Studies on Graphene-Based Nanomaterials for Biomedical Applications (Springer Theses) 9811522324, 9789811522321

Citation preview

Springer Theses Recognizing Outstanding Ph.D. Research

Je Min Yoo

Studies on Graphene-Based Nanomaterials for Biomedical Applications

Springer Theses Recognizing Outstanding Ph.D. Research

Aims and Scope The series “Springer Theses” brings together a selection of the very best Ph.D. theses from around the world and across the physical sciences. Nominated and endorsed by two recognized specialists, each published volume has been selected for its scientific excellence and the high impact of its contents for the pertinent field of research. For greater accessibility to non-specialists, the published versions include an extended introduction, as well as a foreword by the student’s supervisor explaining the special relevance of the work for the field. As a whole, the series will provide a valuable resource both for newcomers to the research fields described, and for other scientists seeking detailed background information on special questions. Finally, it provides an accredited documentation of the valuable contributions made by today’s younger generation of scientists.

Theses are accepted into the series by invited nomination only and must fulfill all of the following criteria • They must be written in good English. • The topic should fall within the confines of Chemistry, Physics, Earth Sciences, Engineering and related interdisciplinary fields such as Materials, Nanoscience, Chemical Engineering, Complex Systems and Biophysics. • The work reported in the thesis must represent a significant scientific advance. • If the thesis includes previously published material, permission to reproduce this must be gained from the respective copyright holder. • They must have been examined and passed during the 12 months prior to nomination. • Each thesis should include a foreword by the supervisor outlining the significance of its content. • The theses should have a clearly defined structure including an introduction accessible to scientists not expert in that particular field.

More information about this series at http://www.springer.com/series/8790

Je Min Yoo

Studies on Graphene-Based Nanomaterials for Biomedical Applications Doctoral thesis accepted by Seoul National University, Seoul, Korea (Republic of)

123

Author Dr. Je Min Yoo Department of Chemistry The Graduate School Seoul National University Seoul, Korea (Republic of)

Supervisor Prof. Byung Hee Hong Department of Chemistry College of Natural Sciences Seoul National University Seoul, Korea (Republic of)

ISSN 2190-5053 ISSN 2190-5061 (electronic) Springer Theses ISBN 978-981-15-2232-1 ISBN 978-981-15-2233-8 (eBook) https://doi.org/10.1007/978-981-15-2233-8 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Supervisor’s Foreword

I met Dr. Je Min Yoo when he joined my research group in 2013 as an integrated Ph.D. student right after he finished his undergraduate at Johns Hopkins University. Since then, I have advised his researches and had a chance to observe the academic capability and achievements very closely. While studying various properties of graphene film, Dr. Yoo was inspired by CVD graphene film’s exceptional ability to donate or withdraw electrons and proposed the use of graphene film to oxidatively destroy phenolic compounds without any additional treatments. Through years of study, he demonstrated that graphene films can be utilized as a novel metal-free catalyst for the destruction of phenols. Dr. Yoo has also validated that the films are recyclable over multiple rounds of treatments. The results of this study were published in Nanoscale, and I strongly believe that the results may be a major technological breakthrough in lowering the costs of wastewater management. Most importantly in April 2013, Dr. Yoo proposed an idea that graphene quantum dots (GQDs)—the smallest and least toxic solution-phase graphene derivatives— may play an interesting role against Parkinson’s disease through unknown interactions with a neuron protein called alpha synuclein, the responsible protein for the pathogenesis of Parkinson’s disease. Since then he had carried out a study with Prof. Han Seok Ko at the Johns Hopkins Institute of Medicine to thoroughly investigate GQDs’ potential therapeutic function against the disease. After 5-year study, our manuscript titled ‘Graphene quantum dots prevent a-synucleinopathy in Parkinson’s disease’ has been published in Nature Nanotechnology, which will probably be the most important publication of Dr. Yoo. I strongly believe that this study can open new venues in clinical drug development against Parkinson’s disease and other neurodegenerative disorders as GQDs showed a number of unique properties including the penetration of the blood-brain barrier. He employed GQDs for other diseases and confirmed that they are effective for intestinal inflammation and Niemann Pick Type C disease as well. Upon introducing Dr. Yoo’s studies through Springer thesis publication, there must be a huge impact by the paradigm-shifting therapeutic applications to neurodegenerative diseases including Parkinson’s & Alzheimer’s diseases as well as a v

vi

Supervisor’s Foreword

novel approach to deal with wastewater treatment. Since the cost of treating neurodegenerative diseases in the United States is tens of billion dollars every year, Dr. Yoo’s new solution to treat the disease will greatly contribute to the healthcare industry not only in the United States but also in the entire world. Based on my experience and as proven by his excellent outcomes, Dr. Yoo is one of the most outstanding scientists in graphene-based biomedical studies not only in Korea but probably all over the world as well. I have no doubt that Dr. Yoo must be one of a few scientists who are able to introduce the world's most leading biomedical technology to the readership of Springer publications.

Seoul, Korea (Republic of) January 2020

Prof. Byung Hee Hong

Preface

Due to many of its unprecedented physical, electrical and chemical properties, graphene has gained enormous attention of researchers since its historic discovery in 2004. Technically, the term ‘graphene’ refers to a two-dimensional singleatom-thick graphite film that once was deemed to plausibly replace indium tin oxide (ITO) as a transparent flexible substrate. In addition to the film type graphene suitable for macroscopic applications, the noticeable features of other forms of graphene-based nanomaterials in solution have been recently revealed for applications in other branches of science including biology and nanomedicine. Along with the current research trends in these areas giving special attention to employing novel nanomaterials, I and my colleagues carried out some innovative studies to demonstrate the importance of graphene and its derivatives for versatile applications in these fields. This dissertation thus highlights the implications of graphene film and graphene-based nanomaterials for human health and the treatment of neurodegenerative diseases. Chapter 1 is an introductory guide to graphene-based nanomaterials, which includes their recent applications in biology and medical science, relevant preparation steps as well as reports on their toxicity/biodegradation analyses. Chapter 2 discusses the use of chemical vapor deposition (CVD) graphene film for degradation of phenolic compounds in industrial wastewater. Phenol and its derivatives are considered as major threatening substances as they can be potentially detrimental to human health. Although previous attempts have successfully destructed these compounds through ferrous ion-based advanced oxidation processes (AOPs), they require complicated and costly procedures including environmentally harmful residual salt removal and pH adjustments. The chapter suggests the unprecedented role of the CVD graphene film in the generation of hydroxyl radicals—the active components in AOPs for phenol destruction—from hydrogen peroxide through immediate electron transfer and demonstrates the application of a recyclable CVD graphene film as an environmentally-friendly catalyst without the need of additional treatments.

vii

viii

Preface

Chapter 3 highlights the therapeutic application of graphene quantum dots (GQDs) for Parkinson’s disease. The pathological hallmark of Parkinson’s disease is closely related to the aggregation of misfolded a-synuclein fibrils. By inhibiting the fibrillation of a-synuclein and inducing disaggregation of mature fibrils as well as blocking their transmission to neighboring neurons, GQDs prevent/rescue the death of dopaminergic neurons in the midbrain through the penetration across the blood-brain barrier (BBB). The outcomes indicate GQDs’ potent therapeutic efficacy and show they are especially promising when compared to current clinical drugs which focus merely on alleviating motor dysfunctions by injecting levodopa, the precursor of dopamine. Seoul, Korea (Republic of)

Dr. Je Min Yoo

Parts of this dissertation have been published in the following journals: 1. J. M. Yoo†, J. H. Kang† and B. H. Hong*. “Graphene-based nanomaterials for versatile imaging studies.” Chemical Society Reviews, 44, 4835–4852 (2015) 2. J. M. Yoo, D. W. Hwang, and B. H. Hong. (2018). “Graphene-Based Nanomaterials.” In: D. Lee, ed., Radionanomedicine: Combined Nuclear and Nanomedicine. Springer International Publishing, pp. 79–103 3. J. M. Yoo†, B. Park†, S. J. Kim, Y. S. Choi, S. Park, E. H. Jeong, H. Lee* and B. H. Hong*. “Catalytic degradation of phenols by recyclable CVD graphene films.” Nanoscale, 10, 5840–5844 (2018) 4. D. Kim†, J. M. Yoo†, H. Hwang, J. Lee, S. H. Lee, S. P. Yun, M. J. Park, M. Lee, S. Choi, S. H. Kwon, S. Lee, S. -H. Kwon, S. Kim, Y. J. Park, M. Kinoshita, Y. -H. Lee, S. Shin, S. R. Paik, S. J. Lee, S. Lee, B. H. Hong* and H. S. Ko*. “Graphene quantum dots prevent a-synucleinopathy in Parkinson’s disease.” Nature Nanotechnology, 13, 812–818 (2018)

ix

Acknowledgements

Upon completing another major stepping-stone in my life, I must acknowledge that I am truly blessed for being surrounded and supported by tremendous people throughout my years at Seoul National University. Foremost, my sincerest gratitude goes to my respected advisor, Prof. Byung Hee Hong, for the opportunities he has provided and for his continued faith in me. It is difficult to imagine the same outcomes without his support, encouragement and inspirational comments on my studies. In addition to academic guidance, he has always emphasized the importance of relationships with colleagues and every individual around me by saying, “No one succeeds alone,” which has become the motto of my life. Further gratitude should go to Prof. Han Seok Ko of Johns Hopkins Medical Institute for everything he has done for me. I never expected my undergraduate research would connect me with a lifelong mentor in this field. Thank you so much for always putting your trust in me. I am profoundly grateful for having Prof. Hyukjin Lee of Ewha Woman’s University as an admirable, friendly role model who provided me so much thoughtful advice over the years. I am also greatly thankful to Prof. Paul Dagdigian of Johns Hopkins University for sparking my interest to pursue chemistry up to this point with his excellent physical chemistry lectures. I would like to extend my appreciation to the dissertation committee: Prof. Seokmin Shin, Prof. Yan Lee, Prof. Jung Pyo Lee and Dr. Young-Ho Lee for their precious time and valued comments on this dissertation. It is indubitable that their insightful feedbacks and advice contributed to improving this work and will positively influence my future studies as well. I also have to express my gratitude to my previous and current collaborators: Dr. Junghee Lee and Prof. Seung R. Paik of the Department of Chemical & Biological Engineering; MinJun Lee and Prof. Seokmin Shin of the Department of Chemistry; Dr. Insung Kang, Dr. Byung-Chul Lee, Dr. Jin Young Lee and Prof. Kyung-Sun Kang of the Department of Veterinary Medicine; Dr. Young-Ho Lee from the Korea Basic Science Institute; Prof. Jung Pyo Lee and his team members of the Department of Nephrology, SNU Boramae Medical Center. A special note of thanks to Dr. Donghoon Kim, who has been the best partner in the development of a novel therapeutic candidate for Parkinson’s

xi

xii

Acknowledgements

disease over the last 6 years. I also wish to express my sincere gratitude to Rebecca Yun for helping me reorganize this dissertation for Springer publications. Withal, I am heavily indebted to the Global Ph.D. Program committee of the National Research Foundation, for 5 years of financial support. As a member of the Graphene Research Laboratory, I have been very fortunate to have had memorable times with my former and current colleagues: Dr. Myung Jin Park, Dr. Sang Jin Kim, Dr. Kyoungjun Choi, Dr. Youngsoo Kim, Dr. Bora Lee, Yong Seok Choi, Bae Kwon Park, Dong Jin Kim, Yuna Kim, Hae-Hyun Choi, Dong Heon Shin, Juhee Kim, Mina Park, Yeon Joon Suh, as well as Wonbae Lee, Dayoung Jeon and Yeseul Joo from Graphene Square Inc. Personally, I am thankful to my lifelong companions from my former soccer club, FCUK: Tae Keun Ane, Sung Woo Kim, Dong Jin Yu, James Kyuyeol Sim, Gwan Lim, Hak Soo Kim and Ikjoon Chang; from Johns Hopkins: Dr. Heeyeul Kwon, Daniel W. Lee, Victor Oh, Sunghyun Joo, Sung Bin Lee, Hyun Sung Park and Seung Hoon Lee; from SNU: Yeonsu Jung, Taeyong Park and Dr. Minwoo Noh; and all others: Dong Joon Shin, Jude Donghun Han, Yejun Yeo and Sumi Kim. I must also credit Jake Hostetler for being a wonderful colleague and helping me going through this dissertation for universal publications. Thank you all for making my life full of cherished moments, happy feelings and laughter. Finally, and most importantly, I wish to convey my deepest gratitude, love and respect to Dr. Young Seon Yoo and Mi Kyoung Lee for being the best parents in the world. I would be remiss to ignore the countless sacrifices they have made to make my life easier. Though I do not often articulate my thoughts, I know by heart that any of this would not have been possible without their unconditional love and support through good and bad. I also want to express heartfelt love towards my dearest little friend, Toto. Thank you very much for being a part of our family, and I will always love and miss you with all my heart. I know we will meet again later someday. See you later my friend.

Los Angeles, California January 2019

Cordially Dr. Je Min Yoo

Contents

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

1 1 2 3 3 5

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

6 6 7 7 8 10 10 12

2 Catalytic Degradation of Phenols by Recyclable CVD Graphene Films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

15 15 15 16 22 22 23 26

3 Graphene Quantum Dots Prevent a-Synucleinopathy in Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 29 29

1 Introduction of Graphene-Based Nanomaterials . . . . . . . . . . . 1.1 Applications in Biology and Medical Science . . . . . . . . . . . 1.1.1 Sensing Applications . . . . . . . . . . . . . . . . . . . . . . . 1.1.2 Therapeutic Applications . . . . . . . . . . . . . . . . . . . . 1.1.3 Fluorescence Bio-imaging . . . . . . . . . . . . . . . . . . . 1.2 Preparation of Graphene-Based Nanomaterials . . . . . . . . . . 1.2.1 Enhancing the Stability Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Reduction of GOs . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Toxicity of Graphene-Based Nanomaterials . . . . . . . . . . . . 1.3.1 In Vitro Toxicity of Graphene-Based Nanomaterials 1.3.2 In Vivo Toxicity of Graphene-Based Nanomaterials 1.3.3 Biodegradation of Graphene-Based Nanomaterials . . 1.4 Perspectives and Other Applications . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

xiv

Contents

3.3 Results and Discussion 3.4 Conclusion . . . . . . . . . 3.5 Experimental . . . . . . . Supplementary Information . References . . . . . . . . . . . . .

. . . . .

30 40 40 48 64

Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

List of Figures

Fig. 1.1 Fig. 1.2

Fig. 1.3

Fig. 1.4

Fig. 1.5

Fig. 2.1

Major characteristics of graphene-based nanomaterials in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graphene based in vivo imaging studies with specific targeting. a Representative in vivo fluorescence images of GQD–HA in mice after tail vein injections. b Fluorescence intensity from major dissected organs. c and d Bright field image and in vivo fluorescence images after GQD-HA injections. e Time-dependent tumor growth curves after photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dynamic tracking of insulin receptors. a Fluorescence images of insulin-conjugated GQDs (green, left), insulin receptor antibodies (IR) (red, middle), and the merge image (yellow, right). b and c Cellular distribution of insulin receptors, apelin and TNF-a after b 10 min or c 1 h of incubation with insulin-GQDs. Scale bar = 10 mm . . . . . . In vivo imaging and bio-distribution studies of carboxylated GQDs. a The in vivo imaging of KB tumor bearing mice after i.v. injection of carboxylated GQDs (5 and 10 mg/kg, upper and lower images, respectively). b and c Representative ex vivo images and quantitative distributions of major dissected organs 24 h post-injection . . . . . . . . . . . . . . . . . . . . Enzymatic degradation of GOs. a TEM and b AFM images of GOs in the presence of horse radish peroxidase (HRP). c Docking simulation between GOs and HRP . . . . . . . . . . . . Preliminary experiments to validate CVD graphene film’s catalytic activity. a Schematic diagram of the conventional phenol degradation with the Fenton’s reagents (top) and with a recyclable graphene film (bottom). b Representative fluorescence intensity of DCFH-DA (2 lM) with increasing hydrogen peroxide concentrations in the presence of 1 cm2 Gr/SiO2 (blue), 1 cm2 SiO2 only (red), and non-treated (black).

..

2

..

4

..

5

..

9

..

11

xv

xvi

Fig. 2.2

Fig. 2.3

Fig. 2.4

Fig. 2.5

List of Figures

c The surface adsorption test of phenols (1 mM) in the presence (black) and absence (red) of 1 cm2 Gr/SiO2. d Representative fluorescence intensity of DCFH-DA (2 lM) in the presence of 1 cm2 Gr/PET (red line), Gr/SiO2 (blue line), PET only (red dot), SiO2 only (black dot), and non-treated (blue dot) and e mono- (red), bilayer Gr/SiO2 (black) and SiO2 only (blue) with the respective Raman spectra. f Time-dependent pH (black) and temperature (blue) changes in the presence of 1 cm2 graphene film, hydrogen peroxide (100 mM), and phenols (1 mM) . . . . . . . . . . . . . . . . . . . . . . . Time-resolved HPLC spectrum after phenol degradation. a Time-resolved HPLC analysis with the assigned peaks exhibiting the detected intermediates in the midst of the complete degradation. b Standard HPLC peaks of phenol and maleic acid. c UV absorption spectra of the labeled intermediates. d Schematic diagram of the identified degradation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Area-dependent catalytic activity and recycle tests. a Actual and schematic representation (red-dot area) of an array of 1 cm2 graphene films to enhance the catalytic efficiency. b Representative area-dependent DCFH-DA fluorescence intensities (2 lM) with 100 mM hydrogen peroxide. c Time-dependent reduction of phenols and d maleic acid using graphene films with different areas. e Time-dependent changes in the amount of phenols through HPLC analysis of the recycled 9 cm2 graphene films after normalization. f Representative optical microscopy (OM) images and Raman spectra of the same graphene film after the first, second, third and fifth cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detailed analysis on the properties of monolayer graphene film. a Representative SEM and b HR-TEM images of the monolayer graphene film with c the SAED pattern. d Representative Cs-corrected STEM image of the monolayer graphene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary HPLC results on the degraded products. a HPLC analysis of hydrogen peroxide (100 mM) and phenols (1 mM). Hydrogen peroxide show strong, broad signals peaking at 7.43 and 8.65 min. b Time-dependent HPLC analysis and c quantified intensities with 9 cm2 graphene film 0, 3, 6, 12 and 24 h post-incubation with 1 mM phenols and 100 mM hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . .

..

17

..

19

..

21

..

24

..

