Fluorine Functionalized Graphene Quantum Dots as Inhibitor against

Feb 23, 2017 - ... Nanosafety, CAS Center for Excellence in Nanoscience, National Center for ... which is important for prevention and treatment of am...
4 downloads 0 Views 2MB Size
Subscriber access provided by University of Newcastle, Australia

Article

Fluorine functionalized graphene quantum dots as inhibitor against hIAPP amyloid aggregation Maryam Yousaf, Huan Huang, Ping Li, Chen Wang, and Yanlian Yang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00015 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Fluorine functionalized graphene quantum dots as inhibitor against hIAPP amyloid aggregation Maryam Yousaf1,2, Huan Huang1, Ping Li1, Chen Wang1,2, Yanlian Yang*1,2 1

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Key

Laboratory of Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China 2

University of Chinese Academy of Sciences, 19 A Yuquan Rd, Shijingshan District, Beijing, P.

R. China 100049

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 2 of 39

Abstract Fibrillar deposits of the human islet amyloid polypeptide (hIAPP) is considered as a root of Type II diabetes mellitus. Fluorinated graphene quantum dots (FGQDs) are new carbon nanomaterials with unique physicochemical properties containing highly electronegative F atoms. Herein we report a single step synthesis method of FGQDs with an inhibitory effect on aggregation and cytotoxicity of hIAPP in vitro. Highly fluorescent and water dispersible FGQDs, less than 3 nm in size were synthesized by the microwave-assisted hydrothermal method. Efficient inhibition capability of FGQDs to amyloid aggregation was demonstrated. The morphologies of hIAPP aggregates were observed to change from the entangled long fibrils to short thin fibrils and amorphous aggregates in the presence of FGQDs. In thioflavin T fluorescence analysis the inhibited aggregation with prolonged lag time and the reduced fluorescence intensity at equilibrium were observed when hIAPP was incubated together with FGQDs. Circular dichroism spectrum results reveal that FGQDs could inhibit conformational transition of the peptide from native structure to β-sheets. FGQDs could also rescue the cytotoxicity of INS-1 induced by hIAPP in dose dependent manner. This study could be beneficial for design and preparation of inhibitors for amyloids, which is important for prevention and treatment of amyloidoses. Key Words:

Fluorinated graphene quantum dots (FGQDs), amyloid peptide, aggregation,

inhibitor, cell viability, hIAPP. 2

ACS Paragon Plus Environment

Page 3 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Introduction The formation and deposition of peptide fibrils and plaques in human body is the hallmark of many degenerative diseases including Alzheimer’s disease (AD), Huntington’s disease (HD), type 2 diabetes mellitus (T2DM), Parkinson’s disease (PD), spongiform encephalopathy, amyotrophic lateral sclerosis etc.

1-3

Most of the amyloid related disease is neurodegenerative

diseases. Different types of amyloid peptides associated with different diseases have the common structural feature, which is abnormal folding followed by aggregation into amyloid fibrils. Human islet amyloid polypeptide (hIAPP) is one of them and could be selected as a model system for study amyloid aggregation because of its faster aggregation dynamics. hIAPP is a neuropancreatic hormone,4 secreted along with insulin by pancreatic β-cells and is involved in glucose homeostasis,

5

under certain conditions hIAPP polymerizes to forms fibrillar amyloid

deposits.6-7 Presence of hIAPP aggregates in β-cells results in their apoptosis and ultimately leads to T2DM.8 In addition, progression of T2DM leads to development of other neurodegenerative diseases. Evidences from clinical studies suggest that T2DM and AD are closely related to each other. People suffering from diabetes have greater chance of developing AD (associated with dysfunction of neurons) in comparison to healthy individuals.9 Hypoglycemia (associated with T2DM) and hyperglycemia in the central nervous system will affect the signaling cascades 3

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 4 of 39

resulting in neuronal and synaptic dysfunction ultimately leading to AD.10 Considering the common amyloid aggregation features, and the correlation of T2DM and AD, we select hIAPP here in this manuscript as a model system representing amyloid peptides. The inhibition of hIAPP aggregation is crucial for developing treatment strategies of T2DM, which is also very crucial for the development of prevention and treatment of other neurodegenerative diseases. In order to reduce cytotoxicity induced by hIAPP, a number of anti-amyloidogenic agents/inhibitors have been screened such as small molecules, polyphenolic structure extracted from herbs,

13

synthetic peptides.

