2 , COOH by MTT assay showed higher sensitivity for BE-2-C (heart), 293 T (brain), and HCM (kidney) respectively. Amine functionalized SPIONs found higher toxic where as acid functionalized SPIONs least toxic. This toxicity effect is concentration dependent on heart, brain, and kidney at 0–36 mM. Confocal microscopybased imaging showed effect on cytoskeleton (Jain et al. 2008). In silico study uses quantitative structure activity properties/toxicity relationship. Current toxicity measurement with accuracy and precision is growing with science and technology uses mostly for in silico models for toxicity to predict which part of the nanomaterial is influencing toxicity. Toxicity database used with QSAR models (Mahmoudi et al. 2011).

7 Discussion and General Comment Nanotechnology has already provided key breakthroughs in medicine especially in cancer diagnostics and therapy applications. The potential benefits of organic and inorganic NPs particularly MNPs for tumor imaging, controlled drug delivery and targeted cancer cell killing can be more specific and may offer clinical therapeutic platforms that simply do not exist today. Current understanding of the effects of human exposure to NPs is very limited and tremendous efforts are still needed to reveal the whole impact of exposure. The recent information from literature indicates that MNPs may have the potential to generate cytotoxic effects. More research on NPs characterization, their biodistribution, toxicity, and clearance is required. A valid universal protocol must have to set to study the biodistribution, toxicity, and clearance of the NPS. At present, insufficient in vivo data is available to know the exact biological effects of the MNPs with respect to cytotoxicity, inflammation, functional alterations at the cellular or whole body level. NPs on entering the body interact with body fluid matrix remaking their structure that results in changes in

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Fig. 8 Cell viability of MTT assay results for a SPIONs-COOH, b uncoated SPIONs, and c SPIONs-NH2 samples on heart (HCM), brain (BE-2-C), and kidney (293 T) cell lines. d Confocal microscopy images showing the effects of the exactly identical amount of negatively charged SPIONs on cellular cytoskeleton as well as compartments of various cell lines (i.e., heart, brain, and kidney). Reprinted with permission from Jain et al. (2008). Copyright 2011 American Chemical Society

their properties; this modifies NPs’ biodistribution, biotransformation and associated toxicity. The understanding of NPs’ processing, biodegradation and bio-assimilation will help predicting potential exposure risks associated with short term and long term fate of NPs. In the future, the main issue is to understand the whole lifecycle of NPs in the body from entry (exposure) to exit (complete clearance). In vivo studies of the long-term fate of the MNPs are still very limited. The available literature showed some aspects of biotransformation of the IONPs, including the release of iron ions and involvement of ferritin. Definitely, many studies examined the behavior of NPs for few days after exposure, but hardly focus on periods of months or years after administration, which can be necessary for the body to eliminate or degrade the MNPs. However, it is difficult to compare these studies with each other to draw a conclusion due to the non-uniformity of the MNPs’ core compositions and the type of animal model used. Although nanomedicine is generally considered a promising field of research with exciting scenarios for the diagnostics and clinical treatment of human diseases, the mechanistic studies from exposure to long term fate

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including biodistribution, biotransformation, toxicological effects, and fate of new nanomaterials must be carefully assessed before actual clinical use. Acknowledgements The author is thankful to Director, Directorate of Forensic Science Laboratories, Kalina, Mumbai and Deputy Director, Regional Forensic Science Laboratory, Nashik, Home Department, M.S. for their constant encouragement, motivation, valuable and kind support. N.D.T. acknowledges funding under the Science Foundation Ireland and Irish Research Council (SFI-IRC) pathway program (21/PATH-S/9634).