25

List of Figures

Fig. 2.6

Fig. 3.1

Fig. 3.2

XPS analysis on graphene pre- and post-phenol degradation. C1s (left) and O1s (right) XPS analysis pre- and post-phenol degradation cycle respectively from the top. The characteristic C1s signals displayed distinctive atomic ratio changes from 0.12 ! 0.10, 0.2 ! 0.22, 0.06 ! 0.04 and 0.62 ! 0.64 respectively at 289 eV (O–C = O), 286.8 eV (C = O), 286.1 eV (C–O) and 285 eV (Csp2). In like manner, the characteristic O1s signals exhibited atomic ratio changes from 0.04 ! 0.04 and 0.96 ! 0.96 at 535 eV (C–O) and 533 eV (H–O–C) . . . . . . . . . Effect of GQDs on a-syn fibrillation and fibril disaggregation. a Schematic diagram of a-syn fibrillation (5 mg/ml a-syn monomers) and disaggregation (5 mg/ml a-syn fibrils) in the presence and absence of GQDs (5 mg/ml). b, c Kinetics of a-syn fibrillation using aliquots of the solution evaluated by ThT fluorescence b and turbidity assays (c). d Representative TEM images of a-syn fibrillation in the absence (left) and presence (right) of GQDs. e, f, Kinetics of preformed a-syn fibrils in the presence of GQDs using aliquots of the solution detected by ThT fluorescence e and turbidity assays at different time points (f). g Quantifications of the end-to-end length and number of a-syn fibrils per lm2. h Amount of remaining a-syn fibrils measured by multiplying the end-to-end length and number of a-syn fibrils at various time points (0, 6, 12, 24 and 72 h) in the presence of GQDs. i Representative TEM images of preformed a-syn fibrils at different time points (6 and 12 h, 1, 3 and 7 days) in the absence (top) and presence (bottom) of GQDs. j Representative dot-blot analysis of a-syn fibrils at various time points (0 and 12 h, 1, 3 and 7 days) with a-syn filament specific antibody. k BN–PAGE analysis of a-syn, prepared with aliquots of the solution at different time points (0, 3, 6 and 12 h, 1, 3 and 7 days) . . . . . . . . . . . . . . . . . . . . . . . Detailed analysis on the interaction between GQDs and a-syn fibrils during the disaggregation process. a TEM images for visualizing the interaction between biotinylated GQDs and a-syn fibrils with low (top) and high (bottom) magnifications. Red arrowheads designate biotinylated GQDs enhanced with ultra-small gold–streptavidin nanoparticles. b Quantification of the average width of a-syn fibrils during the dissociation process after 1 h of incubation. c NMR chemical shift difference plot collected from full 1H–15N HSQC spectra. The decreased intensity ratio of NMR chemical shifts is analyzed for each residue after binding with GQDs. d Time course simulation snapshots during the interaction between GQDs and mature a-syn fibrils, and 65 ns snapshot image (bottom right)

xvii

26

31

xviii

Fig. 3.3

Fig. 3.4

List of Figures

of the interaction between GQDs and mature a-syn fibrils with designated residues contributing to the major binding force. e Time-dependent plots for r.m.s.d. of atomic positions, SASA for the a-syn fibril only group and a-syn fibril and GQDs group, total potential energy (D Utot), electrostatic energy (D Eelec) and van der Waals energy (D Evan) for a-syn fibril and GQDs group, respectively (from the top). f Time-dependent secondary structure plot calculated by the DSSP algorithm. g, CD spectra of a-syn monomers and a-syn fibrils without (black) and with GQDs (red) after 7 days of incubation. h Fractional secondary structure contents ratio of a-syn fibrils and a-syn fibrils disaggregated by GQDs calculated using the algorithm CONTIN/LL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of GQDs on a-syn PFF-provoked neuron death, fibrillation and transmission in vitro. a–c Neuronal death assessed by TUNEL (a), alamarBlue (b) and LDH assays (c) after a-syn PFFs treatments (1 lg/ml) with and without GQDs (1 lg/ml) for 7 days. d Representative immunoblot levels with p-a-syn antibody. e Quantifications of the SDS-insoluble fraction normalized to b-actin levels. f Representative p-a-syn immunostaining images with p-a-syn antibody. g Quantifications of p-a-syn immunofluorescence intensities normalized to the control. h Schematic illustration of the microfluidic device composed of three connected chambers. i Representative images of p-a-syn immunostained neurons in the microfluidic device 14 days post a-syn PFFs addition. j Quantifications of p-a-syn immunofluorescence intensities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of GQDs on a-syn-induced pathologies in vivo. a Schematic illustration of injection coordinates for stereotaxic injection of a-syn PFFs (5 lg) in mice. 50 lg of GQDs were i.p. injected biweekly for 6 months as a treatment. b Representative TH immunohistochemistry images in the substantia nigra of a-syn PFF-injected hemisphere in the absence (top) and presence (bottom) of GQDs. c Stereological counting of the number of TH- and Nissl-positive neurons in the substantia nigra via unbiased stereological analysis after 6 months of a-syn PFF injection with and without GQDs. d Representative TH immunohistochemistry images in the striatum of a-syn PFF-injected hemisphere. e Quantifications of TH-immunopositive fiber densities in the striatum. f Assessments of the behavioral deficits measure use of forepaws in the cylinder test. g Assessments of the behavioral deficits measured by the ability to grasp and descend from a

34

35

List of Figures

Fig. 3.5 Fig. 3.6

Fig. 3.7

Fig. 3.8

pole. h Representative p-a-syn immunostaining images in the striatum (STR) and substantia nigra (SN) of a-syn PFF-injected hemisphere. i Quantifications of p-a-syn immunoreactive neurons in the striatum and substantia nigra. j Distribution of LB/LN-like pathology in the CNS of a-syn PFF-injected hemisphere (p-a-syn positive neurons, red dots; p-a-syn positive neurites, red lines) . . . . . . . . . . . . . . . . . . . . Mean ± S.D. values and P values of Fig. 3.1b, c, e, and f. . . Schematic representation of the collective effects of GQDs on a-synucleinopathy in Parkinson’s disease. Without GQDs, pathological a-syn monomers undergo spontaneous fibrillation to form aggregates, which eventually provokes loss of dopamine producing neurons in the midbrain, LB/LN like pathology and motor dysfunctions. On the other hand, GQDs treatment prevents a-syn fibrillation and disaggregates mature fibrils to monomers and thereby rescues from dopaminergic neuron loss, LB/LN-like pathology and behavior deficits provoked by abnormal a-syn fibrillation . . . . . . . . . . . . . . . . . Synthesis and biotinylation of GQDs and binding assay between GQDs-biotin and a-syn fibrils. a Schematic representation of GQDs synthesis from carbon fiber. b Representative AFM image of synthesized GQDs. c Fractions of GQDs with different height profiles. d, Schematic illustration of biotinylation on GQDs using EDC coupling. e Representative FT-IR spectra of GQDs (black line) and biotinylated GQDs (red line) with peaks and shifts assigned for designated functional groups. f Schematic representation of the experimental procedures for the binding assay between biotinylated GQDs and nanogold-streptavidin tagged a-syn fibrils. g Representative TEM images of preformed a-syn fibrils after 7 days of incubation in the presence of GQDs-biotin-nanogold-streptavidin complex (left) and nanogold-streptavidin only (right) . . . . . . . . . . . . . . . . . . The effect of GQDs on disaggregation of a-syn fibrils. a The distribution of a-syn fibril lengths at various time points (0, 6, 12, 24, and 72 h, n = 50 fibrils at each time point). b Representative AFM images of a-syn fibrils in the presence of GQDs after various time points (0, 24, and 48 h) and the representative line profiles of the designated regions, marked by blue (GQDs) and red (a-syn fibrils) arrows at each time point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

.. ..

37 49

..

49

..

50

..

51

xx

Fig. 3.9

Fig. 3.10

Fig. 3.11

Fig. 3.12

List of Figures

The effect of GQDs on disaggregation of a-syn PFFs. a Representative TEM images of fibrillated a-syn PFFs (5 mg/ml) incubated in the absence of (top) and in the presence of (bottom) GQDs (5 mg/ml) for 1 h. b The end-to-end length of a-syn PFFs after 1 h of incubation. c The amount of remaining a-syn PFFs and disaggregated a-syn PFFs assessed by BN-PAGE after various incubation periods . . . . . . . . . . . . . . 1 H-15N HSQC spectral analysis. a Full, overlapped 1H-15N HSQC spectra of 15N-labeled a-syn monomers (black) and after a week of incubation with GQDs (red). The assigned residues from 15N-labeled a-syn monomers only group. b A full HSQC spectrum of 15N-labeled a-syn monomers incubated with GQDs. Based on the peak intensity of 15 N-labeled a-syn monomers only group, the residues with no or minimal decrease in peak intensity (red), the residues with significant decrease in peak intensity (blue), and undetectable residues (black) are distinctively assigned . . . . . . . . . . . . . . . . . . The effect of GQDs on a-syn PFFs-induced cell death and restricted neurite outgrowth. a Representative TUNEL-positive neurons. Primary cortical neurons were treated with a-syn PFFs (1 lg/ml) in the absence and presence of GQDs (1 lg/ml). TUNEL assay was performed after 7 days of incubation. b Representative micrographs of neurite outgrowth and cell viability assays stained by outer cell membrane (red) and cell-permeable viability indicator (green). c, d Quantifications of neurite outgrowth and neuron viability from the stained images. e Representative immunoblot analysis of SNAP25 and VAMP2 protein levels. Primary cortical neurons were treated with a-syn PFFs (5 lg/ml) in the absence and presence of GQDs (5 lg/ml) and incubated for 7 days. f The expression levels of SNAP25 were normalized to b-actin level and quantified. g VAMP2 were normalized to b-actin level and quantified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of GQDs on a-syn PFFs-induced mitochondrial dysfunction and oxidative stress. a Representative 8-OHG immunostaining images in primary cortical neurons treated with a-syn PFFs (1 lg/ml) in the absence and presence of GQDs (1 lg/ml) and incubated for 7 days. b Quantifications of 8-OHG content by the immunofluorescence levels. c Measurements of the mitochondrial complex I activity after 7 days of a-syn PFFs (1 lg/ml) incubation. d Representative MitoTracker positive micrographs. Primary cortical neurons were treated with a-syn PFFs (1 lg/ml) in the absence and presence of GQDs (1 lg/ml). After 7 days of incubation,

52

53

54

List of Figures

Fig. 3.13

Fig. 3.14

mitochondria were labeled with MitoTracker® Orange CMTMRos (Red). e Quantifications of the length of the stained mitochondria. f Aspect ratio of the stained mitochondria. g Representative TEM images with low and high magnifications after the same preparation steps. h Microplate-based respirometry readings for neurons. The oxygen consumption rate measured in an XF 24 Seahorse analyzer in primary cortical neurons treated with a-syn PFFs (1 lg/ml) in the absence and presence of GQDs (1 lg/ml) for 24 h. i Quantifications of the basal respiratory rate. j Quantifications of the maximal respiratory rates from the respirometry results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of GQDs on a-syn PFFs-induced primary neuronal toxicity and pathology at different treatment points of GQDs and live cell imaging. a Neuronal toxicities assessed by alamarBlue and b LDH assays. Mouse cortical neurons were treated with a-syn PFFs (1 lg/ml) with 3 days pre- (n = 4, Before), simultaneous (n = 4, Simul), and 3 days post-injection (n = 4, After) of GQDs (1 lg/ml) for 7 days. c Representative images of p-a-syn by dot-blot analysis and d quantified intensities normalized to the PBS control. e Representative p-a-syn immunostaining micrographs with p-a-syn antibody after 7 days of incubation. f Quantifications of p-a-syn immunofluorescence intensities normalized to the PBS control. g Representative images showing GQDs and a-syn PFFs are co-localized in the lysosome of primary cultured neuron with live imaging. The LysoTracker (blue), GQDs-biotin-streptavidin Qdot complex (red), and FITC-labeled a-syn PFFs (green). h a-syn PFFs disaggregation process by GQDs monitored by time-lapse fluorescence signals during live imaging . . . . . . . . . . . . . . . . . . . The BBB permeability of GQDs. a Schematic diagram of the in vitro BBB model. b The confluent monolayer formations of the brain microvascular endothelial cells (BMEC) and astrocytes determined by immunofluorescence staining with CD31 (endothelial cell marker) or GFAP (astrocyte marker) antibodies, respectively. c The transepithelial electrical resistance (TEER) measured at various time points. d The permeabilities of endothelial cell monolayer and astrocyte monitored by measuring the ratio between the blood side and the brain side using dextran-fluorescein (3 kDa) and dextran-rhodamine (2,000 kDa) e Quantifications of permeability of GQDs and GQDs-biotin over time. f The a-syn fibril disaggregation abilities of pristine GQDs and

xxi

56

57

xxii

Fig. 3.15

Fig. 3.16

List of Figures

GQDs-biotin monitored by dot-blot. g ThT, and turbidity assays. h Confocal laser scanning microscope images of primary cultured BMEC and astrocyte after 1-hour incubation with GQDs-biotin. GQDs-biotin are shown with Qdot™ streptavidin, whereas the lysosomes labeled by LysoTracker orange. i Quantifications of the exosomal GQDs-biotin from BMEC or astrocytes. The amount of GQDs-biotin measured from the isolated exosome at 24 and 48 h post-endocytosis. j Immunohistochemical analysis of i.p. injected GQDs-biotin (2 mg/kg) by staining the olfactory bulb, neocortex, midbrain, and cerebellum with avidin-biotin complex method or immunogold method at 7 days after injection. The DAB positive stained signals detected in GQDs-biotin-injected mice compared to vehicle-injected control group. Immunogold positive signals observed inside (red triangles) or outside of neurons (blue triangles). k The comparison between intraperitoneal (IP; red bar) and intravenous (IV; blue bar) injections of GQDs-biotin. l The GQDs-biotin concentrations of the plasma measured from the non-injected control after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, and 6 months after biweekly GQDs-biotin injections using biotin quantification kit. m The brain (green bar) and plasma (blue bar) concentrations measured from 1 h, 7 days, and 14 days after single i.p. injection (2 mg/kg of GQDs-biotin. n 3 months and 6 months after multiple biweekly injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of GQDs on a-syn PFFs-induced glial cell activation in the SN. a Representative immunohistochemistry images for Iba-1 in the SN with low and high magnifications. Microglia in the SN of a-syn PFFs-injected hemisphere were stained with specific microglial marker Iba-1 (ionized calcium binding protein; specific marker of microglia/macrophage). b Quantifications of Iba-1-positive microglia. c Representative immunohistochemistry images for GFAP in the SN with low and high magnifications. Astrocytes in the SN of a-syn PFFs-injected hemisphere were stained with GFAP (glial fibrillary acidic protein; specific marker of astrocytes) antibody. d Quantifications of GFAP intensity. The relative GFAP intensities were normalized against the PBS-injected control group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of GQDs on pathologies and glial cell activation of hA53T a-syn transgenic mice. a Schematic illustration of the observing areas of hA53T a-syn Tg model. As a treatment, 50 lg of GQDs or PBS were i.p. injected biweekly for 4 months.

59

60

List of Figures

Fig. 3.17

Fig. 3.18

b Representative p-a-syn immunostaining images in the cortex, ventral midbrain, and brainstem of hA53T a-syn Tg. c Quantifications of p-a-syn immunoreactive neurons in the cortex (Ctx), ventral midbrain (VM), and brainstem (BS). d Representative dot-blot assay of p-a-syn and a-syn fibrils. e Hindlimb clasping dystonic phenotypes of hA53T a-syn Tg mice were compared with GQDs-injected group. f Assessment of the behavioral deficits measured by the clasping scores. g Assessment of the behavioral deficits measured by the pole tests. h Representative immunohistochemistry images of microglia in the LC of the brainstem with low and high magnifications. Microglia in the brainstem of nTg or hA53T a-syn Tg were stained with Iba-1. i Quantifications of Iba-1-positive microglia. j Representative immunohistochemistry images of GFAP in the brainstem with low and high magnifications. k Quantifications of GFAP intensity. The relative GFAP intensities were normalized against the PBS-injected control group. l The effect of GQDs on a-syn aggregate formation in HEK293 cells with hA53T a-syn overexpression. HEK293T cells were transfected with pCMV5-myc-A53T a-syn and the test groups were treated with GQDs (0.1 lg/ml). After 48 h, cells were immunostained with a-syn antibodies. m The number of immuno-positive aggregates per field was quantified and normalized . . . . . . . . . . Long-term in vitro and in vivo toxicity of GQDs. a Cytotoxicity measurements of GQDs on primary cortical neurons after 7 days of incubation with alamarBlue and LDH assays. b Survival curves of GQDs injected mice. 50 lg of GQDs or vehicle were i.p. injected in C57BL/6 mice biweekly for 6 months and in vivo toxicity of GQDs was monitored (n = 20). c Representative hematoxylin and eosin (H&E) staining images of the major organs. The nuclei and eosinophilic structure of the liver, kidney, and spleen of GQDs-injected group were compared to the PBS-injected control group. d Schematic illustration of the injection and sampling methods for in vivo tracking of GQDs-biotin. e 50 lg of GQDs-biotin were i.p. injected in C57BL/6 mice. The concentrations of GQDs-biotin were measured using the brain (red) or urine (blue) of GQDs-biotin-injected mice at various time points (1, 3, 7, and 14 days) . . . . . . . . . . . . . . . . . . . . . . . . The comparative effects of nano-GOs and rGQDs on a-syn PFFs induced primary neuronal toxicity, disaggregation of fibrils and the BBB permeability. a FT-IR spectra of pristine GQDs (black), nano-GOs (green), and reduced GQDs

xxiii

61

62

xxiv

List of Figures

(rGQDs, purple) with designated peaks for the functional groups. b AFM images of nano-GOs and GQDs with representative line profiles. c Quantifications of the cell toxicities after 72 h of incubation with 20 lg/ml of each material. d The cell toxicities monitored in SH-SY5Y cells by alarmarBlue assay at 12, 24, and 72 h with various concentrations of each material (1, 10, and 20 lg/ml). e The comparative dissociation effects of GQDs, nano-GOs, and rGQDs assessed by dot blot. f ThT and turbidity assays. g TEM images after 7 days of incubation (each with 5 mg/ml concentration; n = 3, independent experiments). h In vitro BBB permeabilities of GQDs, nano-GOs, and rGQDs using the in vitro BBB model in Fig. 3.13 . . . . . . . . . . . . . . . . . . . . . .

63

Chapter 1

Introduction of Graphene-Based Nanomaterials

1.1 Applications in Biology and Medical Science The first serendipitous yet monumental discovery of graphene in 2004 immediately aroused considerable attention of researchers from all fields of science due to many of its unprecedented characteristics [1]. In the beginning, the major research focus had been solely on macroscopic applications including the replacement of indium tin oxide (ITO) with chemical vapor deposition (CVD) graphene for flexible thin substrates for transparent electrodes. Relevant studies in the fields of physics, materials science, and electronics have thus been explored as well [2]. Given a growing consensus on graphene and its derivatives’ environment-friendly properties, however, scientists now aim their attention at employing the materials in other areas of science such as biology and nanomedicine. As a result, graphene-based nanomaterials in solution including graphene oxides (GOs), graphene quantum dots (GQDs) are now commonly exploited for various biomedical studies while the term ‘graphene’ technically refers to a two-dimensional atomically thin film. Especially, GOs and GQDs attract huge interests due to their exceptional dispersions in polar solvents and low cytotoxicity both in vitro and in vivo. Figure 1.1 summarizes the major characteristics of GOs, GQDs and rGOs. Generally speaking, GOs are synthesized via the Hummer’s method and GQDs are prepared by thermo-oxidatively cut various carbon precursors. Notably, GOs and GQDs have abundant edge functional groups available for diverse chemical reactions, which can be exploited for desired studies including dynamic tracking and further improve their dispersion in different solvents.

*The contents of this chapter are reproduced from: (1) Je Min Yoo† , Jin Hyoun Kang† and Byung Hee Hong* Chemical Society Reviews (2015) 44, 4835–4852. © The Royal Society of Chemistry and (2) Je Min Yoo, Do Won Hwang and Byung Hee Hong. (2018) Graphene-Based Nanomaterials. In: Lee D. (eds) Radionanomedicine. Biological and Medical Physics, Biomedical Engineering. Springer, Cham. © Springer Nature Singapore Pte Ltd. 2020 J. M. Yoo, Studies on Graphene-Based Nanomaterials for Biomedical Applications, Springer Theses, https://doi.org/10.1007/978-981-15-2233-8_1

1

2

1 Introduction of Graphene-Based Nanomaterials

Fig. 1.1 Major characteristics of graphene-based nanomaterials in solution

1.1.1 Sensing Applications At the early stages, scientists had primarily taken note of the ability of GOs to quench fluorescence and the availability of functional groups for various chemical conjugations for diverse bio-sensing studies. Most notably, Lu et al. carried out real-time monitoring of fluorophore-labeled DNA on and off GOs in 2009 [3]. This work was followed by similar studies with other target molecules including protein kinases, phosphate containing metabolites, neurotransmitters, and trypsin by applying appropriate surface modifications [4–7]. A study by Mei et al. even demonstrated that the discrimination of ferric (Fe3+ ) and ferrous (Fe2+ ) ions in living cells with logically designed GO gates by exploiting their ability to quench fluorescence [8]. Some scientists devoted keen interest on the unusual stem cell growth and differentiation patterns on CVD graphene film, which could develop entirely novel approaches in tissue/stem cell engineering [9, 10].

1.1 Applications in Biology and Medical Science

3

1.1.2 Therapeutic Applications Graphene-based nanomaterials are known to possess relatively high absorbance in the near-infrared (NIR) region, which gave rise to some innovative approaches for selective ablation of cancer cells with NIR laser-triggered local heat generation, known as photo- or hyperthermia [11, 12]. Through relevant modifications with desired moieties, graphene-based nanomaterials can distinctively target malignant tumor cells without affecting the normal cells. What is more, graphene-based nanomaterials’ hydrophobic basal plane composed of sp2 carbon bonds can be utilized to yield synergistic effects. Some researchers exploited the fact that hydrophobic anti-cancer drugs (i.e. doxorubicin) and photodynamic agents (i.e. zinc phthalocyanine) can be readily loaded onto graphene’s basal plane through pi-interactions. By incorporating chemotherapies and/or photodynamic therapies with photothermal ablation, more efficient therapeutic strategies are achievable [13, 14]. In addition, NIR-responsive hyperthermia can not only be employed for the direct excision of tumor, but may also be exploited as an extrinsic cue for controlled drug and gene deliveries by disorganizing endocytosed vesicles or drug-loading matrices [15–17].