14

11

metal complexes,12

Liu et al, found that co-

assembling insulin with hIAPP over negatively charged surface influence hIAPP aggregation process. Increasing concentration of insulin in hIAPP/insulin ratio disrupt the morphologies from hIAPP fibrils to oligomers/monomers and finally ends up in annular fibrils (insulin alone) due to electrostatic interaction between insulin and hIAPP.15 It has also been recognized that dedicated efforts are needed to improve permeability, stability within the body, etc. of these inhibitory materials for their potential application as anti-amyloidogenic agents. 16 In another closely related subject of research, nanomaterials have been drawing great attention in this field because of their small size which allows them to pass through cell membranes and enter different cells and organelles, perturbing self-assembly pathway of proteins.17 Applications of nanomaterials as hIAPP anti-amyloidogenic agents could be promising because of their small 4

ACS Paragon Plus Environment

Page 5 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

size and rich chemistry. Either inhibitory or enhancement effect on the process of aggregation have been reported, while the applications of the nanomaterials in amyloid aggregation modulation is quite limited because of the biocompatibility issues.18, 19 Carbon nanomaterials of different types (carbon nanotubes, C60, fullerenes, and graphene),17, 20

size (graphene oxide)21 and dimensions (zero carbon dots, one dimensional carbon nanotubes

and two dimensional graphene oxide)22 have been studied extensively as amyloid inhibitor because of their unique structure and properties. Among these materials graphene based materials are of special interest because of their biologically friendly nature, possibility of π- π stacking, electrostatic and hydrophobic interactions between graphene and amyloid peptide.

23

Graphene oxide (GO) and graphene quantum dots (GQDs) have been studied to modulate amyloid-β peptide, whereas reports are also available regarding the use of GO about modulation of hIAPP aggregation.

16, 18, 24

N,N/ -Dimethylformamide modified GQDs had been reported to

enhance the aggregation of hIAPP peptide.20 Surface chemistry and electronic properties of GQDs have been reported to be modified by doping with heteroatoms (nitrogen, sulphur, boron, halogens in GQDs).25-27 Interaction between nanoparticles and amyloid proteins varied with physicochemical properties of amyloid proteins and surface functional groups of nanoparticles.

18

GQDs behavior is also expected to be varied

on its surface functionalization with heteroatoms that will change the surface chemistry of 5

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 6 of 39

GQDs. GQDs (with pristine graphene) and its fluorine functionalized form as hIAPP aggregation inhibitor has not been reported so far, F atom was chosen due its importance in medicinal field. From medicinal point of view fluorinated organic compounds are of significant interest as insertion of F atom in a molecule give special properties (enhance selectivity and interaction of drugs with receptor) to molecules, that is difficult to attain by insertion of other elements. Fluorination increase pharmacological properties of molecules, a number antibiotics, anticancer, anti-inflammatory, and anti-viral etc., has been reported.28, 29 Therefore it was speculated that among the possible derivatives of GQDs fluorine (F) doped GQDs could be of great significance and in future will open a new avenue for synthesis of new F based nanomedicine. Doping of GQDs with F alters biological,

30, 31

electrochemical,

32

physico-chemical

properties (lipophilicity or basicity) of GQDs. Introduction of highly electronegative and strong electron withdrawing F atom in GQDs will significantly increase acid nature of carboxylic groups group enhancing the interaction with peptide, whereas CF bond could interact with via hydrophobic and hydrogen bonded interaction. Thus it was speculated that presence of F atom will play a crucial role in enhancing the GQDs ability to inhibit amyloid aggregation. Therefore, we explore the inhibitory effects of fluorinated graphene quantum dots (FGQDs) and compare it with GQDs and nitrogen doped GQDs (N-GQDs).

6

ACS Paragon Plus Environment

Page 7 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

However, it is difficult to synthesize fluorinated graphene quantum dots (FGQDs) using conventional top down methods like acidic exfoliation, microwave-assisted hydrothermal treatment, sonication and electrochemical oxidation (commonly employed for synthesis of GQDs from graphene/ graphene oxide)

33, 34

due to the chemical inertness and hydrophobicity of

fluorinated graphene (FG) sheets. Few reports are available regarding the synthesis of FGQDs using FG sheets but all these reported methods are quite complicated and time consuming. 35, 36 It is therefore highly desirable to develop effective and facile methods to prepare FGQDs and explore their biological applications. Herein we report facile single-step bottom up method (glucose as a precursor) for the synthesis of FGQDs with potential application as a therapeutic agent against hIAPP aggregation. Aggregation of hIAPP is directly related to the death of pancreatic β-cell that leads to insulin deficiency in T2DM, so finding a new anti-amyloidogenic agent like FGQDs with an ability to protect pancreatic β-cells against hIAPP aggregates mediated cellular toxicity will be of great significance. Results and Discussion Morphological characterization of FGQDs. Microwave hydrothermal method, employed for the synthesis of FGQDs that provide dual advantage of microwave and hydrothermal heating. Continuous, fast and homogeneous microwave heating leads to the formation of quantum dots 7

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 8 of 39

with uniform size distribution. Glucose molecules under microwave hydrothermal conditions (180 ˚C, 500 W, 3 h) undergoes dehydration forming in a honeycomb like structure. Hydrofluoric acid catalyses the dehydration reaction under acid conditions and provides fluorine as a dopant. Fig. 1(a) shows that the growth of FGQDs occurs in following three steps. During first step glucose molecules dehydrated to from nucleation crystal of FGQDs. In second step chemically active functional groups attach at the edges or the surface of growing FGQDs, Thirdly, with an increase in time size of FGQDs increases. The HRTEM image (Fig.1b) indicates high crystallinity of the FGQDs, where lattice fringes with an in-plane lattice spacing of 0.214 nm, that is in accordance with data previously reported for FGQDs.35 The most frequent size of FGQDs determined by Gaussian distribution (Fig. 1c) is 2.38±0.04 nm.