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Recent Status of the Current Clinical Trials Going on for Superparamagnetic Materials Priyal Chikhaliwala and Sudeshna Chandra

Abstract Superparamagnetic iron-oxide nanoparticles (SPIONS) due to their intrinsic property of superparamagnetism are widely explored and studied as a promising material in the field of disease diagnosis and nanomedicines or as a combined platform as ‘theranostics’. SPIONS offer easy synthesis, biocompatibility, multifunctionality and possibility for surface modification, which supports diagnostic and therapeutic applications in medicine. Based on recent preclinical and clinical studies, it is evident that they are emerging as promising candidates for cancer research and intervention modality in disease diagnosis and treatment. A brief idea about preclinical and clinical trials is covered in the introduction section of this chapter. A lot of research efforts have been made to study different designs and formulations based on SPIONS towards diagnosis and treatment of various types of cancer. Tunable properties such as size, shape, surface charge, stability in physiological medium and hydrophobicity must be optimized in developing a formulation for a desired function. Majority of formulations of SPIONS are in preclinical stage especially for therapeutics, especially drug delivery to the targeted tumor site. Research shows conjugations of various biomolecules to SPIONS to render therapeutic efficacy in in vitro and in vivo systems. This part of information is provided in the section preclinical intervention of SPIONS of the chapter. Diagnosing tumors in cancer through imaging modality such as magnetic resonance imaging (MRI) is of high significance as it provides crucial information about the metastasis or spread of tumors to the surrounding tissues. This gives a fair idea about the decision for early treatment intervention procedure for surgical removal or adjuvant therapies. Much interest has been devoted to optimizing SPION formulation for use as MRI contrast agents wherein, SPIONS are modified with exterior surface coating and functional probes to investigate their abilities to provide enhancement in MRI contrast. Such particles accumulate at tumor sites and provide accurate information about the tumor extent. Further, to avoid opsonization and rapid clearance from the blood by the reticuloendothelial system, SPIONS are mostly passivated with biocompatible materials such as poly-ethylene glycol, chitosan, dextran and other polymers. This retains the biological behavior of SPIONS for a favorable in vivo effect. Such types P. Chikhaliwala · S. Chandra (B) Institute of Analytical Chemistry, University of Regensburg, 93040 Regensburg, Germany e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Thorat and N. K. Sahu (eds.), Superparamagnetic Materials for Cancer Medicine, Nanomedicine and Nanotoxicology, https://doi.org/10.1007/978-3-031-37287-2_12

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of MRI enhancement studies with SPIONS for cancer detection are in clinical trials. Ferumoxides, ferumoxtran, ferucarbotran etc. as ultra-small SPION formulations are widely investigated as MRI contrast enhancement in clinical trials for their safety, efficacy, and pharmacokinetic parameters in human subjects. This information is summarized mainly from clinicaltrials.gov in the section of clinical considerations and recent clinical status of the SPIONS in this chapter.

1 Introduction 1.1 What Are Clinical Trials? The World Health Organization defines clinical trials as ‘any research study that prospectively assigns human participants or groups of humans to one or more health-related interventions to evaluate the effects on health outcomes’ (https:// www.who.int/clinical-trials-registry-platform). They are also referred to as interventional trials which include drugs, biological products, devices, surgical and radiologic procedures, preventive care, and behavioral therapies. US Food and Drug Administration (FDA) oversees the clinical trials and ensures that they are designed, conducted, analyzed, and reported according to Good Clinical Practice (GCP) regulations (https://www.fda.gov/science-research/science-and-research-spe cial-topics/clinical-trials-and-human-subject-protection). Most importantly, the role of FDA is to protect the rights, safety and welfare of the people who participate in the clinical trials.

1.2 Importance of Clinical Trials in Medicine Clinical trials are essential to develop new treatment modalities and evaluate the above-mentioned new interventions. It involves research using human volunteers where the participants receive specific interventions according to the research protocols of researchers. The research is designed to enhance medical knowledge with respect to a disease, or diagnosis or prevention of ailment. Through clinical trials, a researcher or a medical practitioner can perform the following: . Evaluate one or more interventions that can be used for treating a disease, syndrome, or a medical condition. This can be a drug, a combination therapy, medical devices etc. . Find out ways to prevent recurrence of a disease or a medical condition. This may include development of vaccines, medicines, change in lifestyles etc. . Evaluate one or more interventions that can be used to identify or diagnose a disease or a medical condition . Examine methods for identifying risk factors for a particular condition