1.1.3 Fluorescence Bio-imaging Several drug delivery photothermal ablation strategies for cancer treatments were achieved with the use of diverse graphene-based complexes. Although some approaches were accompanied by intrinsic in situ fluorescence imaging of drug delivery/therapy system, most of these studies additionally attached inorganic quantum dots or other fluorophores to graphene-based materials to enable real-time monitoring. In 2013, Nahain et al. demonstrated two distinct graphene-based anti-cancer drug delivery methods with rGOs and GQDs [18, 19]. For the rGO–hyaluronic acid (HA) complex (avg. size ∼ = 200 nm), spiropyran was tagged as an additional photochromic dye to generate a fluorescent nanocomposite [18]. Shortly after, the researchers carried out similar studies without the use of spiropyran. Alternatively, the authors exploited GQDs’ intrinsic fluorescence (avg. size ∼ = 20 nm) to track the delivery of GQD–HA to the desired receptors [19]. Successful targeting of the GQD–HA complex to overexpressed CD44 receptors was corroborated with the fluorescence images of the tumor tissue in both in vitro and in vivo trials (Fig. 1.2). Anti-cancer treatment was immediately followed by spontaneous doxorubicin release under mild acidic environments, which were loaded on the hydrophobic basal plane of GQDs. Put simply, while previous graphene-based therapy/imaging studies incorporated additional fluorescent molecules, the authors validated that GQDs’ intrinsic fluorescence can be exploited for real-time monitoring of cancer treatment. The outcome was further supported in 2014 by Ge et al., where the authors employed a few nano-meter scale GQDs in highly efficient photodynamic cancer treatment for simultaneous fluorescence imaging [20]. In this report, the authors prepared GQDs with a broad absorption

4

1 Introduction of Graphene-Based Nanomaterials

Fig. 1.2 Graphene based in vivo imaging studies with specific targeting. a Representative in vivo fluorescence images of GQD–HA in mice after tail vein injections. b Fluorescence intensity from major dissected organs. c and d Bright field image and in vivo fluorescence images after GQD-HA injections. e Time-dependent tumor growth curves after photodynamic therapy

and strong deep-red emission peaking at 680 nm. After thorough in vitro and in vivo studies, the authors verified that GQDs can be applied as promising photodynamic agents, with a superior singlet oxygen quantum yield, photo- and pH-stabilities that can even enable real-time fluorescent imaging. While most researchers focused on utilizing GQDs’ fluorescence to monitor in situ drug delivery upon targeting, Zheng et al. suggested that GQDs can be employed as a novel fluorophore that could divulge unidentified biological functions (Fig. 1.3) [21]. In this report, insulin receptors were specifically labeled and dynamically tracked by conjugating insulin receptors with GQDs in adipocytes. By exploiting GQDs’ fluorescence, the authors observed that the internalization and recycling of insulin receptors are regulated by two specific proteins with distinct functions: (1) apelin, which enhances the insulin sensitivity, and (2) TNF-α, which promotes the insulin resistance. While this single study may not be sufficient to fundamentally change the therapeutic approaches to treat diabetes, revealing unidentified cellular/subcellular functions by GQDs’ fluorescence must be beneficial for further researches in relevant areas.

1.2 Preparation of Graphene-Based Nanomaterials

5

Fig. 1.3 Dynamic tracking of insulin receptors. a Fluorescence images of insulin-conjugated GQDs (green, left), insulin receptor antibodies (IR) (red, middle), and the merge image (yellow, right). b and c Cellular distribution of insulin receptors, apelin and TNF-α after b 10 min or c 1 h of incubation with insulin-GQDs. Scale bar = 10 mm

1.2 Preparation of Graphene-Based Nanomaterials As mentioned, GOs and GQDs are now commonly utilized in various biomedical studies due to the large surface area, low cytotoxicity, and excellent dispersion in polar solvents. In most cases, GOs are obtained from graphite via the Hummer’s method and GQDs are synthesized by thermo-oxidative cutting of different carbon precursors including GOs. As these graphene derivatives exhibit exceptional optical properties, they are well-suitable for versatile biomedical applications including bio-imaging. Further modifications of GOs and GQDs could make them even more appropriate for desired applications.

6

1 Introduction of Graphene-Based Nanomaterials

1.2.1 Enhancing the Stability Under Physiological Conditions Well-prepared GOs can be easily dispersed in deionized water without considerable aggregation for several months but are prone to agglomeration in physiological solutions with high concentrations of ionic salts. Two most frequently applied strategies for improving stability under these conditions are size reduction and an introduction of biocompatible hydrophilic polymers. Although the as-synthesized GOs display broad size distributions by nature, they can be narrowed down by tip-sonication. Many previous studies demonstrate that the sonication-assisted methods change the average size of GOs due to the its high energy, where large GO particles can be disintegrated to much smaller fragments, as small as 10 nm [22]. Since small-sized GOs show enhanced stability in various solvents including physiological solutions [23], sub-500 nm GOs are widely employed for bio-imaging studies. For the attachment with hydrophilic polymers, polyethylene glycol (PEG) is the most commonly used. Sun et al. grafted branched PEGs on GOs to improve the dispersion in biological buffers, which is essential for further applications as imaging agents [22]. In that study, PEGs were conjugated to GOs through the reactions with epoxy and carboxylic acid groups yielding PEGylated nano-sized GOs, which exhibited outstanding stability in phosphate buffered saline (PBS). Many researchers have used PEGs to achieve high stability even in solutions with high concentrations of salts (B10% NaCl) [24]. Other biocompatible hydrophilic polymers such as polyacrylic acid (PAA), polyamido amine (PAMAM) and dextran (DEX) can also be utilized as a stabilizer through covalent modifications [25–27].

1.2.2 Reduction of GOs rGOs have been employed for different photothermal and/or photoacoustic studies by exploiting their high absorption cross-section in the NIR region. To obtain highquality rGOs, as-synthesized GOs are reduced through photothermal [28], electrochemical [29], or chemical reduction methods [30], but chemical reduction is considered the most facile way to produce rGOs. In 2007, Stankovich et al. reported that hydrazine can reduce dispersed GOs in aqueous solution [30]. Ever since, hydrazine has been universally exploited to obtain rGOs. Nevertheless, owing to the toxicity of hydrazine and insolubility of rGOs, the process is usually accompanied by additional modifications. Unlike GOs, however, surface modifications with hydrophilic molecules are not sufficient to dramatically alter the solubility of rGOs as only a negligible amount of epoxy and carboxyl acid groups are available. Alternatively, noncovalent modifications between the basal plane of rGOs and hydrophobic molecules are used to change the properties of rGOs based on the strong van der Waals interaction. For instance, highly dispersible rGOs can be produced by attaching hydrophobic alkyl chains with PEGs terminals, as suggested from the report by Shi et al. [31].

1.2 Preparation of Graphene-Based Nanomaterials

7

Recently, different proteins are also employed as a stabilizer and reducing agent of GOs for enhanced stability and biocompatibility. Liu et al. suggested that GOs can be reduced by gelatin and they can remain well-dispersed in physiological solutions [32]. The resulting gelatin-rGO nanosheets can be also internalized in cells without significant cytotoxicity, and used as effective in vitro imaging agents. In addition, Sheng et al. reported the use of bovine serum albumin (BSA) to reduce GOs and simultaneously function as a surfactant, yielding hydrophilic rGOs which can be successfully exploited for in vivo imaging studies [33].

1.3 Toxicity of Graphene-Based Nanomaterials Among different nanoscale carbon allotropes, carbon nanotubes (CNTs) and graphene-based nanomaterials have been extensively studied, which share practically identical chemical compositions. However, recent investigations have revealed entirely different levels of toxicities from these two classes of materials [34, 35]. Unlike the toxicity studies on various types of CNTs, most reports have a general consensus on the negligible toxicity of graphene-based materials, with more emphasis on GQDs, in both in vitro and in vivo studies.

1.3.1 In Vitro Toxicity of Graphene-Based Nanomaterials Within the recent few years, multiple researchers explored cellular internalization and in vitro toxicity of functionalized graphene derivatives—GOs and GQDs—with different types of mammalian cells. In general, these studies agree on graphene derivatives’ insignificant toxicity with respectable cellular uptake, which make them appropriate for different biomedical applications [36–41]. However, some scientists assert that the toxicity of micro-meter or a few hundred nanometer-sized GOs is much more critical than that of GQDs, which must not be neglected in biomedical studies [37, 38]. Notably, some reports suggested that GOs and GO-based nanoflakes can be associated with severe toxicity and relevant lung diseases [42, 43]. Other studies divulged heterogeneous cell-specific cytotoxicity of GOs by carrying out thorough cytotoxicity screening of GOs on multiple cell lines [44]. Collectively, the toxicity of graphene-based materials is strongly affiliated with the particle size, which could partially explain the lower cytotoxicity of a few nano-meter sized GQDs than a few micro-meter sized GOs [38, 43, 45]. On the other hand, Akhavan et al., have persistently suggested that the toxicity of graphene-based materials may be irrelevant to the particle size. Instead, direct interaction of the sharp edges of graphene with the cell membranes is a more plausible argument underlying the observed cytotoxicity [46, 47]. This leads to the authors’ another reasoning that even nano-sized GOs can be detrimental to mammalian cells. Thus, detailed toxicity mechanisms pertaining

8

1 Introduction of Graphene-Based Nanomaterials

to the size and shape of graphene-based materials remain uncertain and thus demand further studies. Furthermore, the effects of edge functionalization in cell permeability and toxicity have been investigated by numerous research groups. It has been commonly discussed that the covalent attachment of hydrophilic molecules including PEGs to the edges of graphene improves its solubility and biocompatibility in biological environments [22]. The possible toxicity alleviation effects by introducing additional functional groups to GQDs such as –COOH, NH2 , CO–N(CH3 )2 , and –PEG were also analyzed [41, 48]. Quantitative data analysis did not indicate any notable toxicity changes in these GQDs derivatives. Meanwhile, the membrane permeability increased in the order of –PEG, –OH, and –NH2 [49]. These results are encouraging for scientists who endeavor to utilize modified graphene derivatives as they show only negligible cytotoxicity levels [50]. In 2011, Sasidharan et al. reported different effects between pristine graphene (hydrophobic) and carboxylated graphene (hydrophilic) in biological environments. In comparison to pristine graphene, carboxylated graphene show pacified hydrophobic interactions with the cell membrane and the consequential toxic effects such as the deformation of cell membrane, increased intracellular ROS level and subsequent apoptosis [39]. Collectively, edge functionalization plays a crucial role not only in cell-nanoparticle interactions, but also in peroxidase enzyme-assisted biodegradation, which will be discussed in a subsequent paragraph.

1.3.2 In Vivo Toxicity of Graphene-Based Nanomaterials In addition to the in vitro toxicity studies addressed above, many scientists have carried out in vivo bio-distribution and toxicology studies of graphene-based materials in recent years. In 2010, Yang et al. reported the long-term in vivo pharmacokinetics and bio-distribution of PEGylated 125 I-labeled nano-graphene sheets (NGS) with systemic toxicology examination [51]. The radioactivity levels of 125 I-NGS-PEG were measured in the blood and different vital organs after intravenous injection were measured over time. Overall, there was a persistent decrease of radioactivity levels of 125 I-NGS-PEG in most organs. The authors presumed that small NGS-PEG particles may be cleared out by renal or fecal excretion. They also explored long-term in vivo toxicology over 3 months by performing blood biochemistry and hematology analysis. Mice injected with NGS-PEG (20 mg/kg) were sacrificed at different periods of time, and all parameters from the blood biochemistry and hematological data did not exhibit any form of toxicity. Soon after, the same group performed in vivo bio-distribution and toxicology studies of functionalized nano-GOs by injecting them through two other major routes: oral feeding and intraperitoneal (i.p.) injection [36]. It was revealed that oral administration did not result in obvious tissue uptake. On the other hand, i.p. injections led to high accumulation of nano-GOs in the reticuloendothelial system (RES) over long periods of time. Despite the results obtained through the i.p. injection, they found that both routes did not provoke abnormal damages to the treated animals.

1.3 Toxicity of Graphene-Based Nanomaterials

9

In 2014, Nurunnabi et al. studied in vivo bio-distribution and organ toxicity of carboxylated GQDs in mice after intravenous (i.v.) injection [52]. The accumulation of GQDs and probable organ damages were evaluated by carrying out a long-term serum biochemical analysis and histological analyses. Notably, the injected GQDs were mainly found in the liver, spleen, lung, kidney, and tumor sites (Fig. 1.4). Overall, the study did not reveal any significant level of in vivo toxicity. A follow-up study also proved that GQDs injection of 5 or 10 mg/kg dosages for 21 days did not result in any appreciable organ damage or lesions in mice. These outcomes were supported by in vivo bio-distribution studies, suggesting fast clearance of GQDs from kidneys without significant accumulation in the main organs [44]. These biocompatibility studies show the potential of GQDs in clinical applications in the impending future. However, these results also clearly suggest that high doses of GOs are toxic and lead to lethal results, on good agreement with the in vitro results.

Fig. 1.4 In vivo imaging and bio-distribution studies of carboxylated GQDs. a The in vivo imaging of KB tumor bearing mice after i.v. injection of carboxylated GQDs (5 and 10 mg/kg, upper and lower images, respectively). b and c Representative ex vivo images and quantitative distributions of major dissected organs 24 h post-injection

10

1 Introduction of Graphene-Based Nanomaterials

1.3.3 Biodegradation of Graphene-Based Nanomaterials As much as different parameters for in vitro and in vivo cytotoxicity assessments are critical, comprehensive understandings on enzyme-assisted degradation processes of graphene-based materials are important for beneficial and universal applications. In 2011, Kotchey et al. examined enzyme-catalyzed oxidation of GOs and rGO by incubating them with low concentrations of hydrogen peroxide and horseradish peroxidase (HRP) [53]. Interestingly, they figured out that the presence of functional groups is directly associated with the magnitude of enzyme-catalyzed degradation. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images showed that the mild enzymatic environment provided by HRP resulted in the generation of holey GOs, which ultimately processed to make fully oxidized debris of GO flakes (Fig. 1.5). On the contrary, the same incubation conditions with rGOs did not induce any notable alterations caused by the enzymatic degradation. The authors suggest that the presence of functional groups promotes looser binding with HRP and allows the enzyme to function more dynamically; the catalytic heme site of HRP can be brought in proximity of GOs during the process. Conversely, more hydrophobic rGOs make tighter binding with HRP, disabling any contacts with the catalytic heme site. A year after the first study, the same group conducted meticulous investigation on the enzyme-catalyzed degradation of CNMs using HRP and myeloperoxidase (MPO) [54]. This report verified promising aspects of functionalized CNMs for in vivo applications because they are supposedly degradable by intracellular enzymes with peroxidase activity such as human MPO (hMPO). It should be noted, however, that the experimental results do not accurately represent the actual biodegradation processes in the human body because the physiological hMPO levels are generally more diluted than the experimental set ups. Moreover, the oxidative debris of GO nanoflakes could provoke unforeseen toxic side effects.

1.4 Perspectives and Other Applications The promising aspects of graphene-based nanomaterials for biomedical applications include: (1) the availability to produce various forms of graphene derivatives for diverse purposes, (2) high dispersibility upon introducing hydrophilic molecules and (3) possibility to transport drugs with specific targeting. The strong intrinsic Raman scattering signals can be exploited for Raman spectroscopy-based analysis, and increased NIR absorbance by chemical and/or thermal reduction is beneficial for successful relevant investigations. The fluorescent properties of GOs and GQDs can be modulated by incorporating different functional groups, and improved quantum yield (QY) enables fluorescence bio-imaging another conceivable approach. Although CNTs manifest some advantageous properties and had been regarded as suitable materials for biological applications early on, the challenging procedures

1.4 Perspectives and Other Applications

11

Fig. 1.5 Enzymatic degradation of GOs. a TEM and b AFM images of GOs in the presence of horse radish peroxidase (HRP). c Docking simulation between GOs and HRP

impede large-scale production of high quality CNTs. Contrarily, the mass production of dispersed graphene derivatives, especially GOs and rGOs are easier to achieve. Solvo-thermal method exploiting sodium already showed its capability to generate gram-scale graphene [55], and shear exfoliation can also be readily used for industry-scale production [56]. Moreover, unlike the CNTs, graphene does not

12

1 Introduction of Graphene-Based Nanomaterials

necessitate additional purification steps, which are important hurdles for CNT commercialization. The produced graphene can be further oxidized in the under oxidative environments to incorporate additional oxygen-containing functional groups, ligands of interest, biomolecules and even targeting moieties can be conjugated to improve graphene derivatives’ dispersibility and enhanced targeting, which are desirable aspects for various biomedical applications. Among different graphene derivatives, CVD graphene is known to exhibit excellent electron conductivity and rapid heat dissipation. CVD graphene film with minimal or practically no defect sites could perfectly trap liquid for preventing vaporization under high vacuum conditions, which are required for scanning or transmission electron microscopy analyses. In other words, graphene film can be exploited as an atomically thin window for in situ electron microscopy for samples in aqueous conditions, including different biomolecules such as proteins and even tissues. In addition, this single-atom thick film enables conformal contacts with non-conductive samples to assure the detailed analysis of the surface. It could be thus employed as a novel coating material for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis where noble metals such as platinum (Pt) are currently used for electron beam damage-resistant coating. In the first chapter, some of the recent biomedical applications, preparation and toxicity of graphene-based nanomaterials were highlighted. As mentioned, graphene has several advantageous properties for different biomedical studies including low toxicity and easily modifiable functional groups. Various graphene derivatives also exhibit unique characteristics including high Raman scattering intensity, large absorption cross section in the NIR region, and sharp photoacoustic contrast with the NIR incident laser. Based on history and current status of graphene-based biomedical applications, it is expected that graphene-based nanomaterials will play vital roles in various important biomedical researches in the impending future.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

K. S. Novoselov et al., Science, 2004, 306, 666. A. K. Geim and K. S. Novoselov, Nat. Mater., 2007, 6, 183. C.-H. Lu et al., Angew. Chem., Int. Ed., 2009, 48, 4785. J.-J. Liu et al., Nanoscale, 2013, 5, 1810. Y. Wang et al., Anal. Chem., 2013, 85, 9148. X. Li et al., Nanoscale, 2013, 5, 7776. S.-J. Jeon et al., J. Am. Chem. Soc., 2014, 136, 10842. Q. Mei et al., Chem. Commun., 2012, 48, 7468. W. C. Lee et al., ACS Nano, 2011, 5, 7334. S. Y. Park et al., Adv. Mater., 2011, 23, H263. J. T. Robinson et al., J. Am. Chem. Soc., 2011, 133, 6825. B. Tian et al., ACS Nano, 2011, 5, 7000. C. Jang et al., Nanoscale, 2015, 7, 18584. L. Zhou et al., J. Photochem. Photobiol., 2014, B 135, 7. H. Kim and W.J. Kim, Small, 2014, 10, 117. P. Matteini et al., Nanoscale, 2014, 6, 7947.

References 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

V. Shanmugam, S. Selvakumar and C. -S. Yeh, Chem. Soc. Rev., 2014, 43, 6254. A.-A. Nahain et al., Biomacromolecules, 2013, 14, 4082. A.-A. Nahain et al., Mol. Pharmaceutics, 2013, 10, 3736. J. Ge et al., Nat. Commun., 2014, 5, 4596. X. T. Zheng et al.en, ACS Nano, 2013, 7, 6278. X. Sun et al., Nano Res., 2008, 1, 203. Z. Liu et al., J. Am. Chem. Soc., 2008, 130, 10876. S. Zhu et al., J. Nanopart. Res., 2014, 16, 2530. P. S. Wate et al., Nanotechnology, 2012, 23, 415101. S. A. Zhang, K. Yang, L. Z. Feng and Z. Liu, ACS Nano, 2011, 49, 4040. G. Gollavelli and Y.-C. Ling, Biomaterials, 2012, 33, 2532. L. J. Cote, R. Cruz-Silva and J. X. Huang, J. Am. Chem. Soc., 2009, 131, 11027. G. K. Ramesha and S. Sampath, J. Phys. Chem. C, 2009, 113, 7985. S. Stankovich et al., Carbon, 2007, 45, 1558. S. Shi et al., Biomaterials, 2013, 34, 3002. K. Liu et al., J. Mater. Chem., 2011, 21, 12034. Z. Sheng et al., Biomaterials, 2013, 34, 5236. A. M. Jastrzebska, P. Kurtycz and A. R. Olszyna, J. Nanopart. Res., 2012, 14, 1320. C. Fisher et al., J. Nanomater., 2012, 2012, 315185. K. Yang et al., Biomaterials, 2013, 34, 2787. Y. Chong et al., Biomaterials, 2014, 35, 5041. H. Zhang et al., ACS Appl. Mater. Interfaces, 2013, 5, 1761. A. Sasidharan et al., Nanoscale, 2011, 3, 2461. C. Wu et al., Adv. Healthcare Mater., 2013, 2, 1613. X. Yuan et al., Nanoscale Res. Lett., 2014, 9, 108. K. Wang et al., Nanoscale Res. Lett., 2011, 6, 1. A. Schinwald et al., ACS Nano, 2012, 6, 736. S. M. Chowdhury et al., Biomaterials, 2013, 34, 283. L. De Marzi et al., J. Biol. Regul. Homeostatic Agents, 2014, 28, 281. O. Akhavan, E. Ghaderi and A. Akhavan, Biomaterials, 2012, 33, 8017. O. Akhavan and E. Ghaderi, ACS Nano, 2010, 4, 5731. W. Kong et al., Nanoscale, 2014, 6, 5116. Kirchner et al., Nano Lett., 2005, 5, 331. L. Yan et al., Nanoscale, 2011, 3, 362. K. Yang et al., ACS Nano, 2011, 5, 516. M. Nurunnabi et al., ACS Nano, 2013, 7, 6858. G. P. Kotchey et al., ACS Nano, 2011, 5, 2098. G. P. Kotchey et al., Acc. Chem. Res., 2012, 45, 1770. M. Choucair, P. Thordarson and J. A. Stride, Nat. Nanotechnol., 2008, 4, 30. 56. K. R. Paton, et al., Nat. Mater., 2014, 13, 6242.