Fig. 1 Microwave-assisted hydrothermal growth process (a), HRTEM image with observed lattice fringe (b) and size distribution histogram (c) of FGQDs. 8

ACS Paragon Plus Environment

Page 9 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Spectroscopic characterization of FGQDs. Bonding composition and functional groups of prepared FGQDs analyzed by FTIR analysis (Fig. 2a) show an obvious peak at 1083 cm-1 is associated with stretching vibrations of C-F indicative of effective doping with fluorine. 37 Peaks positioned at 1402 cm-1 (C-O) and 1594 cm-1 (C=O) confirmed the presence of an oxygen containing functional group in FGQDs, the broad peak centered at 3435 cm-1 reveals presence of O-H bonds. From FTIR spectrum peaks analysis it is concluded that FGQDs composition consists of fluorine atom in the form of C-F bond and a carboxylic group, as C=O, C-O, and O-H bonds, that guarantee the good water dispersibility of FGQDs. Further confirmation of the surface functional groups of FGQDs was carried out using XPS characterization. XPS survey spectra in (Fig. 2b) reveals that FGQD synthesized from glucose contains fluorine along with carbon and oxygen. The measured C1s spectra of FGQDs was deconvoluted into five surface components (Fig. 2c), corresponding to sp2 (C=C) at binding energy 284.7 eV, as C=O at 288 eV and as C-O at 286 eV.38, 39 Peak centered at 289.3 eV is because of covalently linked F atom with sp3 C, this type of peak is most commonly observed in graphite fluorides. While peak at 289.9 eV is assigned to covalent C-F bonds that is consistent with the previously studies.34, 37, 40 F1s spectra of FGQDs at 689.0 eV is because of C-F covalent bonds on a carbon structure (Fig. 2d.)

41

According to XPS analysis, the carbon, oxygen, and

fluorine contents of FGQDs are about 57.36 %, 39.18%, and 2.84%, respectively. Components 9

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 10 of 39

determined by XPS spectra are in agreement with FTIR analysis, suggesting that fluorine atoms have been successfully functionalized over the surface of glucose. PL (photoluminescence) and PLE (photoluminescence excitation) spectra of FGQDs was measured (Fig. 2f). PL spectra of FGQDs excited at various excitation wavelengths is shown in Fig. 2e. A broad emission peak at 497 nm is observed when the sample is excited by 360 nm wavelength. While PLE spectra of FGQDs show a strong peak at 360 nm when FGQDs was monitored at 497 nm wavelength. Fluorescent carbon materials commonly possessed excitationdependent PL emissions.33, exhibit blue fluorescence

44

42, 43

According to previous report GQDs synthesized by glucose

but in our case green fluorescence (inset in Fig. 2f) is observed in

spite of this fact that size of prepared FGQDs is much smaller than reported in the previous report, this red shifted PL of FGQDs as compared to simple GQDs might be due to fluorination. Red shift in FGQDs PL is due to irradiative decay of activated electrons due to entrapment of generated excitons in shallow trap states surface created due to charge separation between graphene and fluorine atom.45-47

10

ACS Paragon Plus Environment

Page 11 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Fig. 2 Spectroscopic characterization of FGQDs a) FTIR spectrum, b) XPS survey spectra, c) Deconvoluted peaks of C1s XPS spectrum, d) F1s XPS spectrum of FGQDs, e) Room temperature PL at different excitation wavelengths varying from 360 to 540 nm and (f) PLE spectra for FGQDs. Effect of FGQDs, GQDs and N-GQDs on hIAPP aggregation process. Quantification of hIAPP amyloid fibrils with and without the addition of FGQDs as a function of reaction time was done by measuring and fitting sigmoidal growth curve on ThT fluorescence intensity. ThT is commonly used fluorescent dye for monitoring amyloid aggregation. Enhancement in ThT fluorescent intensity is observed due to specific binding between ThT and the β-sheet structure of amyloid fibrils.48, 49

11

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 12 of 39

From a typical sigmoidal growth curve of hIAPP aggregation (Fig. 3a) it is obvious that aggregation process occurs in three phases, namely, lag phase, elongation phase, and saturation phase, easily distinguished by variation in ThT fluorescence intensity. During first few minutes of hIAPP incubation (lag phase) ThT fluorescence intensity increased slowly indicating the absence of amyloid fibrils. Lag phase is considered to be the critical and rate limiting step during which monomers undergoes conformational rearrangement to form clusters that act as precursor for fibril formation.50-52 At the end of the lag phase, ThT fluorescence increased rapidly that finally reached to equilibrium. However, in the presence of FGQDs, a noteworthy decrease in ThT fluorescence intensity along with prolonged lag time was observed in the hIAPP aggregation process. FGQDs inhibit hIAPP aggregation process in dose dependent manner (Fig. 3a). hIAPP was incubated with FGQDs in different mass ratios (1:1, 1:2, 1:5, 1:10), at fixed hIAPP concentration of 20 µM. Prolonged lag phase time along (inset of Fig 3a) with a decrease in ThT fluorescence emission in all the three phases was observed on increasing amount of FGQDs in hIAPP solution. Pronounced inhibitory effect of FQGDs was attributed to fluorination of GQDs. To confirm this speculation, inhibitory effect of FGQDs was compared with inhibitory performance of GQDs (no fluorine, contain only –COOH, -OH, negative control) ∽2-3 nm (Fig. S1a) and N-GQDs (contain N heteroatom instead of F and –COOH, -OH) ∽3-6 nm in size (Fig. S1b), taken at 1:1 mass 12