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. Exploring various ways to provide care and improve the quality of patient’s life suffering from chronic illness Clinical trials are carried out for the benefit of humans so that any new interventions can help them to live longer or with minimal health issues. They are usually carried out after in vivo experiments to find out whether new interventions are safe and effective to humans, and acceptable to a large human population. This stage is also known as pre-clinical studies. The potential toxicity and safety of the interventions are first studied in laboratory animals and only those treatments that demonstrate promising results and have an acceptable safety profile are taken forward to clinical studies. Clinical trials help in assessing the effectiveness of new interventions in the following ways: . Whether the new intervention cures larger proportion of patients than the existing intervention . Whether the new intervention has significantly higher efficacy than the existing interventions in terms of better health management and disease control . Whether the new intervention has fewer side-effects, cheaper, safe, or easier to administer so that it promotes better patient compliance Based on the types of interventions, there are seven categories as discussed below (https://www.drugwatch.com/fda/clinical-trials/) . Preventive trials: Used for assessing new medications (drugs, vitamins, supplements, vaccines) that can effectively improve a patient’s medical condition. They are usually carried out in healthy people and individuals who had a known but recovered history of a disease . Screening trials: Used for screening of a particular disease or medical condition. They are usually carried out on patients with certain symptoms . Diagnostic trials: Used for diagnosing or identifying certain conditions by use of test methods that can accurately detect the disease. These studies are carried out in individuals who exhibit symptoms of the disease under study . Treatment (Intervention) trials: Involves drugs, psychotherapy, surgical or other procedures and new devices. These trials are directed towards finding new treatment strategies for disease control and treatment along with safety concerns . Genetic studies: Aims to identify and understand the role of genes in a disease and predict the occurrence of certain disorders. These studies are now gaining importance in adapting various treatment modalities . Quality of Life studies: Aims to evaluate comfort and quality of life of a patient who has a chronic illness. These studies focus on health management of patients suffering from prolonged illness like cancer . Epidemiological studies: These are used by researchers and scientists to understand a disease pattern, cause, and prevalence and how it can be controlled in patients

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1.3 Various Stages of Clinical Trials There are predominantly four phases of clinical trials, namely Phase I, II, III and IV, each of which are briefly discussed in the following section (https://www.nia.nih. gov/health/what-are-clinical-trials-and-studies). Phase I: This stage allows researchers or medical practitioners to evaluate the safety effects of the new intervention in a small number of participants, normally between 20 and 80 healthy volunteers. In many cases, phase I is further split into two steps: Phase Ia and Ib. In Phase Ia, a single dose of intervention is administered to a small group (2–10 participants) and observed over a period. If no adverse effects are seen, then a higher dose is given to a second small group and the effects are monitored. In Phase Ib, multiple low doses are given to participants and fluid samples are collected at various time points and analyzed. In this way, the correct dose of the intervention is found out. Phase II: Once the safety of the intervention is established in Phase I, studies are carried out on a larger group of participants (ranging from 100 to 300) to evaluate the safety and efficacy of the intervention in patients. Phase II can also be divided into two steps: Phase IIa and IIb. In Phase IIa, the amount of intervention that can be given is assessed, while in Phase IIb, how well the intervention works at a certain amount is assessed. In other words, Phase II talks about the effectiveness of the intervention. It also provides preliminary data on whether the intervention works in a certain group of people who have a specific disease or medical condition. Phase II can last several years and is conducted in a variety of patient populations or indications. Phase III: Studies under Phase III are carried out on large patient groups, (300– 3,000 or more) and are designed to confirm the safety and efficacy of the new intervention. In this phase, comparative studies of the new interventions are also carried out with available modalities or are tested in combination with other therapies. In this phase, adverse effects are monitored, and relevant information is collected for establishment of safety and efficacy of the new intervention. Phase IV: This Phase is also known as Post-Marketing Surveillance Trials because in this phase, studies are carried out only after the new intervention has received regulatory approvals or market authorization. They are designed to evaluate longterm risks and benefits of the new intervention with respect to efficacy and safety in large numbers of patients and subpopulations of patients.