13

Chapter 2

Catalytic Degradation of Phenols by Recyclable CVD Graphene Films

2.1 Summary Phenolic compounds are considered as major threatening industrial pollutants as they can be potentially detrimental to human health. To remove these substances, ferrous ion-based catalysts have been commonly exploited for oxidative degradation through advanced oxidation processes (AOPs). Nevertheless, these catalysts imminently accompany a few major disadvantages such as the need of additional purification steps to remove residual salts and pH adjustment for the complete activation. This chapter discusses the use of a chemical vapor deposition (CVD) graphene film as a novel catalyst for the AOP-based destruction of phenols, which does not necessitate further steps for salt removal nor external energy to optimize the process. We have also demonstrated that the catalytic activity is proportional to the surface area of the graphene film and the catalytic efficiency can be considerably enhanced simply by utilizing multiple graphene films. Furthermore, the graphene film’s recyclability has been tested and validated its practical use by carrying out repeated cycles with the same film.

2.2 Introduction While phenol and phenolic compounds are essential substances for many industries, their safety remains questionable as these materials can become potentially carcinogenic and/or mutagenic [1–3]. Destructing these chemicals organic pollutants in aqueous environments has thus been one of the major foci in the field of wastewater treatment and environmental science. Up to now, degradation of phenol and *This chapter is reproduced from: Je Min Yoo† , Baekwon Park† , Sang Jin Kim, Yong Seok Choi, Sungmin Park, Eun Hye Jeong, Hyukjin Lee* and Byung Hee Hong* Nanoscale (2018) 10, 5840– 5844. © The Royal Society of Chemistry. © Springer Nature Singapore Pte Ltd. 2020 J. M. Yoo, Studies on Graphene-Based Nanomaterials for Biomedical Applications, Springer Theses, https://doi.org/10.1007/978-981-15-2233-8_2

15

16

2 Catalytic Degradation of Phenols …

the derivatives has been mostly completed through advanced oxidation processes (AOPs), which utilize the generated secondary radicals (i.e. hydroxyl radicals) as strong oxidants [4–6]. To expedite the process while minimizing drawbacks, various metal-based catalysts such as the Fenton’s reagents (Fe2+ ion-based) have been universally exploited. Nevertheless, these catalysts unavoidably require additional procedures to eliminate residual salts from the system and are fully activated only under mildly acidic conditions. Moreover, other non-ferrous ion catalysts require external sources of energy including electricity, sonication and strong UV irradiation, which limits their practical applications [7–10]. In this chapter, the application of a monolayer CVD graphene film as a metalfree catalyst is reported, which can chemically destroy phenols through AOP. Since its first discovery in 2004, various properties of graphene have been highlighted by researchers from different fields of study, especially focusing on its excellent chemical, optical, and physical properties, as well as its outstanding ability role in redox reactions [11–14]. Based on this, we set up a hypothesis that a monolayer graphene film can be employed to catalyze the secondary radical generation from hydrogen peroxide via spontaneous redox reactions, where the generated secondary radicals can oxidatively degrade phenolic compounds to less harmful linear compounds and/or even to water.

2.3 Results and Discussion For this study, a high quality CVD graphene film was prepared according to the conventional method exploiting copper foil and methane gas (Fig. 2.4), and transferred onto a silicon oxide substrate (Gr/SiO2 ) [15, 16]. In theory, even a small fraction of monolayer graphene can catalyze the AOP-based phenol degradation analogously to the conventional Fenton’s reagents, but could be even more favorable for having different attributes including: (1) no additional procedures to remove residual salts from the system, (2) no restriction pH adjustment for complete activation, (3) no external energies required to further activate the process, and (4) the replenishment of hydrogen peroxide should be solely sufficient to carry out repeated cycles with the same film if the surface of graphene is not significantly damaged (Fig. 2.1a). To test monolayer graphene film’s ability to produce secondary radicals from ambient hydrogen peroxide, the fluorescence intensity of 2 μM DCFH-DA (2 ,7 dichlorofluorescein diacetate) was monitored with increasing hydrogen peroxide levels. DCFH-DA is one of the most widely employed probes the measure the level of reactive oxygen species (ROS) [17, 18], where the fluorescence intensity is strongly corresponded to the abundance and oxidation strength of ambient radicals. Preliminarily, the changes in the fluorescence intensity of non-treated, silicon oxide substrate only, and a monolayer graphene on substrate were measured. While the negative control and substrate only batches respectively led to a 12-fold and 27-fold increases in the fluorescence intensity, the presence of graphene film resulted in a 102-fold increase with 100 mM hydrogen peroxide (Fig. 2.1b).

2.3 Results and Discussion

17

Fig. 2.1 Preliminary experiments to validate CVD graphene film’s catalytic activity. a Schematic diagram of the conventional phenol degradation with the Fenton’s reagents (top) and with a recyclable graphene film (bottom). b Representative fluorescence intensity of DCFH-DA (2 μM) with increasing hydrogen peroxide concentrations in the presence of 1 cm2 Gr/SiO2 (blue), 1 cm2 SiO2 only (red), and non-treated (black). c The surface adsorption test of phenols (1 mM) in the presence (black) and absence (red) of 1 cm2 Gr/SiO2 . d Representative fluorescence intensity of DCFH-DA (2 μM) in the presence of 1 cm2 Gr/PET (red line), Gr/SiO2 (blue line), PET only (red dot), SiO2 only (black dot), and non-treated (blue dot) and e mono- (red), bilayer Gr/SiO2 (black) and SiO2 only (blue) with the respective Raman spectra. f Time-dependent pH (black) and temperature (blue) changes in the presence of 1 cm2 graphene film, hydrogen peroxide (100 mM), and phenols (1 mM)

18

2 Catalytic Degradation of Phenols …

Apparently, the difference in DCFH-DA intensity can be only attributed to the presence of a 1 cm2 monolayer graphene film, which supposedly generated hydroxyl radicals from hydrogen peroxide; the radicals’ strong oxidizing strength is directly indicated by the discernible increment. Interestingly, previous studies with density functional theory (DFT) calculations investigated probable hydrogen peroxide reduction reaction (HPRR) pathways on graphene, where strong secondary radicals are produced en route to the complete reduction to water [19, 20]. Explicitly, ambient hydrogen peroxide molecules become temporarily adsorbed on the basal plane of graphene through the van der Waals interactions. Upon the transient physisorption, the O–O bond in hydrogen peroxide is displaced by the formation of the C–O bond with graphene and become adsorbed radicals, where the temporary hydroxyl radicals on graphene oxidizes the neighboring target molecules like DCFH-DA [21, 22]. While the transient physisorption of hydrogen peroxide is desirable, the adsorption of phenols on the graphene can impede the oxidative destruction as aromatic molecules tend to interact with the basal plane of graphene through hydrophobic interactions [22]. To address this probable obstacle, the adsorption test with phenols on graphene was conducted by introducing 1 cm2 graphene in a 1 mM phenol solution without hydrogen peroxide (Fig. 2.1c). The results clearly showed that practically no phenol molecules were adsorbed onto the graphene films. It can be thus deduced that the pi-pi interactions between aromatic compounds and graphene are either only inconsequential or merely temporary, which was also suggested by a previous report that re-examined the general notion about pi-stacking [23]. To account for any unforeseen effects of the substrates underneath graphene films and the number of graphene layers on the catalytic efficiency, monolayer graphene on polyethylene terephthalate (PET) film and bilayer graphene on silicon oxide wafer were prepared for separate studies (Fig. 2.1d, e). Figure 2.1d shows that the change substrate in only led to indistinguishable alterations in the catalytic performance, as assessed by DCFH-DA fluorescence intensities. As there is no evident difference between these two most widely used substrates, silicon oxide wafers were used for all following assessments to assist the progress of surface characterizations including X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Similarly, monoand bilayer graphene, which display characteristic Raman 2D/G ratios of 4.27 and 1.15, did not elicit meaningful alterations in the catalytic performance (Fig. 2.1e) [24]. The outcome is correlated to a previous study on the electron transfer of monoand bilayer graphene, which essentially does not exhibit any distinct difference [25]. The time-dependent temperature and pH changes were detected by incubating 1 cm2 Gr/SiO2 and 1 mM phenols with 100 mM hydrogen peroxide for 24 h under ambient conditions (Fig. 2.1f). Foremost, there were no critical alterations in the temperature during the incubation except for the minimal fluctuations caused by the changes in the surrounding temperature. Interestingly, the pH started at 6.06 and continuously elevated with time, and saturated at 7.06 24-h post-incubation. The pH increment should be attributed to the gradual depletion of acidic molecules—phenols and hydrogen peroxide—by the generated hydroxyl radicals [26, 27]. Namely, hydrogen peroxide is utilized to generate hydroxyl radicals and phenols are oxidatively destroyed as a result of the process. Eventually, the final pH of graphene-catalyzed

2.3 Results and Discussion

19

solution reaches that of water, which is incomparably more benign than the Fenton’s reagent that are activated in the pH range of 3–4 for the optimal efficiency [5]. Next, the effect of graphene-catalyzed secondary radicals on phenol degradation was explored. It should be noted that the concentration of hydrogen peroxide was equally controlled at 100 mM in all degradation experiments as the DCFH-DA intensity for Gr/SiO2 was essentially saturated from 100 mM onwards. After incubating 1 cm2 Gr/SiO2 with 1 mM phenols for 24 h, the resulting solution was examined with time-resolved ion-exchange high performance liquid chromatography (HPLC) (Fig. 2.2a). Besides the characteristic HPLC peak for phenol at 68.56 min (Fig. 2.2b), four distinct peaks were also detected. To identify the newly emerged substances, a HPLC-coupled organic acid analyzer with a proton column and ultraviolet (UV) absorption spectroscopy were employed. First, the three compounds with distinctive peaks at 16.65, 41.99, and 44.25 min post-injection were identified as they should evidence less degraded molecules than the one emerging at 9.74 min post-injection (Fig. 2.2c). According to the standard UV spectra of phenolic compounds, it was able to reveal that the collected absorption spectra epitomize those of p-benzoquinone, hydroquinone and catechol, for the peaks at 16.65, 41.99, and 44.25 min, respectively

Fig. 2.2 Time-resolved HPLC spectrum after phenol degradation. a Time-resolved HPLC analysis with the assigned peaks exhibiting the detected intermediates in the midst of the complete degradation. b Standard HPLC peaks of phenol and maleic acid. c UV absorption spectra of the labeled intermediates. d Schematic diagram of the identified degradation pathway

20

2 Catalytic Degradation of Phenols …

[28–30]. As predicted, these substances are the aromatic by-products from oxidatively degraded phenols, with different degrees of oxidation. Although the strong and broad signal for hydrogen peroxide peaking at 7.43 and 8.65 min overlapped with the signals at 9.74 and 16.65 min (Fig. 2.5a), the resultants were still able to be identified with the HPLC-coupled organic acid analyzer with the reference spectra. Lastly, the major product detected at 9.74 min was sequentially analyzed. Based on the provided reference HPLC peaks, the signal distinctively represents maleic acid (Fig. 2.2b), which possesses a less hazardous linear structure with four carbons and is known to be biodegradable. Previous reports investigating the phenol degradation pathway also classify maleic acid to be a major product en route to the complete oxidation to carbon dioxide and water [4]. The study also categorizes p-benzoquinone, hydroquinone, and catechol as the key intermediates before the ring opening process to yield maleic acid, while aromatic compounds are not considered to be naturally degradable (Fig. 2.2d). Because 1 cm2 graphene film only resulted in 25% reduction of phenols (1 mM), we simply employed a stack of graphene films to enhance the catalytic performance (Fig. 2.3a). Preliminarily, the DCFH-DA fluorescence intensities with increasing areas of graphene films—1, 2, 4, and 9 cm2 —were evaluated to verify that the number of secondary is proportional to the surface area. As expected, the fluorescence intensities were distinctly elevated as the surface area enlarged, by displaying a 131, 208, and 913-fold increments in the DCFH-DA intensity with 2, 4, and 9 cm2 Gr/SiO2 , respectively (Fig. 2.3b). The same groups were then tested for time-dependent phenol degradation and analyzed with HPLC. Consistent with the results from DCFH-DA assessments, the oxidative degradation of phenols was observed in a time-dependent manner where the magnitude of phenol reduction depends strongly on the area of the graphene film. 24 hours post-incubation, the amount of phenols was decreased by 54%, 60%, and 92% with 2, 4, and 9 cm2 Gr/SiO2 , respectively (Fig. 2.3c). Note that the total amount of non-degradable aromatic compounds—phenol, hydroquinone and catechol—progressively diminished with time. In like manner, time-dependent maleic acid concentrations were detected by the HPLC-coupled organic acid analyzer (Fig. 2.3d). Remarkably for 9 cm2 Gr/SiO2 , which induced the greatest phenol reduction, the amount of maleic acid discernibly elevated for the first 3 h of incubation. Nevertheless, it exhibited a modest downturn for the following 6 h and became saturated until 24 h of incubation. This contradictory trends between the phenol decrements and the maleic acid increments can be attributed to the final degradation of maleic acid into carbon dioxide and water, which was visually revealed by the generation of bubbles. Lastly, the recyclability of the graphene film was studied by performing repeated phenol degradation cycles using the same 9 cm2 Gr/SiO2 film (Fig. 2.3e). After each run, the amount of phenols and intermediates were measured. Strikingly, graphene films exhibited practically the same degradation performance after a series of testing cycles; four separate time-resolved HPLC spectra for phenol degradation by the first, second, third, and fifth recycled 9 cm2 Gr/SiO2 exhibit essentially the same results (Fig. 2.3e). On average, the concentration of the remaining phenol was 6.5 ± 2.3% in

2.3 Results and Discussion

21

Fig. 2.3 Area-dependent catalytic activity and recycle tests. a Actual and schematic representation (red-dot area) of an array of 1 cm2 graphene films to enhance the catalytic efficiency. b Representative area-dependent DCFH-DA fluorescence intensities (2 μM) with 100 mM hydrogen peroxide. c Time-dependent reduction of phenols and d maleic acid using graphene films with different areas. e Time-dependent changes in the amount of phenols through HPLC analysis of the recycled 9 cm2 graphene films after normalization. f Representative optical microscopy (OM) images and Raman spectra of the same graphene film after the first, second, third and fifth cycles

each cycle. This is nearly equivalent to the 92% reduction accomplished with 9 cm2 Gr/SiO2 on the first test run. In addition to the catalytic performance, the intrinsic properties of graphene films were analyzed to confirm physical damages and/or chemical alterations through optical microscopy (OM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) analyses (Figs. 2.3f and 2.6). As shown from the figure, the representative OM images evidently show that the graphene film was not severely scratched or worn out

22

2 Catalytic Degradation of Phenols …

after 5 rounds of test cycles, and only negligible part of stains were detected. The respective Raman spectra further corroborated that the initial property and condition graphene were preserved over the 5 rounds of degradation testes, by displaying respective 2D/G ratios of 3.72, 3.43, 4.30, and 3.84 for the same graphene film respectively after the first, second, third, and fifth tests (2D peak at 2680 cm−1 and G peak at 1580 cm−1 ). Generally, the 2D/G ratio greater than 2 indicates an important feature of high-quality monolayer graphene, and a higher 2D/G ratio is commonly corresponded to better crystallinity. Moreover, XPS analyses on the atomic composition of graphene pre- and post-phenol destruction showed practically the same outcomes, which demonstrated that submerging graphene in an oxidizing aqueous condition does not critically affect the properties of graphene (Fig. 2.6).

2.4 Conclusion Collectively, we have validated the use of a CVD graphene film as novel metal-free catalyst oxidative for AOP-based oxidative degradation of phenols. While its catalytic performance seems relatively weaker and thus necessitates a longer incubation time than the Fenton’s reagents, it eliminates the imminent disadvantages of the conventional approaches such as the need of further steps for residual salt removal, input of external energies, and acidic pH adjustment. Moreover, it should be noted that the catalytic performance can be significantly enhanced by exploiting a large dimension of the film, which is expected to be exploited as a recyclable film with respectably high catalytic effects for industrial wastewater treatments. As graphene can be transferred onto flexible substrates without affecting the performance, the range of probable applications can be expanded to other areas of research that require strong radicals for beneficial purposes.

2.5 Experimental Preparation of CVD Graphene Film. Graphene was synthesized by the chemical vapor deposition (CVD) method on a high-quality copper foil (Alfa Aesar, 99.95%) in accordance with the previous report [13]. The growth was carried out at 1000 °C with flowing 5 sccm methane gas and 40 sccm hydrogen gas to ensure the reducing environment. Followed by the coating process with a poly methyl methacrylate (PMMA) layer on one side of as-synthesized graphene on Cu foil, the uncoated graphene was removed by introducing strong oxygen plasma. The Cu foil was subsequently immersed in ammonium persulfate (APS) etching solution (20 g/L) for 5 h. The PMMA/Gr layer was then transferred onto SiO2 wafer and baked at 80 °C for 24 h to ensure the adhesion. Finally, the PMMA layer was cleaned off by immersing in acetone.

2.5 Experimental

23

Characterization. Optical microscopy (OM) images were captured by a NIKON ECLIPSE LV 100 ND OM installed with NIS Elements D 4.20.00 software. A RENISHAW Raman spectrometer allows the evaluation of the quality and uniformity of the synthesized graphene films. The Raman spectra were collected using an Ar laser (514 nm) with 1 mm spot size. The graphene samples were analyzed by high resolution—transmission electron microscope (HR-TEM, JEM-3010, JEOL Ltd.) and Cs corrected STEM with Cold FEG (JEM-ARM200F, JEOL Ltd.) and the images were captured by Gatan Digital Camera (MSC-794) coupled to the electron microscope. XPS spectra for Gr/SiO2 were collected by Kα XPS system (Thermo-Scientific) with an X-ray mono-chromator (Al Kα Micro-focused depth profiling EX06 Ion Source) with an average spot size of 30–400 μm. DCFH-DA Fluorescence Measurements. The amount of reactive oxygen species (ROS) was detected with 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) fluorescence probe (Sigma Aldrich, Ex/Em: 495 nm/529 nm, 2 μM) [16]. The measurements for all test groups were carried out after a fixed time period of incubation with 5 ml of total volume. Ion-exchange HPLC Analysis. A hydrogen column (Aminex 87H column, 300 × 10 mm, Bio-Rad, USA) ion-exchange high performance liquid chromatography (HPLC, Dionex Ultimate3000) was utilized for the analysis of oxidative degradation of phenols. In all test samples, the injection volume was set to 10 μl with 0.01 N H2 SO4 eluent and the flow rate of 0.5 ml/min. Throughout 90 min of injection, time-resolved HPLC analyses were carried out, and the intermediate structures were identified with the RI detector (ERC, RefractoMAX520, Japan). The produced organic acid was identified based on the reference peaks and the retention times by the organic acid analysis coupled to the HPLC. pH and Temperature Measurements. Throughout the graphene-based phenol degradation, the pH and temperature changes were observed using a pH meter with a digital thermometer (Trans instruments, BP3001). Any alterations in both parameters were recorded after 0, 1, 3, 6, 12, 24 h of incubation. For stabilization, the measured values were recorded after 5 min of each time point. X-ray Photoelectron Spectroscopy Analysis. XPS spectra for Gr/SiO2 were collected by Kα XPS system (Thermo-Scientific) with an X-ray mono chromator (Al Kα Micro-focused depth profiling EX06 Ion Source) with an average spot size of 30–400 μm.

Supplementary Information See Figs. 2.4, 2.5 and 2.6.