ACS Paragon Plus Environment

Page 13 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

ratio. Fig. 3b shows a comparative effect of GQDs, N-GQDs and FGQDs over the hIAPP aggregation kinetics. Inhibitory performance was in the following order FGQDs˃NGQDs˃GQDs. Noticeable elongation in the lag phase of hIAPP aggregation up to 100 minutes was observed on premixing FGQDs with 20 µM of hIAPP in 1:1 mass ratio. Whereas in case of N-GQDs and GQDs augmentation of 20 and 10 minutes in lag phase was observed, confirming the potent role of F atom in enhancement of inhibitory efficiency of GQDs. Furthermore, we speculated that FGQDs interacts with hIAPP monomers/oligomers during the early stage of nucleation and inhibit the formation of oligomers/aggregation of oligomers that later on aggregated to form hIAPP fibrils. To support this speculation, FGQDs was added in hIAPP solution (10 µM) in 1:1 mass ratio at different time points i.e., 0 and 5 minutes of incubation (Fig. 3c). Samples were taken out at different time points, mixed with ThT solution and fluorescence emission was measured. In the absence of FGQDs aggregation process was very rapid without any lag time but after the addition of FGQDs at 0 minute it was increased to approximately 20 minutes. However, FGQDs were added after 5 minutes of incubation no change in lag time could be seen (Fig. 3c). These results indicate that FGQDs have a greater inhibiting effect on hIAPP aggregation when added at initial stages of nucleation. This observation suggests that the inhibition of amyloid formation is probably due to the interaction of FGQDs with the hIAPP monomeric or oligomeric species before critical nucleation 13

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 14 of 39

concentration is reached. Once critical nucleation concentration is reached, the addition of FGQDs could not inhibit amyloid fibril formation but may disintegrate the pre-formed fibrils. Data was analyzed statistically by ANNOVA. Differences among the variables were significant at P < 0.05 (Fig. 3d).

Fig. 3 (a) Growth kinetic of hIAPP aggregation monitored at different time interval via ThT fluorescence assay. (b) Comparison of inhibitory performance of FGQDs with inhibitory effects of GQDs and N-GQDs (c) Variation in lag phase of hIAPP aggregation upon addition of FGQDs 14

ACS Paragon Plus Environment

Page 15 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

at different time intervals. (d) ANNOVA fitted over 20µM hIAPP aggregation with and without FGQDs incubated in different mass ratio. (**** = level of significance is higher, *** = Significant difference from control (P < 0.05), *= difference is not very much significant from control. Morphological characterization of fibrils. Inhibitory effect of FGQDs was confirmed by a morphological study of hIAPP amyloid fibrils after incubating 20 µM hIAPP in the presence and absence of FGQDs (1:1 mass ratio) using TEM. For hIAPP alone, we observed a dense cluster of thick fibrils after 100 minutes of incubation (Fig. 4a). Fibrils are ∼22 nm wide and ∼90 nm long and some of them are branched. Addition of FGQDs in hIAPP markedly reduce fibril formation, as even after 100 minutes of incubation only short length, thinner fibrils (Fig.4b) along with amorphous aggregates with no particular structure (Fig.4b & c), some monomers ∼2.5 nm (Fig. 4d) and oligomers of different size with most frequent size of ∼12 nm (Fig.4e) were observed. Formation of shorter and thinner fibrils in the presence of inhibitor is due to improper association of the protofilament subunits that forms amyloid fibrils and/or due to enhancement in fibrils breakage.5 These observations suggest that FGQDs could have significantly reduced amyloid fibrils formation either by enhancing fibrils breakage or by impairing attachment of protofilament subunits to mature amyloid fibers. Whereas reduction in the amount of hIAPP fibrils is due to the interaction of 15

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 16 of 39

FGQDs with hIAPP monomers/oligomers as it is clear from (Fig.4d & e), hence preventing selfaggregation of oligomers into amyloid. TEM results are consistent with ThT kinetics results.