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2 Preclinical Interventions of SPIONs Research on superparamagnetic iron oxide nanoparticles (SPIONs) continues to grow in the direction of biomedical application, ranging from molecular therapeutics to diagnostics to imaging. Properties of SPIONs such as size, shape, magnetism, crystallinity, and flexibility can be tailored to make it multifunctional by attaching fluorescence, therapeutic agents and ligands that can be used for varied applications (Fig. 1). Numerous works have been reported on surface modification of SPIONs to achieve desirable properties so that SPIONs can be used for magnetic drug targeting, magnetic fluid hyperthermia, magnetic resonance imaging, magnetofection and gene delivery, and magnetic stem cell imaging. However, only a handful of SPIONs are considered for clinical trials right now. The potentials of SPIONs are large, however, to take advantage of the potentials, it is also important to evaluate the long-term efficacy and scaling up of the SPIONs with desired properties. According to Cortajarena et al. (Cortajarena et al. 2014a), successful translation of SPIONs into clinical settings is dependent on the toxicity, physicochemical properties, and functionality of the SPIONs. With respect to drug delivery in cancer therapy, SPIONs are typically found to be useful for magnetic targeting and increasing drug concentration at the desired site. And, on the other hand, targeting ligands or cancer biomarkers can be attached to the SPIONs to improve specific binding to cancer cells. Thus, both the strategies can be used to selectively target cancer cells and increase the therapeutic efficacy and reduce side-effects. Though there are several reports on potential clinical applications of functionalized SPIONs, only few of them are in clinical trials.

Fig. 1 Representative image of multimodal SPIONs integrated with a radionuclide, optical tag, targeting moiety and therapeutic load

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The most frequently investigated applications of SPIONs for clinical setup are (i) as MRI contrast agent (ii) magnetic hyperthermia (iii) magnetic drug delivery and (iv) a combination of either of these modules. Besides this, the SPIONs are now reported for screening, monitoring and diagnosis of cancer. Recently, Onishi et al. (Onishi et al. 2022) reported the use of magnetic beads to detect extracellular vesicles in serum of patients having pancreatic cancer. The efficacy of magnetic hyperthermia using SPIONs for preclinical studies are demonstrated in ex vivo and in vivo experiments in which biocompatibility and depot stability have been assessed. Local hyperthermia was demonstrated in preclinical glioma models using SPIONs which also induced potent antitumor immune responses (Grauer et al. 2019). SPIONs have also been looked upon as efficient carriers for combination therapy, particularly a combination of magnetic hyperthermia and radiotherapy. In such interventions, SPIONs are used as a controllable heat source that can raise the temperature of the tumor cell environment to 40–42 °C, thereby denaturing the proteins and damaging the DNA, ultimately resulting in cell death. Unlike conventional hyperthermia, tumor cells exposed to SPIONs are heated in an alternating magnetic field that overcomes the problem associated with conventional hyperthermia i.e., generating therapeutic temperature deep in the tumors. Magnetic hyperthermia also complements radiotherapy by reaching the poorly perfused tumor environment that is resistant to the ionizing radiation. Thus, magnetic hyperthermia is believed to act as a radiosensitizer to the tumor cells that show resistance to radiation therapy (Janko et al. 2019; Russell et al. 2021). When it comes to SPIONs-based imaging, it has convincingly been proved better than Gd-based imagers due to their ability to perform as dual interventions. For example, Shevtso et al. (Shevtsov et al. 2015) functionalized the SPIONs with recombinant IL-1 receptor to target glioma cells where hypotensive T2weighted images were obtained due to retention of SPIONs in the tumor. This proved that the SPIONs can be used as negative contrast agents. Moreover, the SPION-IL-1 conjugates could reduce the peritumoral edema depicting its therapeutic efficacy. The study opened avenues for theranostic approach in neuro-oncology wherein the SPIONs-IL conjugates can be used for diagnosis of glioma cells as well as management of peritumoral edema. Figure 2 represents the therapeutic and imaging efficacy of SPION-IL-1. Figure 2a shows obvious dark regions of magnetic conjugates in the glioma on T2 scans in comparison to baseline images (on TurobRARE-T2 and FLASH regimens. Figure 2b shows the Kaplan–Meier curves depicting a two-fold increase in the life span of those rats treated with SPION–IL-1Ra in comparison to control animals. Conjugation of IL-1Ra with SPIONs mediated targeted delivery of the particles in the tumor, thereby depicting the potential of the SPIONs to be used as both imaging and therapeutic agents. Zhang et al. (2016) used folic-acid coated SPIONs as a T2-negative contrast agent in MRI and detected intraperitoneal human ovarian cancer in a xenograft tumor model. Due to the presence of folic acid, the SPIONs are specifically bound to the ovarian cancer cells. Preclinical assessment of magnetic targeting by SPIONs were established by many researchers wherein systemic injection of SPIONs, concentration of nanoparticles, choice of animal models, intensity of magnetic field and various other parameters