24

2 Catalytic Degradation of Phenols …

Fig. 2.4 Detailed analysis on the properties of monolayer graphene film. a Representative SEM and b HR-TEM images of the monolayer graphene film with c the SAED pattern. d Representative Cs-corrected STEM image of the monolayer graphene

Supplementary Information

25

Fig. 2.5 Supplementary HPLC results on the degraded products. a HPLC analysis of hydrogen peroxide (100 mM) and phenols (1 mM). Hydrogen peroxide show strong, broad signals peaking at 7.43 and 8.65 min. b Time-dependent HPLC analysis and c quantified intensities with 9 cm2 graphene film 0, 3, 6, 12 and 24 h post-incubation with 1 mM phenols and 100 mM hydrogen peroxide

26

2 Catalytic Degradation of Phenols …

Fig. 2.6 XPS analysis on graphene pre- and post-phenol degradation. C1s (left) and O1s (right) XPS analysis pre- and post-phenol degradation cycle respectively from the top. The characteristic C1s signals displayed distinctive atomic ratio changes from 0.12 → 0.10, 0.2 → 0.22, 0.06 → 0.04 and 0.62 → 0.64 respectively at 289 eV (O–C = O), 286.8 eV (C = O), 286.1 eV (C–O) and 285 eV (Csp2 ). In like manner, the characteristic O1s signals exhibited atomic ratio changes from 0.04 → 0.04 and 0.96 → 0.96 at 535 eV (C–O) and 533 eV (H–O–C)

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

E. Halfon, Environ. Sci. Technol., 1986, 20, 1173–1179. M. Pera-Titus et al., Appl. Catal., B, 2004, 47, 219–256. P. R. Gogate and A. B. Pandit, Adv. Environ. Res., 2004, 8, 501–551. J. A. Zazo et al., Environ. Sci. Technol., 2005, 39, 9295–9302. J. J. Pignatello et al., Crit. Rev. Environ. Sci. Technol., 2006, 36, 1–84. R. Andreozzia, A. Insolab and R. Marottac, Catal. Today, 1999, 53, 51–59. P. V. Nidheesh, RSC Adv., 2015, 5, 40552–40577. T. Soltani and M. H. Entezari, Chem. Eng. J., 2014, 251, 207–216. C. C. Winterbourn, Toxicol. Lett., 1995, 82–83, 969–974. M. L. Kremer, J. Phys. Chem. A, 2003, 107, 1734–1741. M. E. Ragoussi et al., J. Am. Chem. Soc., 2014, 136, 4593–4598. H. Sun et al., ACS Nano, 2014, 8, 6202–6210. P. L. Levesque et al., Nano Lett., 2011, 11, 132–137. T. Hu and I. C. Gerber, J. Phys. Chem. C, 2013, 117, 2411–2420. S. J. Kim et al., Nano Lett., 2015, 15, 3236–3240. S. Bae et al., Nat. Nanotechnol., 2010, 5, 574–578. Y. Oyama, T. Uehab and K. Maekawac, Brain Res., 1994, 635, 113–117. K. Setsukinai et al., J. Biol. Chem., 2003, 278, 3170–3175.

References 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

S. Amirfakhri et al., J. Power Sources, 2014, 257, 356–363. P. Wu et al., Phys. Chem. Chem. Phys., 2013, 15, 6920–6928. H. Tachikawa, T. Iyama and H. Kawabata, Jpn. J. Appl. Phys., 2013, 52, 01AH01. S. Akca et al., PLoS One, 2011, 6, e18442. C. R. Martinez and B. L. Iverson, Chem. Sci., 2012, 3, 2191–2201. S. Virendra, J. Daeha and Z. Lei, Prog. Mater. Sci., 2011, 56, 1178–1271. M. Velicky et al., ACS Nano, 2014, 8, 10089–10100. A. Kütt et al., J. Org. Chem., 2008, 73, 2607–2620. M. G. Harris et al., Am. J. Optom. Physiol. Opt., 1988, 65, 527–535. T. Wilke et al., Open J. Phys. Chem., 2013, 3(2), 97–102. A. Kiss, J. Molnar and C. Sandorfy, Bull. Soc. Chim. Fr., 1949, 16, 275–280. M. J. S. Dewar, V. P. Kubba and R. Pettit, J. Chem. Soc., 1958, 3076–3079.

27

Chapter 3

Graphene Quantum Dots Prevent α-Synucleinopathy in Parkinson’s Disease

3.1 Summary While growing evidence suggests that the pathogenesis of Parkinson’s disease is heavily associated with the accumulation [1, 2] and transmission [3, 4] of α-synuclein (α-syn) aggregates in the midbrain, no anti-fibrillation agents have been developed to treat the disease for the clinical practice. In this chapter, we highlight that graphene quantum dots (GQDs) prevent fibrillation of α-syn and directly interact with mature fibrils, inducing their dissociation into monomers. In addition, GQDs can inhibit the loss of neurons and synaptic proteins, reduce the formation of Lewy body and Lewy neurite, alleviate mitochondrial dysfunctions and sequential subcellular damages, and block neuron-to-neuron transmission of α-synucleinopathy induced by preformed α-syn fibrils [5, 6]. We also fount out, in vivo, that GQDs pass through the blood–brain barrier (BBB) and protect against the loss of dopamine producing neurons provoked by exogenous α-syn preformed fibrils, innate α-syn fibrillation by transgenic deficits, Lewy body/Lewy neurite formation and behavioral dysfunctions.

3.2 Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder. It is characterized by degeneration of dopaminergic neurons, accumulation of pathological α-syn aggregates in the midbrain and accompanied by motor dysfunctions. The emerging evidence indicates that polymerization of α-syn monomers into fib*This chapter is reproduced from: Donghoon Kim† , Je Min Yoo† , Heehong Hwang, Junghee Lee, Su Hyun Lee, Seung Pil Yun, Myung Jin Park, MinJun Lee, Seulah Choi, Sang Ho Kwon, Saebom Lee, Seung-Hwan Kwon, Sangjune Kim, Misaki Kinoshita, Young-Ho Lee, Seokmin Shin, Seung R. Paik, Sung Joong Lee, Seulki Lee, Byung Hee Hong* and Han Seok Ko* . Nature Nanotechnology (2018) 13, 812–818. © Springer Nature. © Springer Nature Singapore Pte Ltd. 2020 J. M. Yoo, Studies on Graphene-Based Nanomaterials for Biomedical Applications, Springer Theses, https://doi.org/10.1007/978-981-15-2233-8_3

29

30

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

rils is a critical step in the pathogenesis of PD. Numerous therapeutic candidates including small organic molecules, peptide mimics and polymers have been shown to prevent the fibrillation of mono/oligomeric α-syn. Nevertheless, the major drawbacks of these candidates are the exhibition of only moderate inhibitory effects on the fibrillation of α-syn mono/oligomers, weak disaggregating ability on fibrils, as well as considerable in vitro and in vivo toxicity. In recent years, a surge of interest in graphene-based nanomaterials has led to their various applications in biomedical sciences. Notably, a few studies have discussed graphene oxides’ (GOs) potential therapeutic role against Alzheimer’s disease (AD) by inhibiting the fibrillation of beta-amyloid (Aβ) peptides, mainly by virtue of their amphiphilic property. Moreover, computational evidence suggests that hydrophobic graphene sheet could cause destruction of the amyloid fibrils. None of these reports, however, have fully investigated graphene-based nanomaterials’ possible therapeutic function based on adequate in vitro and in vivo studies or their efficacy against PD. Previous studies have suggested that pathological α-syn spreads and propagates along anatomically connected brain regions and thereby contributes to the progression of PD. Likewise, the addition of exogenous sonicated α-syn preformed fibrils (PFFs) provokes endogenous α-syn to form aggregates, and thus leads to transmission and propagation in α-syn PFFs neuron/mouse models. In this study, potential therapeutic efficacy of GQDs in PD as an anti-aggregation agent by (i) investigating their effects on α-syn fibrillation and fibril disaggregation in vitro (ii) employing the α-syn PFFs transmission model to study the antiaggregation efficacy of GQDs on the pathogenesis caused by the transmission and propagation of pathological α-syn aggregates both in vitro and in vivo.

3.3 Results and Discussion After the preparation and characterization of graphene quantum dots (GQDs) (Fig. 3.6a–c, e), we explored their probable function in preventing α-synuclein (αsyn) fibrillation and dissociating mature fibrils (Fig. 3.1a). Thioflavin T (ThT) fluorescence (Fig. 3.1b), turbidity assays (Fig. 3.1c) and transmission electron microscopy (TEM) analysis (Fig. 3.1d) revealed that α-syn monomers spontaneously assembled into mature fibrils without GQDs. Meanwhile, the same measurements displayed that GQDs practically suppressed the entire fibrillation process. In addition, the presence of GQDs leads to the disaggregation of α-syn fibrils into smaller fragments (Figs. 3.1e–i and 3.7a). The average length of the shortened fractions changed from 1 μm to 235 nm and 70 nm after 6 and 24 h of incubation, respectively (Fig. 3.1g, i). The number of the fragments shortened elevated for the first 24 h of incubation, indicating that the fibrils were dissociated from the inner parts of the α-syn fibrils (Fig. 3.1h). The number of shortened fragments, however, started to exhibit a downturn at day 3 and they were no longer observable after 7 days, illustrating a complete disaggregation of fibrils with time (Fig. 3.1i). Time-dependent atomic

3.3 Results and Discussion

31

Fig. 3.1 Effect of GQDs on α-syn fibrillation and fibril disaggregation. a Schematic diagram of α-syn fibrillation (5 mg/ml α-syn monomers) and disaggregation (5 mg/ml α-syn fibrils) in the presence and absence of GQDs (5 mg/ml). b, c Kinetics of α-syn fibrillation using aliquots of the solution evaluated by ThT fluorescence b and turbidity assays (c). d Representative TEM images of α-syn fibrillation in the absence (left) and presence (right) of GQDs. e, f, Kinetics of preformed α-syn fibrils in the presence of GQDs using aliquots of the solution detected by ThT fluorescence e and turbidity assays at different time points (f). g Quantifications of the end-to-end length and number of α-syn fibrils per μm2 . h Amount of remaining α-syn fibrils measured by multiplying the end-to-end length and number of α-syn fibrils at various time points (0, 6, 12, 24 and 72 h) in the presence of GQDs. i Representative TEM images of preformed α-syn fibrils at different time points (6 and 12 h, 1, 3 and 7 days) in the absence (top) and presence (bottom) of GQDs. j Representative dot-blot analysis of α-syn fibrils at various time points (0 and 12 h, 1, 3 and 7 days) with α-syn filament specific antibody. k BN–PAGE analysis of α-syn, prepared with aliquots of the solution at different time points (0, 3, 6 and 12 h, 1, 3 and 7 days)

32

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

force microscopy (AFM) results displayed essentially the same disaggregation patterns, where GQDs and fibrils are distinguished by their distinctive height profiles (Fig. 3.7b). Dot-blot assay with α-syn filament specific antibody and blue native polyacrylamide gel electrophoresis (BN–PAGE) analysis also clearly showed that the presence of GQDs in the same system induced a continuous reduction of fibrils in a time-dependent manner (Fig. 3.1j, k). Corresponding outcomes were also detected with sonicated α-syn preformed fibrils (PFFs) and GQDs, which generated a higher population of monomers as the co-incubation period prolonged (Fig. 3.8a–c). To visually corroborate the interaction between GQDs and α-syn fibrils, the carboxyl groups on GQDs were conjugated with PEGylated biotin through amide coupling reaction (Fig. 3.6d–g). Note that owing to steric issues, we expect that only one or two carboxyl groups changed to PEG-biotin terminals after the coupling. Unreacted carboxyl groups that were transiently coupled to EDC reagents but spontaneously go back to the original conditions to yield carboxyl-rich biotinylated GQDs as the final product. Using streptavidin-tagged ultra-small gold particles that are easily visualized through TEM, the direct interaction between GQDs and α-syn fibrils was detected (Fig. 3.2a, b). Two-dimensional 1 H–15 N heteronuclear single-quantum coherence correlation (HSQC) NMR spectroscopy was exploited for more detailed analysis of the interaction site of 15 N-labeled α-syn [7, 8] with GQDs, namely the molecular interactions at residue level in solution. Notably, co-incubation with GQDs resulted in many amino acids with evident chemical shifts, which represent the major sites of the interaction (Fig. 3.9). Meanwhile, the peaks for some residues became completely unobservable, indicating the ones that are deeply associated with the interaction between GQDs and α-syn. Interestingly, these residues were centered on the N-terminal region, suggesting that the initial binding is chiefly driven by the charge interaction between the negatively charged carboxyl groups of the GQDs (Fig. 3.6e) and the positively charged domain of α-syn fibrils (Fig. 3.2c). A 200 ns molecular dynamics (MD) simulation was performed in attempt to further comprehend and elucidate the detailed mechanism of α-syn fibril disaggregation by GQDs. The α-syn fibril structure was adopted from the recently reported ssNMR (solid-state nuclear magnetic resonance) structure of pathological human α-syn [9]. To expedite the simulation, α-syn fibril with four β-sheet monomer strands consisted of the non-amyloid-β component (NAC) domain (residues 71 to 82) that is essential for α-syn fibrillation [10] was solely taken into account. After the binding between of the GQDs to the N terminal cross-β part of α-syn at 1 ns, the β-sheet structure of the outermost monomer was entirely disrupted after 50 ns; its C-terminal part was released from the core and made interactions with the other side of basal plane of the GQDs (Fig. 3.2d). The time-dependent secondary structure plot, obtained using the dictionary of secondary structure of proteins (DSSP) algorithm, also depicted an evident decrease in the β-sheet component of the outer monomer, implying pivotal structural alteration in the fibril from 50 ns onwards (Fig. 3.2f). Changes in the rootmean-square deviation (r.m.s.d.) of atomic positions, solvent accessible surface area (SASA) and various interaction energies of the fibril were plotted against time for further analysis (Fig. 3.2e). While the SASA and r.m.s.d. underwent notable changes

3.3 Results and Discussion

33

34

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.2 Detailed analysis on the interaction between GQDs and α-syn fibrils during the disaggregation process. a TEM images for visualizing the interaction between biotinylated GQDs and α-syn fibrils with low (top) and high (bottom) magnifications. Red arrowheads designate biotinylated GQDs enhanced with ultra-small gold–streptavidin nanoparticles. b Quantification of the average width of α-syn fibrils during the dissociation process after 1 h of incubation. c NMR chemical shift difference plot collected from full 1 H–15 N HSQC spectra. The decreased intensity ratio of NMR chemical shifts is analyzed for each residue after binding with GQDs. d Time course simulation snapshots during the interaction between GQDs and mature α-syn fibrils, and 65 ns snapshot image (bottom right) of the interaction between GQDs and mature α-syn fibrils with designated residues contributing to the major binding force. e Time-dependent plots for r.m.s.d. of atomic positions, SASA for the α-syn fibril only group and α-syn fibril and GQDs group, total potential energy ( U tot ), electrostatic energy ( E elec ) and van der Waals energy ( E van ) for α-syn fibril and GQDs group, respectively (from the top). f Time-dependent secondary structure plot calculated by the DSSP algorithm. g, CD spectra of α-syn monomers and α-syn fibrils without (black) and with GQDs (red) after 7 days of incubation. h Fractional secondary structure contents ratio of α-syn fibrils and α-syn fibrils disaggregated by GQDs calculated using the algorithm CONTIN/LL

after 50 ns, the total potential energy ( U tot ) has a gradual yet continuous decline. The major stabilizing/disaggregating force is attributed to hydrophobic interactions, as the van der Waals energy ( E van ) declines while the electrostatic energy ( E elec ) stays nearly consistent. The results correspond to the 65 ns snapshot image that depicts strong interactions between the GQDs’ basal plane and hydrophobic valine residues in α-syn NAC domain (Fig. 3.2d). The changes in the secondary structure were further examined using circular dichroism (CD) spectroscopy. The collected spectra were exploited to provide detailed fractions of secondary structures using the algorithm CONTIN/LL [11, 12] (Fig. 3.2g, h). 7 days post-incubation, the changes in the secondary structure were monitored. While the β-sheet component diminished from 53.3 ± 3.5% to 29.8 ± 3.4%, the combined fractions of α-helix and random coil increased from 4.2 ± 1.2% to 19.8 ± 1.5% and 20.1 ± 5.7% to 24.6 ± 3.6%, respectively. In sum, the interaction between GQDs and α-syn is initially driven by the charge interactions, and fibril disaggregation is mainly induced by hydrophobic interactions and accompanied by major structural changes. The probable role of GQDs in inhibiting α-syn PFF-induced pathology was subsequently investigated in primary neurons. Various cell viability assessments such as terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) (Figs. 3.3a and 3.10a), alamarBlue (Fig. 3.3b), lactate dehydrogenase (LDH) (Fig. 3.3c) and neurite outgrowth assays (Fig. 3.10b–d) showed that α-syn PFF treatment provokes significant cell death. On the other hand, α-syn PFF-provoked cytotoxicity was markedlymitigated by the presence of GQDs. Moreover, α-syn PFFs treatment results in a reduced synaptic protein levels including SNAP25 and VAMP2, indicating critical dysfunction in neuronal networks [5], which was prevented by the presence of GQDs (Fig. 3.10e–g). Another major pathological hallmark during the progression of Parkinson’s disease [13] is mitochondrial dysfunction and consequential cellular/subcellular damages. GQDs effect on these pathological features was explored by carrying out oxidative stress marker 8-hydroxyguanosine (8-OHG) staining, mitochondrial complex I activity assay, MitoTracker staining, TEM analysis and seahorse

3.3 Results and Discussion

35

Fig. 3.3 Effect of GQDs on α-syn PFF-provoked neuron death, fibrillation and transmission in vitro. a–c Neuronal death assessed by TUNEL (a), alamarBlue (b) and LDH assays (c) after α-syn PFFs treatments (1 μg/ml) with and without GQDs (1 μg/ml) for 7 days. d Representative immunoblot levels with p-α-syn antibody. e Quantifications of the SDS-insoluble fraction normalized to βactin levels. f Representative p-α-syn immunostaining images with p-α-syn antibody. g Quantifications of p-α-syn immunofluorescence intensities normalized to the control. h Schematic illustration of the microfluidic device composed of three connected chambers. i Representative images of p-α-syn immunostained neurons in the microfluidic device 14 days post α-syn PFFs addition. j Quantifications of p-α-syn immunofluorescence intensities

assay (Fig. 3.11). While the α-syn PFFs treatment caused mitochondrial damage distinguished by morphological shrinkage, declined oxygen consumption rate, and increased level of reactive oxygen species (ROS) in neurons, GQDs addition relieved these detrimental phenomena. Thereafter, the effect of GQDs on α-syn PFF-provoked Lewy body (LB)/Lewy neurite (LN)-like pathology was investigated. 7 days post-α-syn PFF injection, western blot analysis exhibited accumulation of α-syn in the SDS-insoluble fraction

36

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

(Fig. 3.3d, e) and increased phosphorylated α-syn (p-α-syn) immunoreactivity in primary neurons (Fig. 3.3f, g) On the contrary, the presence of GQDs markedly reduced the population of SDS-insoluble α-syn and p-α-syn immunoreactivity is hardly distinguishable in primary neurons treated with GQDs. To study the effect of treatment points on α-syn pathology, GQDs were injected simultaneously with and 3 days pre/post α-syn PFF treatments. The effects on inhibition of p-α-syn accumulation and cytotoxicity alleviation were slightly weaker when GQDs were treated 3 days post-α-syn PFF injection. However, this can be also attributed to the fact that a week of incubation is not enough to result in critical differences and does not reflect actual alterations in the pathological features. Taking this into account, all three different GQDs treatment points yielded practically consistent results (Fig. 3.12a–f). To identify the subcellular location of interaction between GQDs and α-syn PFFs, these two substances and the lysosome were individually labeled with fluorescent probes for live imaging (Fig. 3.12g, h). Equivalent to the time-course snapshots, the fluorescence signals for α-syn PFFs and GQDs are co-localized within the lysosome. Interestingly, the signal for α-syn PFFs progressively becomes faint and that of GQDs intensifies with time. It can thus be assumed that both GQDs and α-syn PFFs are endocytosed, and the dissociation of α-syn fibrils occurs in the lysosome by the ambient GQDs. It should be also noted that the rapid degradation of α-syn fibrils can be attributed to lysosome’s acidic environment, whereas GQDs’ intrinsic resistance to acids enable them not to be degraded throughout the process and the two components are separated afterwards. One the most important pathological features of the α-syn PFFs neuron model involves neuron-to-neuron transmission of α-syn aggregates [5]. To study GQDs’ inhibitory role against this transmission, a triple-compartment microfluidic culture device was employed, where sequential transmissions among neurons occur from chamber 1 to chamber 3 (Fig. 3.3h). After 14 days of α-syn PFF treatment, the relative levels of p-α-syn were quantified by immunostaining. Interestingly, the levels of pα-syn immunoreactivity were markedly diminished in chamber 2 and chamber 3 when GQDs were added in the first chamber, which indicates that GQDs inhibit de novo fibrillation of endogenous α-syn from assembling into pathological aggregates (Fig. 3.3i, j). Likwise, when GQDs were treated in the second chamber, the levels of p-α-syn immunoreactivity were decreased in chamber 2 and 3, demonstrating that GQDs prevent the α-syn transmission to adjacent neurons. To investigate any neuroprotective effects of GQDs against α-syn PFF-provoked transmission and toxicity in vivo, α-syn PFFs were stereotaxically injected into the striatum of wild-type (WT) mice and GQDs were biweekly administered via intraperitoneal (i.p.) injection as a treatment (Fig. 3.4a). Prior to any in vivo analyses, we tested the blood–brain barrier (BBB) permeability of GQDs with a wellestablished in vitro BBB model [14] (Fig. 3.13a, b). The formation and functionality of the BBB model were verified by measuring the transepithelial electrical resistance (TEER) and dextran fluorescence intensity, respectively (Fig. 3.13c, d). Namely, the TEER measurement results show the integrity of cultured cells. Dextran fluorescence intensity is a great indicator of the functionality as only dextran-fluorescein (3 kDa) can pass through the BBB and 100% of dextran-rhodamine (2000 kDa)