Fig. 4 Dense cluster of hIAPP fibrils alone (a). Formation of thin short fibrils (b), amorphous aggregates (c), monomeric (∼2.5 nm in size) (d) and oligomeric species (∼12 nm in size) after addition of FGQDs in 20µM hIAPP solution. Arrows in (d) and (e) pointed towards the attached FGQDs whereas yellow circles outline monomers and oligomers. FGQDs possess strong potential to inhibit hIAPP aggregation, as evident from the results. In addition to its inhibitory potential we further check whether FGQDs could disassemble already present hIAPP fibrils. Disassembly of pre-formed hIAPP fibrils by FGQDs was also observed by adding FGQDs (1:1 mass ratio) into the fibrils of 20 µM hIAPP. From results we have found some indication that the disassembly do happen, while we still need more experiment to verify (results not shown). From ThT results it is clear that GQDs and N-GQDs also possess inhibitory effect against hIAPP aggregation but not as pronounced as FGQDs. GQDs only slow down the process of hIAPP aggregation but did not reduce the length of formed fibrils Branched and elongated fibrils was 16

ACS Paragon Plus Environment

Page 17 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

observed in GQD-hIAPP system similar to control hIAPP (Fig.S3a) but less in amount in Fig.S3b. While in case of N-GQDs shorter thin fibrils (Fig.S3c) than control samples were observed along with some amorphous aggregates (inset of Fig.S3c). Greater inhibition than GQDs might be due to the increased electrostatic interaction between peptide and N-GQDs resulted from the higher electron density of the graphene sheet in presence of N atom. While these effects are less pronounced than FGQDs, where the electron withdrawing effect of F will enhance the electrostatic interaction, additional hydrophobic interaction and π-π interaction because of F atom will also enhance the binding ability of FGQDs with peptides. Effect of FGQDs on secondary structure. Far UV CD spectroscopy is an outstanding technique for quick evaluation of secondary structure, binding and folding properties of proteins.53-55 The CD spectra of a freshly prepared solution of 20µM hIAPP alone displayed negative minima at ∼203 nm (Fig. 5) characteristics of the random coiled structure.56 Deviation from random coil conformation could be clearly seen in CD spectra of hIAPP alone after an incubation time of 40 minutes with ∼38.14 % β-strand and∼ 0.8% α-helix secondary components. After 100 minutes of incubation, a deep negative peak at ∼218 nm appears confirming the induction of β-sheet, characteristic of typical amyloid fibrils. CD spectra of 20µM hIAPP containing FGQDs (1:1 mass ratio) also show a negative minimum at ∼203 nm (Fig. 5) at 0 minutes of incubation. After 20 minutes of incubation, 17

ACS Paragon Plus Environment

ACS Chemical Neuroscience

∼95.36 % of α-helical character was observed. Interestingly, this α-helical structure was still stable even after 100 min of incubation with two negative minima at ∼ 211 nm and ∼ 219 nm (Fig. 5 inset). These observations suggest that morphological conversion of hIAPP monomers to hIAPP fibrils follow helical transition (α-helix oligomers are formed as intermediate) as reported previously.

57 58 59

Probably, FGQDs block the α-helix oligomers and prevent their aggregation

in to β-sheets containing amyloid fibrils even after 100 minutes of incubation, although hIAPP alone show a typical β-sheet character. These results are consistent with TEM images. It is clear from the results that process of fibril formation via aggregation of the α-helical intermediate state is halted by FGQDs. 40 30

CD(mdeg)

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 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 57 58 59 60

Page 18 of 39

20

211 nm 219 nm

10 0

-10 -20 190

200

210

220

230

240

Wavelength (nm)

250

260

270

hIAPP (0 min)

hIAPP+FGQDs (0 min)

hIAPP (40 min)

hIAPP+FGQDs (40 min)

hIAPP (100 min)

hIAPP+FGQDs (100 min)

Fig. 5 Far-UV CD spectrum representing a conformational change in hIAPP at different time points in the presence and absence of FGQDs. 18

ACS Paragon Plus Environment

Page 19 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

To see whether other inhibitors GQDs and N-GQDs could halt hIAPP conformational transformation, CD spectra of hIAPP in the presence of GQDs and N-GQDs at different time point (0, 40 and 100 minutes) was recorded (Fig.S4). It is clear that after 40 minutes of incubation, conformational transformation of hIAPP starts with induction of 49% and 30% βstrands for GQDs and N-GQDs respectively, followed by complete induction of β-sheet, characteristic of typical amyloid fibrils (Fig.S4b). Whereas in case of FGQDs, even after 40 minutes of incubation α-helical structures persist, which indicates that FGQD shows greater effect on α-helical conformational stabilization. This α-helical conformational stabilization effect could also be reflected in the inhibitory effect of FGQDs. Reduced cytotoxicity of hIAPP fibrils by FGQDs. Effect of FGQDs toxicity was evaluated using INS-1 cell line. Cell viability of the FGQDs treated INS-1 cells is greater than 90% at 0.3µg/mL (Fig. 6a). 80% cell survival rate even after the addition of 100 µg/mL (FGQDs) proves FGQDs as less toxic and biocompatible nanomaterial. From preceding results, it is clear that FGQDs possess inhibitory effect against hIAPP aggregation. To evaluate the effect of FGQDs over hIAPP fibrils induced cytotoxicity hIAPP (20 µM) was incubated with FGQDs in equal mass ratio and in five folds’ higher concentration. 20 µM concentration of hIAPP induces significant toxicity, with cell viability of only about 60% of the hIAPP untreated control. Increment of only 20 % in cell survival rate than that of the hIAPP alone was observed on 19