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Fig. 2 a MRI of the C6 glioma 24 h following i.v. treatment with SPIONs and SPION–IL-1Ra conjugates. MR scans at RARE-T1, TurboRARE-T2, FLASH, and MSME regimens show retention of SPIONs as hypotensive zones at T2-weighted images and in the regimen of the gradient echo (red arrows) b Kaplan–Meier survival curves of control groups and animals treated with BSA, dexamethasone, and SPION–IL-1Ra conjugates at 1.25 and 2.5 mg/kg of IL-1Ra (Adapted from https://doi.org/10.1016/j.neo.2014.11.001123)

were optimized. Aiming the SPIONs for targeting the tumor environment needs precise control over tumor induction in different body parts by using different cell lines and cell concentration. Method of administration and dosage of SPIONs also require standardization with respect to location, size, and volume of the tumor. Efficacy and success of magnetic targeting is also dependent on the strength (intensity) of the magnetic field and duration of the applied field, and physico-chemical properties and stability of the SPIONs in the bloodstream (Aguiar et al. 2017). Physico-chemical properties such as size, shape, and surface chemistry of SPIONs can affect the interaction of the nanoparticles with plasma proteins and therefore considered important for cellular uptake and toxicity. Generally, inorganic Fe3 O4 core is surface modified with hydrophilic polymer layers [such as polysaccharides and poly-ethylene glycol (PEG)] to achieve a good colloidal stability (Alkhayal et al. 2021; Zhu et al. 2009). Suciu et al. (2021) demonstrated the in vivo biocompatibility and biodistribution of 50 nm SPIONS-PEG nanoclusters. The particles dispersed well through the bloodstream with no tissue damage. Further, surface modification helps to conjugate the targeting moieties (Fig. 1) for enhancing therapeutic effect. For example, in a study, SPION modified with ß-cyclodextrin and conjugated to anti-prostate specific membrane antigen was used to deliver docetaxel as a therapeutic option for prostate cancer (Nagesh et al. 2016). In this way, a carefully tailored therapeutic SPIONS can overcome the biological barriers inside the human body system. Overall, such modifications ensure biocompatibility, biodistribution, stability and blood half-life to reach the targeted cells. In vivo biodistribution and clearance profiles were studied by Pham et al. (2018) where it was found that smaller sized SPIONs (10 nm) had higher accumulation in tissues and the 25 nm SPIONs were cleared faster. Silva et al. (2017) developed a