3.3 Results and Discussion

37

Fig. 3.4 Effect of GQDs on α-syn-induced pathologies in vivo. a Schematic illustration of injection coordinates for stereotaxic injection of α-syn PFFs (5 μg) in mice. 50 μg of GQDs were i.p. injected biweekly for 6 months as a treatment. b Representative TH immunohistochemistry images in the substantia nigra of α-syn PFF-injected hemisphere in the absence (top) and presence (bottom) of GQDs. c Stereological counting of the number of TH- and Nissl-positive neurons in the substantia nigra via unbiased stereological analysis after 6 months of α-syn PFF injection with and without GQDs. d Representative TH immunohistochemistry images in the striatum of α-syn PFF-injected hemisphere. e Quantifications of TH-immunopositive fiber densities in the striatum. f Assessments of the behavioral deficits measure use of forepaws in the cylinder test. g Assessments of the behavioral deficits measured by the ability to grasp and descend from a pole. h Representative p-α-syn immunostaining images in the striatum (STR) and substantia nigra (SN) of α-syn PFF-injected hemisphere. i Quantifications of p-α-syn immunoreactive neurons in the striatum and substantia nigra. j Distribution of LB/LN-like pathology in the CNS of α-syn PFF-injected hemisphere (p-α-syn positive neurons, red dots; p-α-syn positive neurites, red lines)

38

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

remain in the blood side. Then, the in vitro permeability of GQDs was determined by the fluorescence intensity on the brain side. Remarkably, the fluorescence signal progressively elevated with time, and eventually reached 100% permeability after 24 h of incubation (Fig. 3.13e). To visualize the penetration process and study the mechanism, GQD–biotin was prepared and conjugated to streptavidin–quantum dots for live fluorescence imaging. Prior to the analysis, the functional behavior of GQD–biotin was tested, which exhibited practically the same BBB penetration rates and fibril disaggregation results as those of as-synthesized GQDs (Fig. 3.13e–g). Snapshots from the live fluorescence imaging displayed that the GQD–biotin complex is internalized in the lysosome of both brain microvascular endothelial cells (BMECs) and astrocytes, and undergoes exosomal release (Fig. 3.13h, i). In other words, GQDs are endocytosed into BMECs on the blood side and released to pass through the BBB, and sequentially endocytosed by astrocytes on the brain side to release them through the exosome again. The in vivo permeability of the BBB was also examined by immunohistochemical analysis of the brain using GQD–biotin DAB staining. Following i.p. injection, a significant amount of GQD–biotin was found throughout the entire central nervous system (CNS) area, including the olfactory bulb, neocortex, midbrain and cerebellum, corroborating that GQDs have the ability to pass through the BBB in vivo (Fig. 3.13j– n). As expected, GQDs did not exhibit any specificity towards the midbrain and spread out the entire brain as they passed through the BBB. Although this may be discouraging if Parkinson’s disease is the only consideration but can be also encouraging to apply for other diseases that share similar pathogenesis mechanisms with Parkinson’s disease. This will be discussed in more detail in the conclusion section. 180 days following α-syn PFF injection, decreased number of tyrosine hydroxylase (TH)- and Nissl-positive neurons were detected in the substantia nigra of WT mice. Nevertheless, mice administered with GQDs showed notably more number of viable dopaminergic neurons in the same region, protected against α-syn PFFprovoked damages (Fig. 3.4b, c). In addition, α-syn PFFs injection led to a loss of TH-positive striatal fiber neurons, which was also prevented by the presence of GQDs (Fig. 3.4d, e). Lastly, any alterations in the motor function were evaluated by cylinder and pole tests, which are well-established behavior tests for Parkinson’s disease. Impressively, mice treated with GQDs displayed alleviated motor deficits by showing balanced use of both forepaws and decreased pole descending time in the cylinder and pole tests, respectively (Fig. 3.4f, g). The levels of p-α-syn, a marker of LBs/LNs, were quantified in the striatum and substantia nigra of α-syn PFF-administered mice. While stereotaxic injection of αsyn PFFs provoked accumulation of p-α-syn in the striatum and substantia nigra, GQDs injection markedly reduced the levels of p-α-syn (Fig. 3.4h, i). Moreover, the injection of α-syn PFFs in the striatum results in the propagation of α-syn aggregates throughout the CNS, whereas GQDs prevent the pathological α-syn aggregates from spreading out (Fig. 3.4j). GQDs injection also mitigated gliosis in the substantia nigra accompanying decreased microglia density and glial fibrillary acidic protein (GFAP)

3.3 Results and Discussion

39

levels in astrocytes, which are provoked by α-syn PFF injection in the absence of GQDs (Fig. 3.14). To further verify the therapeutic potential of GQDs against a transgenic model, 6month human A53T α-syn transgenic mice [15] were treated with GQDs biweekly for 4 months (Fig. 3.15a). Consistent to the outcomes in the α-syn PFF-induced model, GQDs treatment reduced p-α-syn levels in the CNS (Fig. 3.15b–d) and microglia density and GFAP levels in astrocytes (Fig. 3.15h–k). GQDs injection also decreased the number of α-syn aggregates intrinsically provoked by overexpressing human A53T α-syn in HEK293 cells [16] (Fig. 3.15l, m). Likewise, GQDs ameliorated motor dysfunctions in the hA53T α-syn Tg mice, monitored by the pole and clasping tests, specific behavior assessments for transgenic animal models (Fig. 3.15e–g). It should be also noted that 6 months of prolonged GQDs injection did not result in loss of dopaminergic neurons, activation of glial cells, any abnormal behaviors and organ damages in mice. Collectively with the urinary excretion data, it can be concluded that GQDs manifest no appreciable long-term in vitro and in vivo toxicity and can be readily cleared from the body (Fig. 3.16). Previous reports explored the therapeutic potential of graphene oxides (GOs) and GQDs against Alzheimer’s disease through inhibition of beta-amyloid (Aβ) peptides fibrillation [17–19]. Computational results also demonstrated that a hydrophobic graphene sheet may induce severe structural disruption of amyloid fibrils [20]. To examine and compare the therapeutic efficacy of GQDs to other graphenebased nanomaterials, nano-sized GOs (nano-GOs, ranging from ~5 to 20 nm) and reduced GQDs (rGQDs) were synthesized (Fig. 3.17a, b). As the Fourier-transform infrared (FT–IR) spectra depict, pristine GQDs possess abundant carboxyl groups (1730 cm−1 ), while nano-GOs and rGQDs exhibit significantly lower carboxyl group: aromatic carbon double bond (1620 cm−1 ) ratios. The difference in the abundance of carboxyl groups is reflected in toxicity assessments, where nano-GOs and rGQDs show severe cytotoxicities, unlike the results by GQDs (Fig. 3.17 c,d). Indeed, several toxicity studies with graphene-based nanomaterials highlighted the importance of functional groups that play pivotal role in mitigating hydrophobic interactionmediated cytotoxicity. In addition, nano-GOs and rGQDs possess noticeably weaker fibril-disaggregation effects than GQDs (Fig. 3.17e–g), which can be attributed to the presence of fewer carboxyl groups as well, for the 1 H–15 N HSQC analysis demonstrated that the positively charged N-terminal region is the initial, but major binding site with GQDs. Also importantly, the BBB permeability of nano-GOs is considerably lower than those of GQDs and rGQDs. Therefore, size may be a decisive factor for the BBB permeability of graphene-based nanomaterials (Fig. 3.17h). Altogether, our results show that GQDs is an optimal candidate against Parkinson’s disease and related α-synucleinopathies, for having no appreciable in vitro and long-term in vivo toxicity, yet possessing respectable fibril disaggregation effects, and the ability to pass through the BBB.

40

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

3.4 Conclusion A desirable therapeutic candidate against Parkinson’s disease must encompass outstanding anti-aggregation and dissociation properties towards α-syn aggregates without severe toxicity. GQDs bind to α-syn fibrils, inhibit its transmission, and possess distinctive neuroprotective effects against the neuropathological α-syn aggregates/fibrils in in vitro and in vivo models. Therefore, GQD-based drugs with appropriate modifications may open a new venue in clinical drug development for treating Parkinson’s disease and other abnormal protein aggregation-related neurological disorders.

3.5 Experimental Preparation of GQDs. GQDs were synthesized by adding 0.9 g of carbon fibers (Carbon Make) into a mixture of strong acid (300 ml sulfuric acid and 100 ml nitric acid; Samchun Chemical) at 80 °C for 24 h. After acid removal, the solution was vacuum-filtered through a porous inorganic membrane filter (cat. no. 6809-5002, Whatman-Anodisc 47; GE Healthcare) to remove large particles. Then, the final product was yield in powder form by to rotary evaporation. AFM imaging. 5 μl of GQDs (10 μg/ml) and α-syn fibrils (10 μg/ml) were prepared on a silicon substrate. The analyses were performed with an XE-100 AFM (Park Systems) under non-contact mode (scan size: 25 μm2 and scan rate: 0.8 Hz). The images were obtained through the provided XE data acquisition program (XEP 1.8.0). FT–IR measurements. The samples were prepared through the conventional KBr pellet method (no. of scans, 32; resolution, 4; wavenumber range, 4,000– 40 cm−1 ). The analysis was performed on a Nicolet 6700 FT–IR spectrometer (Thermo Scientific). ThT and turbidity assays. The number of α-syn fibrils were measured through Turbidity and ThT assays. For the ThT assay, 50 μl of each sample was centrifuged at 16,000 g for 30 min. The pellet was re-suspended in 200 μl of ThT assay solution, consisting 25 μM ThT (cat. no. T3516, Sigma-Aldrich) dissolved in 10 mM glycine buffer (pH 9.0). The number of α-syn fibrils was calculated by a fluorescence spectrophotometer with the ThT fluorescence measured at 482 nm (excitation at 440 nm). For the turbidity assay, α-syn fibrils were diluted with PBS (1:10). The diluted α-syn fibrils were loaded into the wells of Corning 96-well plates. The turbidity of each sample was determined by a microplate reader (absorbance at 360 nm). TEM imaging. The α-syn fibrils were adsorbed to glow discharge grids (EMS, 400 mesh carbon-coated copper grids) for 2 min and washed with 3 drops of TrisHCl (50 mM, pH 7.4). The washed grids were floated with 2 drops of 0.75% uranyl formate. After they were dried, the images were acquired by a Phillips CM 120 TEM (80 kV) with an AMT ER-80 charge-coupled device (8 megapixel). Primary cultured

3.5 Experimental

41

neurons were seeded at a density of 100,000 cells per well in a poly-d-lysine-coated 35 mm dish. At days in vitro (DIV) 10, the cells were treated with 1 μg/ml of PFFs with and without 1 μg/ml of GQDs. At DIV 17, the cells were washed with 1% sodium nitrite containing PBS (pH 7.4) and fixed in 3% PFA, 100 mM cacodylate, 1.5% glutaraldehyde, 2.5% sucrose containing fixative (pH 7.4) for 1 h. TEM images were captured by a Philips EM 410 TEM on a Soft Imaging System Megaview III digital camera. Dot-blot assays. Tris-buffered saline (TBS)-wetted nitrocellulose membrane (NC membrane, 0.45 μm pore) was placed on the Bio-Dot microfiltration apparatus (cat. no. 1706545, Bio-Rad), and the samples were loaded into the wells of the microfiltration apparatus under the condition of mild vacuum. After washing the NC membranes with TBS, the membranes were blocked with 0.5% Tween-20 and 5% non-fat dry milk containing TBS blocking buffer and incubated with anti-α-syn filament antibody (cat. no. ab209538, 1: 1,000, abcam) at 4 °C for 12 h. Then, the membrane was incubated with horseradish peroxidase (HRP)-conjugated rabbit second antibodies (GE Healthcare) at room temperature for 1 h. The signals from α-syn aggregates were then visualized using enhanced chemiluminescence (ECL) solution. The data were captured and analyzed with ImageJ software (http://rsb.info.nih.gov/ij/, NIH). BN–PAGE and SDS–PAGE. For BN–PAGE, α-syn fibrils and α-syn PFFs were prepared using a Native PAGE sample prep kit (cat. no. BN2008, Life Technologies) and run on Native PAGE Novex 4–16% bis–tris protein gels (cat. no. BN1002Box, Life Technologies) at 200 V for 90 min. Then, 50 mM tricine, 15 mM Bis–Tris, 0.02% Brilliant Blue G (pH 7.0) containing cathode buffer and 50 mM Bis-Tris (pH 7.0) containing anode buffer were used for BN–PAGE. After, the gels were stained using a SilverQuest silver staining kit (cat. no. LC6070, Life Technologies), according to the manufacturer’s protocols. For SDS–PAGE, DIV 10 cortical neurons were treated with α-syn PFFs (5 μg/ml) in the conditions with and without GQDs (5 μg/ml). At DIV 17, the soluble proteins were extracted from the neurons using a 1% TX-100, protease inhibitors and phosphatase inhibitors cocktail (cat. no. PPC1010, Sigma-Aldrich) in sample buffer. For insoluble protein isolation, the lysates were sonicated and centrifuged at 12,000 g for 30 min at 4 °C. After the supernant was removed, the pellet was washed and re-suspended in PBS with 2% SDS. Laemmli sample buffer (2 × , cat. no. 1610737, Bio-Rad) was used to dilute both soluble and insoluble protein samples. For SDS–PAGE, 20 μg of the proteins was loaded in the wells of Novex 8–16% tris-glycine gel (cat. no. XP08160BOX, Life Technologies) and electrophoresis was performed at 130 V for 85 min. The proteins were transferred onto a NC membrane and blocked with 0.1% Tween-20 and 5% non-fat dry milk containing TBS for 1 h. Then, the membrane was incubated in SNAP25 (cat. no. 111-002, 1: 2,000, Synaptic Systems), VAMP2 (cat. no. ab3347, 1: 1,000, abcam) or anti-pS129-α-syn antibodies (cat. no. ab59264, 1: 1,000, abcam) overnight at 4 °C. The following day, NC membranes were incubated with HRP-conjugated mouse or rabbit secondary antibodies (GE Healthcare) for 1 h at room temperature and visualized using ECL solution. The signals were captured using ImageJ software (http://rsb.info.nih.gov/ij/, NIH).

42

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Preparation of sonicated α-syn PFFs. The α-syn PFFs were prepared using the method published in ref. 21. Full-length mouse recombinant α-syn was cloned using the pRK172 bacterial expression vector. The plasmid was transformed into BL21(DE3)RIL-competent Escherichia coli (cat. no. 230245, Life Technologies). After bacterial growth, the α-syn monomers were purified through several steps including dialysis, anion exchange and size exclusion chromatography. In vitro αsyn fibrils were assembled in an Eppendorf orbital mixer (cat. no. 538400020) under specifically determined agitation conditions (1,000 r.p.m., 37 °C, 7 days). α-syn fibrils were sonicated with 60 pulses (20% vibration amplitude, 0.5 s on, 0.5 s off) with a 1/8” probe sonicator to form preformed fibrils Biotinylation of GQDs and binding assay. GQDs (50 mg) were dissolved in conjugation buffer (pH 4.7). Subsequently, 12.5 mg of EDC reagent (N-(3dimethylaminopropyl)-N”-ethylcarbodiimide hydrochloride; cat. no. 03449, SigmaAldrich) was added to replace the carboxyl groups. After 1 h of vigorous stirring, 25 mg of EZ-Link amine-PEG3-biotin (cat. no. 21347; Thermo Scientific) was added to EDC-activated GQDs. Then, dialysis and rotary evaporation were performed to yield the final product in powder form. To measure the binding assay between GQDs and α-syn fibrils, 5 mg/ml of α-syn fibrils were incubated with 5 mg/ml of biotinylated GQDs and streptavidin-conjugated 0.8 nm ultra-small gold particles (cat. no. 800.099, Aurion) for 1 h. Next, the streptavidin-conjugated ultra-small gold particles bound to biotinylated-GQDs with high affinity were enhanced with GoldEnhance EM Plus solution (cat.no. 2114, Nanoprobes) for 5 min. Non-reacted solution was removed by spin column with 100 kDa molecular weight cut-off (cat. no. UFC510024, Millipore-Sigma). Then, TEM analysis was conducted. Purification of 15 N-labeled α-syn. The α-syn gene cloned in pRK172 vector was transformed into E. coli BL21 (DE3) for α-syn overexpression. For the preparation of isotope-labeled α-syn, cells were fostered at 37 °C in M9 minimal medium containing 0.5 g of 15 NH4 Cl and 1 g of 13 C glucose (Cambridge Isotope Laboratory) per liter treated with 100 μg/ml ampicillin. After induction with isopropyl β-d-1 thiogalactopyranoside (IPTG), the heat-treated cell lysate was purified consecutively through DEAE-Sephacel anion-exchange, Sephacryl S-200 size-exclusion and S-Sepharose cation-exchange chromatography. The α-syn was then dialyzed against 12 L of fresh 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5) for a total of three times, and preserved at-80 °C in aliquots of 1 mg/ml concentration. The sample was concentrated to 5 mg/ml using Nanosep 10 K membrane (Pall Gelman) at 4 °C immediately before conducting the experiment. NMR spectroscopy analysis. A950 MHz spectrometer equipped with a cryogenic probe (Bruker) was used to perform a detailed NMR analysis of the interaction between α-syn and GQDs. 15 N-labeled α-syn (5 mg/ml, 100 μl) and GQDs (5 mg/ml, 100 μl) were incubated at 37 °C at 1,000 r.p.m. for 3 days. 15 N-labeled α-syn samples were prepared using 20 mM MES buffer (pH 6.5) containing 10% for 1 H–15 N HSQC measurements. 1 H–15 N HSQC spectra of α-syn and GQD-reacted α-syn were obtained at 37 °C. The acquired data was processed and analyzed by NMRPipe [22] and Sparky [23].

3.5 Experimental

43

Simulation details. The MS simulation (200 ns) was performed with Gromacs 5.1 to observe the interaction between GQDs and α-syn fibrils on a molecular level [24]. The initial structure of the hydrophobic NAC domain (residues 71–82) of α-syn was adapted from the ssNMR structure (PDB ID: 2N0A) with CHARMM force field [25], The GQD structure was designed with CGenFF [26] following the protocols from https://cgenff.paramchem.org. CD measurements. The α-syn fibrils (5 mg/ml, 100 μl) were added to GQDs solution (5 mg/ml, 100 μl) and depolymerized on a shaking incubator at 1,000 r.p.m. at 37 °C for 7 days. To take far-ultraviolet CD measurements, the solution was diluted (1/2) with deionized water. CD spectra between 190 and 260 nm were measured at 0.5 nm intervals using a J-815 spectropolarimeter (Jasco) and a 0.2-mmpath-length quartz cuvette. The spectrum of the buffer solution was deducted from the sample spectra. CD signals were normalized to the mean residue ellipticity, [θ], deg cm2 d mol−1 . The fractional secondary structure contents of the α-syn fibrils and depolymerized α-syn fibrils were calculated using the algorithm of CONTIN/LL provided on http://dichroweb.cryst.bbk.ac.uk. Reference set 7 on Dichroweb was used for calculations using CONTIN/LL, which is optimized for the wavelength range between 190 and 240 nm. Primary neuron culture. Primary cortical neurons were isolated from the brain of C57BL/6 mice (Charles River) at embryonic day 15 and seeded onto poly-dlysine-coated dishes (cat. no. P6407, Sigma-Aldrich). The neurons were maintained in complete culture medium composed of B27 supplement (cat. no. 17504044, Life Technologies), L-glutamine (cat. no. 25030149, Life Technologies) and neurobasal medium (cat. no. 21103049, Life Technologies) at 37 °C with CO2 level of 7%. To prevent the growth of glial cells in the neuron culture, the culture was treated with 30 μM of 5-fluoro-2’-deoxyuridine (cat. no. F0503, Sigma-Aldrich) at day 5 of culture. Cell viability and cytotoxicity assays. Primary cortical neurons were seeded onto poly-d-lysine-coated glass coverslips at a density of 10,000 cells per cm2 in complete neuronal culture medium. The cells were then incubated in 7% CO2 at 37 °C. The cytotoxicity was measured with α-syn PFFs (1 μg/ml) in the presence and absence of GQDs (1 μg/ml) in 10 DIV primary mouse cortical neurons for 7 days using a LDH cytotoxicity assay kit (cat. no. 88954, Pierce) and TUNEL assay kit (cat. no. 12156792910, Roche). The viability of mouse primary cortical neurons and SH-SY5Y cells was measured using an alamarBlue cell viability assay kit (cat. no. DAL1025, Molecular Probes) and a neurite outgrowth staining kit (cat. no. A15001, Molecular Probes), abiding by the manufacturers’ guidelines. In vitro immunofluorescence. Mouse primary cortical neurons were seeded onto poly-d-lysine-coated glass coverslips at a density of 20,000 cells/cm2 . They were fixed with 4% PFA and subsequently, blocked for 1 h at room temperature with 5% normal donkey serum (NDS; cat. no. 017-000-121, Jackson ImmunoResearch), 2% bovine serum albumin (BSA; cat. no. A7030, Sigma-Aldrich) and 0.1% Triton X-100 (cat. no. T8787, Sigma-Aldrich) in PBS. Thereafter, they were incubated in anti-8-OHG (cat. no. ab62623, 1: 1,000, abcam), anti-pS129-α-syn (cat. no. ab59264, 1: 1,000, abcam) and anti-MAP2 (cat. no. MAB3418, 1: 1,000, Millipore)