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 20 of 39

addition of hIAPP and FGQDS in 1:1 mass ratio (Fig. 6b). Increase in cell survival rate occurs in dose dependent manner, at a higher ratio of FGQDs (5 folds higher than hIAPP), FGQDs rescued up to 90 % cells by decreasing hIAPP mediated cellular toxicity. From results is concluded that FGQDs could reduce the hIAPP induced toxicity by suppressing the formation of toxic, cell membrane penetrating hIAPP oligomers. As reported previously hIAPP monomers form toxic oligomers that have the ability to penetrate the cell membrane and ultimately kill the cells, later on, these oligomers self-aggregated to forms mature amyloid fibrils60. Therefore, we can conclude from above results that reduced fibrillation of hIAPP by FGQDs indeed mitigate cytotoxicity induced by hIAPP fibrils.

Fig. 6 (a) Cytotoxicity of FGQDs for INS-1 cell. (b) FGQDs reduced 20 µM hIAPP fibrils mediated cellular toxicity for INS-1 in dose dependent manner (ratio is mass ratio of hIAPP and FGQDs). Discussion 20

ACS Paragon Plus Environment

Page 21 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

hIAPP fibrils aggregation in the pancreas is considered as a hallmark in progression of T2DM because pancreatic β-cells are impaired or killed by these fibrils, any factor that increases the death rate of β-cells is responsible For T2DM.61 hIAPP fibrillation process involves a number of intermediate steps starting from nucleation to oligomerisation that finally ends up in to fibril formation. Hydrogen bonding, π-π stacking interaction and intermolecular interaction between hydrophobic regions are considered as main forces involved in fibril formation.56 Progression of T2DM leads to development of another neurodegenerative disease AD and this fact is confirmed from epidemiologic and clinical studies. Therefore it is highly desirable to find out therapeutic agent that have an ability to block active sites of monomer/protofibrils of hIAPP so that formation and deposition of these amyloid plaques can be prohibited. It is still very challenging to find out therapeutic agents to interfere with the interactions involved in fibril formation between peptides. Graphene based materials are of significant importance in biomedicine and material sciences, mainly GO sheets and GQDs. Due to low toxicity, high biocompatibility, GQDs have been widely used in nano-biomedicines and also reported as amyloid inhibitor. Fluorination of GQDs could probably enhance the inhibitory effects of GQDs, therefore we synthesized fluorine (F) functionalized GQDs.

21

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 22 of 39

Fluorinated organic compounds are of special interest in medicinal aspect because of unique properties of the fluorine atom, organic compounds are being fluorinated to enhance their metabolic activity by replacing H with F atom. Fluorinated materials could induce α-helix character in amyloid proteins, a significant contributing factor for the inhibition of amyloid aggregation. Fluorinated solvent e.g., 1, 1,1,3,3,3-hexafluoro-2-propanol (HFIP) etc. strongly promotes α-helical structure, thus inhibiting peptide aggregation.62, 63 Therefore, we synthesize GQDs containing F atom in their structure, water dispersible FGQDs of approximately 2.38 nm in size were synthesized by single step bottom up method. Good water dispersibility of FGQDs is because of the presence of hydrophilic groups (–COOH and –OH) in their structure. Whereas the presence of F atoms provides slight hydrophobicity to FGQDs that allows its interactions with the hydrophobic regions of hIAPP involved in oligomerisation. Limited literature is available regarding toxicity of fluorine containing nanomaterials,64 among these reports Rebeca Romero-Aburto et al, found that fluorinated graphene oxide was almost nontoxic to human breast cancer cell (MCF-7).31 Reports are available regarding little to no acute toxicity on inhalation of fluorosubstituted alkanes/alkenes, therefore, it was speculated that the toxicity of fluorinated compounds depend upon their molecular characteristics, atomic % of fluorine, and physiochemical properties that can alter the interaction between cell and nanomaterials ultimately effecting their degree of cytotoxicity.65, 66 66, 67 68 Teo et al found in their study that it 22

ACS Paragon Plus Environment

Page 23 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

is the molecular form of fluorine that played a potent role in the toxicity of fluorinated graphene materials and one with mono-fluorosubstitution is more toxic than one with di/trifluorosubstitution.