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pharmacokinetic model based on primary macrophages to evaluate the pharmacokinetics of the SPIONs in a clinical setup. Simulations of distribution and accumulation of SPIONs were made considering their penetration through capillary walls and their uptake in various organs by the macrophages. The developed model was validated against in vivo pharmacokinetics and the authors envisaged that the modeling technique can be used to bridge the pharmacokinetics of preclinical and human trials. Janikowska et al. (2017) demonstrated the magnetic capture of SPIONs in an ex vivo model of the human umbilical artery. They noted that precise positioning of external magnets can efficiently draw the SPIONs to superficial arteries, however, more sophisticated magnetic field geometries are required for deep-seated arteries. The rheological behavior of blood cells also influences the capture efficiency and thus, it is important to evaluate the accumulation of SPIONs under arterial flow conditions. Magnetic drug targeting was studied by Al-Jamal et al. and co-workers (Al-Jamal et al. 2016). In this work, oil-cored polymeric magnetic nanocapsules with tunable SPION loadings were used to understand magnetic drug targeting after intravenous administration in CT26-tumor bearing mice model. The oil core gave a thrust to increased loading of hydrophobic anticancer drug docetaxel. High SPIONS loading resulted in a reduced blood circulation time and plateauing of magnetic targeting. Further, to translate the results from mice to human, mathematical modeling was used to compute the in vivo magnetic, viscoelastic, convective, and diffusive forces acting on the SPIONS nanocapsules in accordance with the Nacev-Shapiro construct. The extrapolation from mice study helped to predict the expected behavior in humans. The authors in this study tried to bridge the gap between preclinical experiments and clinical translation in the field of magnetic targeting. A study by Balk et al. also investigated the impact of magnetic drug targeting based on SPIONS on head and neck cancer cell lines of squamous cell carcinoma of the pharynx, tongue, and parotid gland (Balk et al. 2021). The authors investigated the effect of two SPION systems; first SPIONS coated with lauric acid and second SPIONS coated with lauric acid and human serum albumin. The cellular uptake and biocompatibility were studied in both the SPION systems in the presence and absence of external magnetic fields. SPIONS coated with lauric acid and human serum albumin exhibited lower cytotoxicity in comparison to SPIONS coated with lauric acid. The study showed that applying an external magnetic field does not affect cell growth and viability. The results indicated that SPIONS are biocompatible and suitable system agents with potential of magnetic drug targeting for head and neck cancers. Similarly, Poller et al. demonstrated the influence of different surface coated SPIONS for future preclinical applications in vivo (Poller et al. 2017). SPIONS coated with dextran, lauric acid and human serum albumin and only lauric acid were evaluated for cytotoxic effect on four different breast cancer cell lines (T-47D, BT-474, MCF7, MDA-MB-231). The study indicated that SPIONS coated with dextran exhibits high stability and less toxicity and can be utilized as MRI contrast agents. Further, SPIONS coated with lauric acid and human serum albumin were suggested for hyperthermia, MRI, and magnetic drug targeting, thus enabling future theranostic applications. Gobbo et al. presented biodistribution and pharmacokinetic properties of SPIONS using different techniques such as inductively coupled plasma mass spectrometry

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(ICP-MS), particle electron paramagnetic resonance (pEPR) and MRI (Gobbo et al. 2015). A comparative assessment for these techniques were made for in vitro and in vivo studies. The study indicated that pEPR technique has great potential in comparison to MRI to detect SPIONS in both preclinical and clinical studies and for theranostic treatment of cancer. According to the authors, pEPR can sensitively evaluate SPIONS accumulation in organs in vivo rather than qualitative indication by MRI. In a study, low molecular weight hyaluronic-acid conjugated SPIONs were evaluated for cytotoxic effect for their application in cancer diagnosis and treatment (Chang et al. 2022). The authors compared the effect of fabricated SPIONS in U87MG human glioblastoma and NIH3T3 normal fibroblast cell lines (control). A three-day experimental duration showed a viability inhibition rate of 34% in cancer cell lines with no significant cytotoxic effect in control. The study indicated the potential of the system to be used in MRI applications as well as an injectable anticancer agent. Yu et al. reported a strategy for combining imaging and therapeutic modules by synthesizing thermally cross-linked SPIONS (Yu et al. 2008). Doxorubicin as an anticancer drug was loaded into the synthesized system. The system’s ability to detect tumor, antitumor efficacy and systemic toxicity was examined in a tumor-bearing mice model. The results demonstrated that the prepared SPION system could efficiently reach the tumor sites without exhibiting any significant systemic toxic side-effects and need for magnetic field. In a study, Branca et al. demonstrated the detection of sub-millimeter sized metastasis in lung by employing two MRI technologies, one based on hyperpolarized (HP) 3 He and second using functionalized SPIONS (Branca et al. 2010). Imaging is more particularly challenged in lung tissues as air-tissue interfaces give rise to weak signals. The signal decays rapidly on the order of 1 ms for humans at 1.5 T and