44

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

antibodies overnight at 4 °C. After washing the coverslips with 0.1% Triton X-100 containing PBS, they were incubated in a mixture of second antibodies consisting FITC-conjugated (donkey anti-rabbit FITC, cat. no. 711-095-152; donkey antimouse FITC, cat. no. 715-095-151, Jackson ImmunoResearch) and Cy3-conjugated (donkey anti-rabbit CY3, cat. no. 711-165-152; donkey antimouse CY3, cat. no. 715165-151, Jackson ImmunoResearch) at room temperature for 1 h. The fluorescence images were captured through a Zeiss LSM 710 confocal microscope. Microfluidic chambers. The rectangular glass coverslips were prepared following the method indicated in ref. 5 before they were mounted on the microfluidic devices (triple-chamber microfluidic devices, cat. no. TCND1000, Xona). Each chamber was seeded at a density of 100,000 cells per chamber. At DIV 7, chamber 1 (C1) or chamber 2 (C2) was treated with 0.5 μg of GQDs. Then, only C1 was treated with 0.5 μg α-syn PFFs. In order to regulate the direction of flow of the channels, the chambers were set with a 50 μl difference in media volume. At DIV 21, the chambers were fixed with 4% PFA containing PBS. After blocking the chambers with 0.1% Triton X-100, 5% NDS and 2% BSA containing PBS at room temperature for 1 h, they were incubated in anti-pS129-α-syn (cat. no. ab59264, 1: 1,000, abcam) and anti-MAP2 (cat. no. MAB3418, 1: 1,000, Millipore) primary antibodies at 4 °C overnight. Thereafter, the chambers were washed in 0.1% Triton X100 containing PBS and incubated in a second antibody mixture of FITC-conjugated (Jackson ImmunoResearch) and Cy3-conjugated (Jackson ImmunoResearch) for 1 h at room temperature. The fluorescent images were captured through a LSM 710 Zeiss confocal microscope. Complex I activity assay. The commercial microplate assay kit (Cat#: ab109721; abcam) was used to measure the activity of complex I enzyme, according to the manufacturer’s instruction. Primary cortical neurons were plated onto poly-d-lysine coated 6 cm dishes at a density of 1,000,000 cells/dish. On DIV 10, 1 μg/ml of α-syn PFFs were added to each dish of neurons either with or without 1 μg/ml of GQDs. After 7-day treatment, the cytosolic proteins were extracted using 1/10 volume detergent in PBS. The supernatant was removed by centrifuging the samples at 12,000 × g for 20 min from 5.5 mg/ml of samples. The isolated samples were loaded, incubated for 3 h at room temperature, washed in washing buffer 2 times, and mixed with 200 μl of assay solution. Then, the mitochondrial complex 1 enzyme activity was measured at approximately 1-min intervals for a total of 30 min. Mitochondrial morphology assessment. Primary cultured cortical neurons were plated at a density of 10,000 cells/cm2 onto poly-d-lysine coated glass coverslips. At days in vitro (DIV) 10, the neurons were treated with 1 μg/ml of α-syn PFFs with or without 1 μg/ml of GQDs. At DIV 17, the mitochondria in the neurons were stained with 100 nM of MitoTracker® Orange CMTMRos probes (Cat#: M7510; Life technologies) for 30 min. After washing the stainings with live cell imaging solution (Cat#: A1429DJ; Life technologies), the morphological characteristics were visualized through a Zeiss confocal microscope (LSM 710). The length or aspect ratio (AP, the ratio between the major and minor axis) were evaluated by ImageJ software (http://rsb.info.nih.gov/ij/, NIH).

3.5 Experimental

45

Determination of oxygen consumption rate. Primary cultured cortical neurons were plated onto 24-well culture plate at a density of 500,000 cells/well. At DIV 10, 1 μg/ml of α-syn PFFs was administered with or without 1 μg/ml of GQDs. At DIV 17, the neurons were washed with heated up PBS and incubated in complete Seahorse assay medium at 37 °C for 1 h. Then, the assay plate was loaded in an XF24 analyzer (XF24 EX extracellular flux analyzer, Seahorse Bioscience) and the oxygen consumption rate (OCR) was quantified by the Seahorse XF cell mito stress test kit (Cat#: 103015; Agilent) through a protocol with specific measurements (1-min mix, 1-min wait, and 2-min measurement). Oligomycin, carbonyl canide mchlorophenyllhydrazone (CCCP), and rotenone were sequentially injected into the Seahorse assay plate to analyze the coupling of respiratory, chain, the basal respiration, and mitochondrial respiratory capacity. The measured OCRs were normalized in respect to the protein concentration of each well. Data is representative of the percentage change in comparison to the control. Live imaging. Primary cortical neurons were plated onto poly-d-lysine coated glass bottom dish (Cat#: 150682; NuncTM) at a density 10,000 cells/cm2 and maintained in an incubator set to 37 °C and 7% CO2 . At DIV 7, the neurons were treated with a mixture of 1 μg/ml FITC labeled α-syn-PFFs, 1 μg/ml GQDs-biotin and streptavidin Qdot complex, and 100 nM LysoTracker™ Blue DND-22 (Cat#: L7525; Life technologies) containing live cell imaging solution for a total of 1 h. The culture dish was mounted on a temperature controlled CO2 incubation system equipped confocal microscope to capture time-lapse confocal live images at the indicated intervals with 488 nm and 561 nm laser excitations. In vitro BBB permeability of GQDs. 10-pups of C57BL/6 mouse were used to prepare primary mouse astrocytes as previously explained for in vitro BBB experiments [29]. The cerebral cortex was isolated from 1-d-old C57BL/6 mice, and the meninges were removed. The cerebral cortex was mechanically disrupted using a 30-ml syringe with a 19-gauge needle. The isolated cells were plated onto 75 cm2 T-flasks and cultured in complete DMEM culture medium. After 14 days, the 95% pure astrocytes were isolated using an astrocyte isolation kit (Cat#: 130-096-053; Miltenyl Biotec). The primary brain microvascular endothelial cells (BMEC) from C57BL/6 mice were purchased from Cell Biologics. Fluorescence staining with cell specific marker GFAP (for astrocyte) and CD31 (for BMEC, Cat#: ab28364; 1:500; abcam) verified the purities of cultured astrocytes (> 95%) and BMEC (> 95%). To create an in vitro BBB, the isolated astrocytes were seeded onto the underside of the inserts (collagen-coated 0.4 μm transwell inserts; Cat#: CLS3491; Sigma-Aldrich) at a density of 106 cells/ml and placed in a 37 °C and 5% CO2 incubator. After 48 h of incubation, the inserts were transported into a 6-well plate and BMEC were seeded on top of the inserts at a density of 106 cells/ml. The epithelial volt/ohm (TEER) meter (Cat#: 300523; EVOM2; world precision instruments) measured the transepithelial electrical resistance (TEER) at 0, 2, 4, 6, and 8 days after BMEC seeding to confirm the structural integrity of the in vitro BBB. BBB-impermeable 3 kDa dextranfluorescein (Cat#: D3306; Life technologies) were loaded on the blood side, and the ratio was computed using fluorescence spectrophotometer (Ex = 494 nm/Em = 521 nm for dextran-fluorescein; Ex = 555 nm/Em = 580 nm for dextran rhodamine). The

46

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

concentrations of remaining GQDs, GQDs-biotin, nano-GOs, and rGQDs of both the blood and brain side of the inserts were measured at 520 nm (Ex = 310 nm) using the FluoroBriteTM DMEM media (Cat#: A1896701; Life technologies). Exosome isolation. To determine the concentration of GQDs-biotin from the released exosome, BMEC or astrocytes plated on 6 cm dishes were treated with 5 μg of GQDs-biotin. After 12 h, the culture medium was changed. 24 and 48 h thereafter, the exosomes were isolated using an exosome isolation reagent (Cat#: 4478359; ThermoFisher). Animals. All animal-based experimental procedures were conducted following the guidelines of the Laboratory Animal Manual of the NIH Guide to the Care and Use of Animals, and the procedures were approved by the Animal Care and Use Committee of Johns Hopkins Medical Institute. Human α-syn-A53T transgenic mice (stock no. 006823, B6.Cg-Tg, Prnp-SNCA*A53T;23Mkle/J) were purchased from the Jackson Laboratory [15]. In vivo BBB permeability of GQDs. For in vivo immunostaining of GQDsbiotin, C57BL/6 mice at week 8 were i.p. injected with 2 mg/kg dosage of GQDsbiotin. The brains were removed and fixed with 4% PFA for 6 h. The brains were then stored in 30% sucrose for 48 h. The biotin signals of the olfactory bulb, midbrain, neocortex, and cerebellum were visualized using DAB staining kit. The GQDs-biotin positive signals were further confirmed by immune-EM staining using GoldEnhanceTM EM Plus solution with 20-min incubation, according to the provided manufacturer’s instructions. For in vivo BBB experiments, C57BL/6 mice at week 8 were i.p. or i.v. injected with GQDs-biotin (2 mg/kg) or vehicle. At day 7 and day 14 following the injection, the brain and blood were harvested. The brain was homogenated in PBS with 1% TX-100. In preparation of the plasma, the whole blood was stored at RT for 30 min, and centrifuged at 2,000 × g for 10 min, removing all clots. The GQDs-biotin concentrations were measured using QuantTag Biotin Kit (Cat# BDK-2000, Vector Laboratories). The brain/plasma concentration ratio of GQDs-biotin was compared to the brain/plasma ratio. α-syn aggregation formation assay. HEK293T cells were purchased from American Type Culture Collection (ATCC) and authenticated through STR analysis. In addition, HEK293T cells were routinely tested to confirm for negative mycoplasma contamination. The cells were plated on glass coverslides and transfected the pCMV5 vectors with myc-tagged A53T α-syn mutant (a kind gift from Dr. Thomas C. Südof). They were treated with either PBS (pH 7.4) or GQDs (0.1 μg/ml). 48 h later, cells were washed three times with PBS and fixed in 4% PFA containing PBS for 20 min at room temperature. The fixed cultures were permeabilized in 0.1% Triton X-100 containing PBS for 4 min. The expression of α-syn was monitored by immunostaining with α-syn antibody (Cat#: 610787; 1:1,000; BD Biosciences). The α-syn aggregates were localized on a subcellular level by confocal microscopy with 550 nm and 570 nm serial excitations. The quantity of α-syn aggregates per field was counted using ImageJ software (http://rsb.info.nih.gov/ij/, NIH). Stereological assessments. C57BL/6 mice (8- to 10-week-old males) were purchased from the Jackson Laboratory. After anaesthetizing the mice with pentobarbital sodium solution (dosage of 60 mg/kg), mice were injected with either PBS (2 μl)

3.5 Experimental

47

or PFFs (5 μg/2 μl) by stereotaxic instruments (cat. no. Model 900, David KOPF instruments). The designated coordinate of the striatum injection site was + 2.0 mm from the midline, + 2.6 mm below the dura, and + 0.2 mm relative to the bregma. As a means of treatment, 50 μl of GQDs (50 μg per mouse) were i.p. injected biweekly (every two weeks) for 6 months. After 6 months of GQDs injection, the mice were perfused, fixed with 4% PFA containing PBS for 12 h, and stored in 30% sucrose for cryoprotection. To conduct TH and Nissl staining, 50 μm coronal brain slices were made using a cryostat (Bio-Rad). Every fourth section underwent stereological assessment. Sections were incubated in rabbit polyclonal anti-TH (cat. no. NB30019, 1: 1,000, Novus Biologicals) or rabbit polyclonal anti-pS129-α-syn (cat. no. ab59264, 1: 1,000, abcam) with 5% NDS, 0.1% TX-100 containing PBS blocking solution. Then, they were incubated in streptavidin-conjugated HRP and biotinylated secondary antibodies (cat. no. PK-6101, Vector Laboratories). Immune-positive signals were imaged using a DAB kit (cat. no. SK-4100, Vector Laboratories). To distinguish the Nissl substance, the TH-stained tissues were counterstained with staining solution containing thionin. The total number of TH- and Nissl-positive neurons in the SN region was numbered by microscopy with Stereo Investigator software (MBF Bioscience). In vivo immunohistochemistry. The isolated brains were perfused and fixed with 4% PFA containing PBS. After cryoprotection with 30% sucrose, mouse brain sections were incubated with anti-GFAP (cat. no. Z0334, 1: 2,000, Dako) or anti-Iba-1 (cat. no. 019-19741, 1: 1,000, Wako) antibodies in blocking solution. Then, the slices were incubated with biotin-tagged anti-rabbit second antibody. After ABC solution (cat. no. PK-6101, Vector Laboratories) incubation, the slices were developed using a DAB peroxidase substrate kit (cat. no. SK-4100, Vector Laboratories). The density of astrocytes and total count of microglia in the substantia nigra were quantified by ImageJ software (http://rsb.info.nih.gov/ij/, NIH). To perform histopathology on major organs such as the liver, kidney and spleen, 8- to 10-week-old male C57BL/6 mice were i.p. injected with 50 μg of GQDs biweekly for 6 months. After the 6 months, they were perfused with 4% PFA containing PBS. The liver, kidney and spleen were stained using an H&E staining kit (cat. no. H-3502, Vector Laboratories). Behavior analyses. The cylinder test [27] was devised to discern asymmetry in forelimb movement and utilization. Monitoring a video clip, a blinded observer scored every physical contact made by a forepaw in a 20-cm-wide clear glass cylinder. The mice were completely isolated from the test cylinder before actual testing. The mice had no form of habituation to the testing cylinder before recording the video clips. Each animal made approximately 20 to 30 wall touches made with full extension of the forelimbs. The degree of impaired forelimb touches was quantified in percent by comparing it to the total forelimb use. The control group scored roughly 50%. Before conducting the pole test [28], the mice were allowed to adapt to the behavioral room for 30 min. A metal pole with a height of 75 cm and diameter of 9 mm was wrapped in bandage gauze. To initiate the test, the mouse was placed 7.5 cm distant from the top of the pole facing upwards. The scores were calculated based on the time elapsed until the mice reached the bottom. The animals were trained for

48

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

2 successive days before the actual trial. Each training consisted of 3 trials with a maximum time limit of 60 s for each animal. The results for the turn down, climb down and the total time elapsed (in seconds) were recorded. For behavior test of hA53T α-syn mice, the hindlimb clasping function was tested [30]. The test animals were lifted by their tails near the base, and their hindlimb positions were monitored for 10 secs. All surroundings were cleared of any objects. The scores were calculated based on the following criteria: (1) Score 0: hindlimbs are spread outwards and away from the abdomen. (2) Score 1: a single hindlimb is drawn back towards the abdomen for more than 5 secs. (3) Score 2: both hindlimbs are partially drawn back towards the abdomen for more than 5 secs. (4) Score 3: both hindlimbs are fully drawn back towards the abdomen for more than 5 secs. Preparation of nano-GOs and rGQDs. Pristine GOs were synthesized by a modified Hummer’s method from previous literature [31] and tip-sonicated in DI water (10 mg/ml) for 3 h to yield nano-GOs. The reduction of GQDs was yield by performing the autoclave-based hydrothermal method at 200°C for 2 h. The viability assay of human neuroblastoma cell line. SH-SY5Y (ATCC® CRL2266™) was purchased from ATCC and authenticated through STR analysis. Cells were routinely tested and confirmed for negative mycoplasma contamination. SH SY5Y cells were plated at a density 50,000 cells/well in 48-well plate and cultured in 5% CO2 and 37 °C incubator. The viability of SH-SY5Y cells was quantified by the manufacturer’s instructions of the alamarBlue cell viability assay kit (Cat#: DAL1025; Molecular Probes™). Statistics. Mean ± S.D. values were computed from more than three independent experiments. P values were determined by Student’s t-test or ANOVA with Bonferroni correction. Statistics analysis was performed on the Prism6 software.

Supplementary Information See Figs. 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17 and 3.18.

Supplementary Information

49

Fig. 3.5 Mean ± S.D. values and P values of Fig. 3.1b, c, e, and f

Fig. 3.6 Schematic representation of the collective effects of GQDs on α-synucleinopathy in Parkinson’s disease. Without GQDs, pathological α-syn monomers undergo spontaneous fibrillation to form aggregates, which eventually provokes loss of dopamine producing neurons in the midbrain, LB/LN like pathology and motor dysfunctions. On the other hand, GQDs treatment prevents α-syn fibrillation and disaggregates mature fibrils to monomers and thereby rescues from dopaminergic neuron loss, LB/LN-like pathology and behavior deficits provoked by abnormal α-syn fibrillation

50

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.7 Synthesis and biotinylation of GQDs and binding assay between GQDs-biotin and αsyn fibrils. a Schematic representation of GQDs synthesis from carbon fiber. b Representative AFM image of synthesized GQDs. c Fractions of GQDs with different height profiles. d, Schematic illustration of biotinylation on GQDs using EDC coupling. e Representative FT-IR spectra of GQDs (black line) and biotinylated GQDs (red line) with peaks and shifts assigned for designated functional groups. f Schematic representation of the experimental procedures for the binding assay between biotinylated GQDs and nanogold-streptavidin tagged α-syn fibrils. g Representative TEM images of preformed α-syn fibrils after 7 days of incubation in the presence of GQDs-biotin-nanogoldstreptavidin complex (left) and nanogold-streptavidin only (right)

Supplementary Information

51

Fig. 3.8 The effect of GQDs on disaggregation of α-syn fibrils. a The distribution of α-syn fibril lengths at various time points (0, 6, 12, 24, and 72 h, n = 50 fibrils at each time point). b Representative AFM images of α-syn fibrils in the presence of GQDs after various time points (0, 24, and 48 h) and the representative line profiles of the designated regions, marked by blue (GQDs) and red (α-syn fibrils) arrows at each time point

52

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.9 The effect of GQDs on disaggregation of α-syn PFFs. a Representative TEM images of fibrillated α-syn PFFs (5 mg/ml) incubated in the absence of (top) and in the presence of (bottom) GQDs (5 mg/ml) for 1 h. b The end-to-end length of α-syn PFFs after 1 h of incubation. c The amount of remaining α-syn PFFs and disaggregated α-syn PFFs assessed by BN-PAGE after various incubation periods

Supplementary Information

53

Fig. 3.10 1 H-15 N HSQC spectral analysis. a Full, overlapped 1 H-15 N HSQC spectra of 15 N-labeled α-syn monomers (black) and after a week of incubation with GQDs (red). The assigned residues from 15 N-labeled α-syn monomers only group. b A full HSQC spectrum of 15 N-labeled α-syn monomers incubated with GQDs. Based on the peak intensity of 15 N-labeled α-syn monomers only group, the residues with no or minimal decrease in peak intensity (red), the residues with significant decrease in peak intensity (blue), and undetectable residues (black) are distinctively assigned

54

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.11 The effect of GQDs on α-syn PFFs-induced cell death and restricted neurite outgrowth. a Representative TUNEL-positive neurons. Primary cortical neurons were treated with α-syn PFFs (1 μg/ml) in the absence and presence of GQDs (1 μg/ml). TUNEL assay was performed after 7 days of incubation. b Representative micrographs of neurite outgrowth and cell viability assays stained by outer cell membrane (red) and cell-permeable viability indicator (green). c, d Quantifications of neurite outgrowth and neuron viability from the stained images. e Representative immunoblot analysis of SNAP25 and VAMP2 protein levels. Primary cortical neurons were treated with α-syn PFFs (5 μg/ml) in the absence and presence of GQDs (5 μg/ml) and incubated for 7 days. f The expression levels of SNAP25 were normalized to β-actin level and quantified. g VAMP2 were normalized to β-actin level and quantified

Supplementary Information

55

56

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.12 The effect of GQDs on α-syn PFFs-induced mitochondrial dysfunction and oxidative stress. a Representative 8-OHG immunostaining images in primary cortical neurons treated with α-syn PFFs (1 μg/ml) in the absence and presence of GQDs (1 μg/ml) and incubated for 7 days. b Quantifications of 8-OHG content by the immunofluorescence levels. c Measurements of the mitochondrial complex I activity after 7 days of α-syn PFFs (1 μg/ml) incubation. d Representative MitoTracker positive micrographs. Primary cortical neurons were treated with α-syn PFFs (1 μg/ml) in the absence and presence of GQDs (1 μg/ml). After 7 days of incubation, mitochondria were labeled with MitoTracker® Orange CMTMRos (Red). e Quantifications of the length of the stained mitochondria. f Aspect ratio of the stained mitochondria. g Representative TEM images with low and high magnifications after the same preparation steps. h Microplate-based respirometry readings for neurons. The oxygen consumption rate measured in an XF 24 Seahorse analyzer in primary cortical neurons treated with α-syn PFFs (1 μg/ml) in the absence and presence of GQDs (1 μg/ml) for 24 h. i Quantifications of the basal respiratory rate. j Quantifications of the maximal respiratory rates from the respirometry results