69

Similar type of toxicity behavior could be expected for FGQDs (in our

case). From our CCK8 assay it is clear that FGQDs are almost non-toxic for the cells up to 500 µg/mL, which demonstrates the good biocompatible properties of FGQD as a potent candidate for biomedical applications. FGQDs contains both mono-fluoro and difluorosubstituted groups, so slight toxicity of FGQDs is speculated to be because of mono-fluorosubstituted groups as clear from the XPS spectra (Fig.2c). From literature survey and our own findings we can say that in fluorinated compounds that contain fluorine in low % and in di/tri substituted form are less toxic and suitable for biological applications. Inhibitory effect of FGQDs over hIAPP aggregation was monitored by ThT fluorescence assay. Experimental results show that FGQDs effectively inhibit hIAPP aggregation in a dosedependent manner, increasing the dose of FGQDs increases the inhibiting potential. Inhibition of hIAPP aggregation is most likely due to the interaction between FGQDs and the hIAPP species i.e., monomers and oligomers. The most probable interaction could be electrostatic interaction or H-bonding between polar groups of hIAPP peptide and FGQDs. Due to highly electronegative F atoms, the polarity of FGQDs is expected to increase as well as the positive charge over the 23

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 24 of 39

aromatic ring of FGQDs. Because of the positive mesomeric and negative inductive effect of F atom, leading to the enhanced hydrogen bond formation and electrostatic interaction with the hIAPP peptide containing polar groups. Another possible interaction between peptide and FGQDs could be π-π stacking interaction. The presence of aromatic structured amino acids like phenylalanine and tyrosine in hIAPP also promotes formation and stabilization of amyloid fibrils via π-π stacking interaction.70,

71

Conjugated graphene structure of FGQDs can form π-π stacking interaction with the aromatic amino acids, which leads to the preferred adsorption of hIAPP molecule on FGQDs surface and inhibit the bulk hIAPP aggregation because of the depletion of peptides. ThT results are consistent with TEM images, where we can only see short thin fibrils, amorphous aggregates, and oligomers suggesting that FGQDs interact with hIAPP monomers and slowdowns oligomerisation or with oligomers, preventing them to self-aggregate into mature fibrils. These results are further supported by a reduction in cellular toxicity of hIAPP in the presence of FGQDs. According to previous reports, hIAPP oligomers and hIAPP amyloids may cause cell apoptosis, reduction in hIAPP mediated cellular toxicity by FGQDs is attributed to a reduction in oligomer concentration that ultimately reduced fibril formation or presence of nontoxic α-helical monomers. From CD results it is clear that even after 100 minutes of incubation α-helical character of hIAPP persist, this α-helical character might be due to α-helical 24

ACS Paragon Plus Environment

Page 25 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

monomers or α-helical oligomers (fibrillation process intermediate) confirming the inhibitory effect of FGQDs. It was hypothesized that functionalization of GQDs with fluorine enhanced its inhibitory effects. Among three type of inhibitors FGQDs have higher binding potential as compared to GQDs and N-GQDs. According to previous reports carbon based nanomaterials inhibits amyloid aggregation through hydrophobic, electrostatic interactions.72 Therefore any atom that could increase hydrophobic and electrostatic interactions between inhibitor and peptide could possibly break the binding between peptides leading to reduced amyloid aggregation.73 Inhibitors other than carbon based inhibitor e.g., resveratrol, curcumin, inorganic nanoparticles, dendrimers, and myricetin have been reported to have inhibitory effect against hIAPP aggregation, while some drawbacks such as the low water solubility, high toxicity, and need of surface passivating agents hinder their applications in medicines.74 Apart from these chemicals some peptides e.g., insulin have also been reported as hIAPP aggregation inhibitor, but Cui et al, reported that insulin could only inhibits hIAPP aggregation for certain time and after that promotes the hIAPP aggregation via attachment with the hIAPP fibrils.75 Herein, we used insulin as positive control against FGQDs because of its most frequent use in T2DM (a disease that is progressed due to hIAPP aggregation) treatment. Insulin does not increase lag time on mixing with hIAPP in 1:1 ratio as shown in ThT results (Fig.S5). This 25

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 26 of 39

increment is not as pronounced as observed on mixing FGQDs with hIAPP in same mass ratio, which indicates the superiority of FGQDs over insulin. Insulin reduces the hIAPP aggregation for certain time after that it promotes the hIAPP aggregation process for incubating it 300 minutes (Fig.S5). This enhancement in fibrillation is because of copolymerization of insulin with hIAPP might be caused by interaction of insulin with monomeric or oligomeric form not with fibrils.75 To remove the ambiguity that enhancement in ThT fluorescence intensity is not because of insulin fibrils but due to hIAPP aggregation, control experiment on insulin was also conducted. Until 300 minutes fluorescence intensity did not increase, which confirmed that the measured ThT fluorescence signals are because of hIAPP aggregation not due to insulin. The TEM images in Fig.S6 showing a dense cluster of short fibrils slightly less than control hIAPP samples indicates that FGQDs possess greater potential of inhibiting hIAPP aggregation than insulin. From the above results, it could be concluded that the FGQDs is a very promising antiamyloidogenic inhibitors and have many advantages over commonly reported inhibitors. Conclusion Herein, we prepared F atom containing GQDs (FGDQs) and investigated their inhibitory effect against peptide aggregation. Less toxic FGQDs with size less than 3 nm was prepared by “bottom-up” approach through a microwave-assisted hydrothermal method using glucose and HF as a precursor. Inhibitory effects of FQGDs was investigated on hIAPP aggregation and 26