Supplementary Information

57

Fig. 3.13 The effect of GQDs on α-syn PFFs-induced primary neuronal toxicity and pathology at different treatment points of GQDs and live cell imaging. a Neuronal toxicities assessed by alamarBlue and b LDH assays. Mouse cortical neurons were treated with a-syn PFFs (1 μg/ml) with 3 days pre- (n = 4, Before), simultaneous (n = 4, Simul), and 3 days post-injection (n = 4, After) of GQDs (1 μg/ml) for 7 days. c Representative images of p-α-syn by dot-blot analysis and d quantified intensities normalized to the PBS control. e Representative p-α-syn immunostaining micrographs with p-α-syn antibody after 7 days of incubation. f Quantifications of p-α-syn immunofluorescence intensities normalized to the PBS control. g Representative images showing GQDs and α-syn PFFs are co-localized in the lysosome of primary cultured neuron with live imaging. The LysoTracker (blue), GQDs-biotin-streptavidin Qdot complex (red), and FITC-labeled α-syn PFFs (green). h αsyn PFFs disaggregation process by GQDs monitored by time-lapse fluorescence signals during live imaging

58

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Supplementary Information

59

Fig. 3.14 The BBB permeability of GQDs. a Schematic diagram of the in vitro BBB model. b The confluent monolayer formations of the brain microvascular endothelial cells (BMEC) and astrocytes determined by immunofluorescence staining with CD31 (endothelial cell marker) or GFAP (astrocyte marker) antibodies, respectively. c The transepithelial electrical resistance (TEER) measured at various time points. d The permeabilities of endothelial cell monolayer and astrocyte monitored by measuring the ratio between the blood side and the brain side using dextran-fluorescein (3 kDa) and dextran-rhodamine (2,000 kDa) e Quantifications of permeability of GQDs and GQDs-biotin over time. f The α-syn fibril disaggregation abilities of pristine GQDs and GQDs-biotin monitored by dot-blot. g ThT, and turbidity assays. h Confocal laser scanning microscope images of primary cultured BMEC and astrocyte after 1-hour incubation with GQDs-biotin. GQDs-biotin are shown with Qdot™ streptavidin, whereas the lysosomes labeled by LysoTracker orange. i Quantifications of the exosomal GQDs-biotin from BMEC or astrocytes. The amount of GQDs-biotin measured from the isolated exosome at 24 and 48 h post-endocytosis. j Immunohistochemical analysis of i.p. injected GQDs-biotin (2 mg/kg) by staining the olfactory bulb, neocortex, midbrain, and cerebellum with avidin-biotin complex method or immunogold method at 7 days after injection. The DAB positive stained signals detected in GQDs-biotin-injected mice compared to vehicle-injected control group. Immunogold positive signals observed inside (red triangles) or outside of neurons (blue triangles). k The comparison between intraperitoneal (IP; red bar) and intravenous (IV; blue bar) injections of GQDs-biotin. l The GQDs-biotin concentrations of the plasma measured from the non-injected control after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, and 6 months after biweekly GQDs-biotin injections using biotin quantification kit. m The brain (green bar) and plasma (blue bar) concentrations measured from 1 h, 7 days, and 14 days after single i.p. injection (2 mg/kg of GQDs-biotin. n 3 months and 6 months after multiple biweekly injections

60

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.15 The effect of GQDs on α-syn PFFs-induced glial cell activation in the SN. a Representative immunohistochemistry images for Iba-1 in the SN with low and high magnifications. Microglia in the SN of α-syn PFFs-injected hemisphere were stained with specific microglial marker Iba-1 (ionized calcium binding protein; specific marker of microglia/macrophage). b Quantifications of Iba-1-positive microglia. c Representative immunohistochemistry images for GFAP in the SN with low and high magnifications. Astrocytes in the SN of α-syn PFFs-injected hemisphere were stained with GFAP (glial fibrillary acidic protein; specific marker of astrocytes) antibody. d Quantifications of GFAP intensity. The relative GFAP intensities were normalized against the PBS-injected control group

Supplementary Information

61

Fig. 3.16 The effect of GQDs on pathologies and glial cell activation of hA53T α-syn transgenic mice. a Schematic illustration of the observing areas of hA53T α-syn Tg model. As a treatment, 50 μg of GQDs or PBS were i.p. injected biweekly for 4 months. b Representative p-α-syn immunostaining images in the cortex, ventral midbrain, and brainstem of hA53T α-syn Tg. c Quantifications of p-α-syn immunoreactive neurons in the cortex (Ctx), ventral midbrain (VM), and brainstem (BS). d Representative dot-blot assay of p-α-syn and α-syn fibrils. e Hindlimb clasping dystonic phenotypes of hA53T α-syn Tg mice were compared with GQDs-injected group. f Assessment of the behavioral deficits measured by the clasping scores. g Assessment of the behavioral deficits measured by the pole tests. h Representative immunohistochemistry images of microglia in the LC of the brainstem with low and high magnifications. Microglia in the brainstem of nTg or hA53T α-syn Tg were stained with Iba-1. i Quantifications of Iba-1-positive microglia. j Representative immunohistochemistry images of GFAP in the brainstem with low and high magnifications. k Quantifications of GFAP intensity. The relative GFAP intensities were normalized against the PBS-injected control group. l The effect of GQDs on α-syn aggregate formation in HEK293 cells with hA53T α-syn overexpression. HEK293T cells were transfected with pCMV5-myc-A53T α-syn and the test groups were treated with GQDs (0.1 μg/ml). After 48 h, cells were immunostained with α-syn antibodies. m The number of immuno-positive aggregates per field was quantified and normalized

62

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

Fig. 3.17 Long-term in vitro and in vivo toxicity of GQDs. a Cytotoxicity measurements of GQDs on primary cortical neurons after 7 days of incubation with alamarBlue and LDH assays. b Survival curves of GQDs injected mice. 50 μg of GQDs or vehicle were i.p. injected in C57BL/6 mice biweekly for 6 months and in vivo toxicity of GQDs was monitored (n = 20). c Representative hematoxylin and eosin (H&E) staining images of the major organs. The nuclei and eosinophilic structure of the liver, kidney, and spleen of GQDs-injected group were compared to the PBS-injected control group. d Schematic illustration of the injection and sampling methods for in vivo tracking of GQDs-biotin. e 50 μg of GQDs-biotin were i.p. injected in C57BL/6 mice. The concentrations of GQDs-biotin were measured using the brain (red) or urine (blue) of GQDs-biotin-injected mice at various time points (1, 3, 7, and 14 days)

Supplementary Information

63

Fig. 3.18 The comparative effects of nano-GOs and rGQDs on α-syn PFFs induced primary neuronal toxicity, disaggregation of fibrils and the BBB permeability. a FT-IR spectra of pristine GQDs (black), nano-GOs (green), and reduced GQDs (rGQDs, purple) with designated peaks for the functional groups. b AFM images of nano-GOs and GQDs with representative line profiles. c Quantifications of the cell toxicities after 72 h of incubation with 20 μg/ml of each material. d The cell toxicities monitored in SH-SY5Y cells by alarmarBlue assay at 12, 24, and 72 h with various concentrations of each material (1, 10, and 20 μg/ml). e The comparative dissociation effects of GQDs, nano-GOs, and rGQDs assessed by dot blot. f ThT and turbidity assays. g TEM images after 7 days of incubation (each with 5 mg/ml concentration; n = 3, independent experiments). h In vitro BBB permeabilities of GQDs, nano-GOs, and rGQDs using the in vitro BBB model in Fig. 3.13

3 Graphene Quantum Dots Prevent α-Synucleinopathy …

64

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Dawson, T. M. & Dawson, V. L. Science 302, 819–822 (2003). Spillantini, M. G. et al. Nature 388,839–840 (1997). Li, J. Y. et al. Nat. Med. 14, 501–503 (2008). Desplats, P. et al. Proc. Natl Acad. Sci. USA 106, 13010–13015 (2009). Volpicelli-Daley, L. A. et al. Neuron 72, 57–71 (2011). Luk, K. C. et al. Science 338, 949–953 (2012). Varela, L. et al. Biomol. NMR Assign. 10, 373–378 (2016). Bodner, C. R. et al. J. Mol. Biol. 390, 775–790 (2009). Tuttle, M. D. et al. Nat. Struct. Mol. Biol. 23, 409–415 (2016). Giasson, B. I. et al. J. Biol. Chem. 276, 2380–2386 (2001). van Stokkum, I. H. et al. Anal. Biochem. 191, 110–118 (1990). Sreerama, N. & Woody, R. W. Anal. Biochem. 287, 252–260 (2000). Lin, M. T. & Beal, M. F. Nature 443, 787–795 (2006). Czupalla, C. J., Liebner, S. & Devraj, K. Methods Mol. Biol. 1135, 415–437 (2014). Lee, M. K. et al. Proc. Natl Acad. Sci. USA 99, 8968–8973 (2002). Brahmachari, S. et al. J. Clin. Invest. 126, 2970–2988 (2016). Li, Q. et al. Chem. Eur. J. 20, 7236–7240 (2014). Mahmoudi, M. et al. Nanoscale 4, 7322–7325 (2012). Liu, Y. et al. Nanoscale 7, 19060–19065 (2015). Yang, Z. X. et al. Nanoscale 7, 18725–18737 (2015). Volpicelli-Daley, L. A., Luk, K. C. & Lee, V. M. Nat. Protoc. 9, 2135–2146 (2014). Delaglio, F. et al. J. Biomol. NMR 6, 277–293 (1995). Lee, W., Tonelli, M. & Markley, J. L. Bioinformatics 31, 1325–1327 (2015). Abraham, M. J. et al. SoftwareX 1–2, 19–25 (2015). Mackerell, A. et al. J. Comput. Chem. 25, 1400–1415 (2004). Yu, W. et al. J. Comput. Chem. 33, 2451–2468 (2012). Peelaerts, W. et al. Nature 522, 340–344 (2015). Mao, X. et al. Science 353, aah3374 (2016). Park, C. et al. Glia 53, 248–256, https://doi.org/10.1002/glia.20278 (2006). Guyenet, S. J. et al. J Vis Exp, https://doi.org/10.3791/1787 (2010). Marcano, D. C. et al. ACS Nano 4, 4806–4814, https://doi.org/10.1021/nn1006368 (2010).

Appendix

Education Sep. 2009–Dec. 2012, Department of Chemistry, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, MD, USA. Bachelor of Arts in Chemistry. Mar. 2013–Feb. 2019, Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Republic of Korea. Doctor of Philosophy in Physical Chemistry. Professional Experience Feb. 2010–Dec. 2012, Department of Neurology, Institute for Cell Engineering, Program in Neurorogeneration and Stem Cell Program, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. Undergraduate Research Assistant. Mar. 2019–Jul. 2019, Research & Development Team, BioGraphene Inc., Senior Researcher. Aug. 2019–Current, BioGraphene Inc. US Branch, Regional Director. Awards and Honors Distinguished Poster Award. “Highly Efficient Nucleic Acid Drug Delivery by Graphene Quantum Dots (GQDs)-Mediated Hyperthermia.” The 2nd Korean Graphene Symposium (2015). Global Ph.D. Fellowship. Therapeutic Applications of Graphene Quantum Dots (GQDs) for Neurodegenerative Diseases. The National Research Foundation of Korea (NRF), Ministry of Education (Mar. 2014–Feb. 2019). Best Ph.D. Dissertation Award. “Studies on Graphene-based Nanomaterials for Biomedical Applications.” College of Natural Sciences, Seoul National University (February 2019). United States National Interest Waiver (NIW) Green Card Beneficiary. Lawful permanent residency granted for an alien of exceptional ability. United States Citizenship and Immigration Services (USCIS) (June 2019). © Springer Nature Singapore Pte Ltd. 2020 J. M. Yoo, Studies on Graphene-Based Nanomaterials for Biomedical Applications, Springer Theses, https://doi.org/10.1007/978-981-15-2233-8

65

66

Appendix

Peer-Reviewed Publications († denotes equal contribution; * denotes corresponding author). 9. M. J. Park, H. –H. Choi, B. Park, J. Y. Lee, C. –H. Lee, Y. S. Choi, Y. Kim, J. M. Yoo, H. Lee, B. H. Hong* . “Enhanced Chemical Reactivity of Graphene by Fermi Level Modulation.” Chemistry of Materials, 30, 5602–5609 (2018) 8. D. Kim† , J. M. Yoo† , H. Hwang, J. Lee, S. H. Lee, S. P. Yun, M. J. Park, M. Lee, S. Choi, S. H. Kwon, S. Lee, S. -H. Kwon, S. Kim, Y. J. Park, M. Kinoshita, Y. -H. Lee, S. Shin, S. R. Paik, S. J. Lee, S. Lee, B. H. Hong* and H. S. Ko* . “Graphene quantum dots prevent α-synucleinopathy in Parkinson’s disease.” Nature Nanotechnology, 13, 812–818 (2018) 7. J. M. Yoo† , B. Park† , S. J. Kim, Y. S. Choi, S. Park, E. H. Jeong, H. Lee* and B. H. Hong* . “Catalytic degradation of phenols by recyclable CVD graphene films.” Nanoscale, 10, 5840–5844 (2018) 6. J. B. Park† , Y. -J. Kim† , S. -M. Kim, J. M. Yoo, Y. Kim, R. Gorbachev, I. I. Barbolina, S. J. Kim, S. Kang, M. -H. Yoon, S. -P. Cho, K. S. Novoselov and B. H. Hong* . “Non-destructive electron microscopy imaging and analysis of biological samples with graphene coating.” 2D Materials, 3, 045004 (2016) 5. S. J. Kim† , T. Choi† , B. Lee, S. Lee, K. Choi, J. B. Park, J. M. Yoo, Y. S. Choi, J. Ryu, P. Kim, J. Hone* and B. H. Hong* . “Ultraclean Patterned Transfer of Single-Layer Graphene by Recyclable Pressure Sensitive Adhesive Films.” Nano Letters, 15, 3236–3240 (2015) 4. Y. Kim† , J. Park† , J. Kang, J. M. Yoo, K. Choi, E. S. Kim, J. -B. Choi, C. Hwang, K. S. Novoselov and B. H. Hong* “A highly conducting graphene film with dual-side molecular n-doping.” Nanoscale, 6, 9545–9549 (2014) 3. S. J. Kim† , J. Ryu† , S. Son, J. M. Yoo, J. B. Park, D. Won, E. -K. Lee, S. -P. Cho, S. Bae, S. Cho and B. H. Hong* . “Simultaneous Etching and Doping by Cu-Stabilizing Agent for High-Performance Graphene-Based Transparent Electrodes.” Chemistry of Materials, 26, 2332–2336 (2014) 2. Y. Kim† , J. Ryu† , M. Park, E. S. Kim, J. M. Yoo, J. Park, J. H. Kang and B. H. Hong* . “Vapor-Phase Molecular Doping of Graphene for High-Performance Transparent Electrodes.” ACS Nano, 8, 868–874 (2014) 1. Y. Kim, J. M. Yoo, H. R. Jeon* , B. H. Hong* . “Efficient n-doping of graphene films by APPE (aminophenyl propargyl ether): a substituent effect.” Physical Chemistry Chemical Physics, 15, 18353–18356 (2013) Reviews and Book Chapters 2. J. M. Yoo, D. W. Hwang, and B. H. Hong. (2018). “Graphene-Based Nanomaterials.” In: D. Lee, ed., Radionanomedicine: Combined Nuclear and Nanomedicine. Springer International Publishing, pp. 79–103. 1. J. M. Yoo† , J. H. Kang† and B. H. Hong* . “Graphene-based nanomaterials for versatile imaging studies.” Chemical Society Reviews, 44, 4835–4852 (2015)

Appendix

67

Manuscripts Under Review/In Preparation 3. I. Kang† , J. M. Yoo† , D. Kim, D. J. Kim, J. Kim, B. –C. Lee, J. Y. Lee, J. –J. Kim, N. Shin, Y. –H. Lee, B. H. Hong* , K. -S. Kang* . “Graphene Quantum Dots Alleviate Impaired Functions in Niemann-Pick Type C Disease In Vivo.” 2. D. Kim, H. Hwang, S. Choi, S. H. Kwon, S. –U. Kang, T. –I. Kam, K. Kim, S. S. Karuppagounder, B. –G. Kang, S. Lee, J. M. Yoo, H. Park, S. Kim, W. Yan, Y. –S. Li, S. –H. Kuo, O. Pletnikova, J. C. Troncoso, X. Mao, V. L. Dawson, T. M. Dawson* and H. S. Ko* . “TRIP12 Ubiquitination of GBA1 Contributes to Neurodegeneration in Parkinson’s Disease.” 1. B. -C. Lee† , J. Y. Lee† , J. M. Yoo, I. Kang, J. -J. Kim, N. Shin, D. J. Kim, S. W. Choi, B. H. Hong and K. -S. Kang* . “Nano-sized graphene oxides attenuate intestinal inflammation by modulating immune homeostasis.” Selected Presentations The 2019 MRS Spring Meeting and Exhibit (Contributed Talk). Graphene Quantum Dots Prevent α-Synucleinopathy in Parkinson’s Disease. Phoenix, AZ, USA (April 2019). The 2018 MRS Spring Meeting and Exhibit (Poster). Catalytic degradation of phenols by recyclable CVD graphene film. Phoenix, AZ, USA (April 2018). The 4th Korea Global Ph.D. Fellowship Conference. Therapeutic Applications of Graphene Quantum Dots (GQDs) for Parkinson’s Disease. Busan, Republic of Korea (December 2017). The 4th Korean Symposium on Graphene and 2D Materials (Poster). Catalytic degradation of phenols by recyclable CVD graphene film. Buyeo, Republic of Korea (April 2017). The Seawater Utilization Plant Research Center: Korea Research Institute of Ships and Ocean Engineering (Invited Talk). Graphene: A New Class of Materials for Various Future Applications. Goseong, Republic of Korea (March 2017). The 1st International Society for Nanomedicine (ISNM) Congress (Poster). Therapeutic Effects of Functionalized Nanographene: Parkinson’s Disease. Seoul, Republic of Korea (October 2016). 8th International Conference on Recent Progress in Graphene/2D Research (Contributed Talk). Therapeutic Application of Graphene Quantum Dots (GQDs) for Parkinson’s Disease. Seoul, Republic of Korea (October 2016). The 3rd Korean Graphene Symposium (Contributed Talk). Therapeutic Applications of Graphene Quantum Dots (GQDs) for Parkinson’s Disease. Buyeo, Republic of Korea (April 2016). Graphene Flagship—Graphene Week 2015 (Poster). Highly Efficient Nucleic Acid Drug Delivery by Graphene Oxides (GOs)-Mediated Hyperthermia. Manchester, UK (June 2015). The 2nd Korean Graphene Symposium (Poster). Highly Efficient Nucleic Acid Drug Delivery by Graphene Quantum Dots (GQDs)-Mediated Hyperthermia. Buyeo, Republic of Korea (March 2015).

68

Appendix

Patents Registered • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Registration: Republic of Korea. “Pharmaceutical Composition Based on Graphene Nanostructure for Preventing or Treating Neurodegenerative Diseases.” Registration Number: KR10-1747147. Date of Registration: 2017.06.08. Filed • K. -S. Kang, B. H. Hong, I. Kang, J. M. Yoo. Country of Application: Republic of Korea. “Pharmaceutical Composition for Preventing or Treating Lysosome Storage Disorder.” Provisional Filing Number and Date: T-01280, 2018.12.13. • B. H. Hong, J. M. Yoo, S. S. Kim. Country of Application: Republic of Korea. “Graphene quantum dots as treating agent for diseases related fibrosis or aggregation of neural proteins.” Provisional Filing Number and Date: SNU-2018-09021, 2018.11.25. • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Application: United States. “Graphene Nanostructure-based Pharmaceutical Composition for Preventing or Treating Neurodegenerative Diseases.” Application Number: 16/004,744., Date of Application: 2018.10.11. • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Application: Europe. “Graphene Nanostructure-based Pharmaceutical Composition for Preventing or Treating Neurodegenerative Diseases.” Application Number: 15 774 194.3., Date of Application: 2016.10.28. • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Application: China. “Graphene Nanostructure-based Pharmaceutical Composition for Preventing or Treating Neurodegenerative Diseases.” Application Number: 2015 800183448, Date of Application: 2016.10.24. • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Application: Japan. “Graphene Nanostructure-based Pharmaceutical Composition for Preventing or Treating Neurodegenerative Diseases.” Application Number: 110000877, Date of Application: 2016.10.03. • B. H. Hong, J. M. Yoo, H. S. Ko, D. Kim. Country of Application: PCT (All designated states). “Pharmaceutical Composition Based on Graphene Nanostructure for Preventing or Treating Neurodegenerative Diseases.” Application Number: PCT/KR2015/003385, Date of Application: 2015.04.03.