ACS Paragon Plus Environment

Page 27 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

compared with controls (GQDs and N-GQDs). hIAPP aggregation is strongly correlated with hIAPP conformational changes, fluorinated compounds possess the ability to induce α-helical structure thus preventing fibril formation, whereas GQDs and N-GQDs did not possess this ability. Observed reduced β-sheet structure with increased α-helical structure in CD results supports this fact that F atom of FGQDs is inhibiting conformational transition of hIAPP, hence preventing the formation of mature fibrils as confirmed by TEM images. Inhibition of hIAPP aggregation showed that FGQDs binds with hIAPP monomer/oligomers because of the presence of hydrophobic groups, higher charge density, and an aromatic ring. Furthermore, FGQDs protected INS-1 cells from hIAPP mediated toxicity either through reducing oligomers concentration or disrupting hIAPP fibrillation In summary, current in vitro hIAPP aggregation, study suggests that FGDQs could potentially reduce the risk of T2DM by preventing toxic hIAPP aggregation. Development of fluorine based antidiabetic drugs could be useful. In future, this study could be beneficial for design and preparation of inhibitors for amyloids related to other degenerative diseases. Experimental section Synthetic peptide. hIAPP with a sequence of KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY ((1–37), disulfide bridge: C2 and C7), purity > 98 % (confirmed with Mass Spectrum and High 27

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 28 of 39

Performance Liquid Chromatography (HPLC)), purchased from Science Biological Technology Co. (Shanghai, China). Synthesis and characterization of FGQDs. FGQDs were synthesized by a microwave-assisted hydrothermal method, using glucose and hydrofluoric acid (HF) as a precursor material. Briefly, 4 mL of HF acid was added in glucose solution (0.1 g of glucose dissolved in 10 mL of deionized water). The whole reaction mixture was transferred into autoclaves (50 mL) and placed in microwave hydrothermal reactor at 180 ˚C and kept for about 3 hr for the producing water soluble brown color dispersion. The dispersion was filtered through 0.2 µm filter to remove lager sized particles and pH was neutralized with the help of NaOH. Resulted light brown solution was dialyzed using dialysis bag (MWCO 1000 D). Morphological characterization of FGQDs was performed with Tecnai G2 F20 U-TWIN transmission electron microscope (TEM), (FEI, USA) at an acceleration voltage of 200 kV. Spectroscopic characterizations were carried out using Fourier transform infrared (FTIR) spectroscopy (Perkin Elmer Instruments, USA) and X-ray photoelectron spectroscopy (XPS) ESCALAB 250Xi spectrometer (Thermo Fisher Scientific, USA). Peptide stock solution. For preparation of peptide stock solution hIAPP was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (1 mg/mL) with overnight shaking. Aliquot was taken, dried in a flow of nitrogen and re-dissolved in Milli-Q water as required. 28

ACS Paragon Plus Environment

Page 29 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Thioflavin T fluorescence assay. Kinetics of hIAPP fibrils were studied by thioflavin T (ThT) binding assay, ThT fluorescence depends upon the concentration of hIAPP amyloid fibrils. For ThT assay 20 µM hIAPP solution was incubated (37 ˚C, 150 rpm) with FGQDs in different mass ratio (1:1, 1:2, 1:5, 1:10), samples were withdrawn at regular interval of time and ThT fluorescence intensity at 485 nm at excitation wavelength of 450 nm was measured using microplate reader (SpectraMax i3, Molecular Devices, USA). Circular dichroism. Circular dichroism spectra of 20 µM hIAPP with or without FGQDs, NGQDs and GQDs after incubation at different time intervals (0, 20 and 100 minutes) were recorded on Jasco-J810 spectrophotometer (Jasco, Japan). Spectra were recorded in the range of 190 to 260 nm. K2D, the most widely used artificial intelligence program (a neural network), that use neural network of proteins and generates secondary structure contents information at each wavelength, was used for the determination of total β sheets, helical structure and random coil confirmation within samples. 55 Morphological characterization. Effect of FGQDs over the morphology of aggregates and interaction with different morphological states (monomers/oligomers) of hIAPP was studied using TEM. To see the morphology of aggregates before and after addition of FGQDs, N-GQDs and GQDs sample solution was prepared by incubating hIAPP (20 µM) with or without FGQDs, N-GQDs and GQD (in1:1mass ratio) for 100 minutes. For fibrillar disassembly assay: 20 µM of 29

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 30 of 39

hIAPP was incubated for 3hr and after that FGQDs were added (1:1 mass ratio) and incubated for further 3 hr and 5 hr. 7 µL of this sample solution was taken out and dropped over carbon coated copper grids and allowed to adsorb at least for 10 minutes. Excess sample was removed using filter paper and allowed to dry, dried sample was stained with 2% uranyl acetate for 3-5 minutes, and excess stain was removed by washing with Milli Q water and allowed to dry. Dried sample was analyzed by HT7700 biology TEM (Hitachi, Ltd., Tokyo, Japan with 80 kV acceleration voltage). To see interaction between FGQDs and monomers and oligomers sample was withdrawn at 0 and 10 minutes of incubation of 1:1mass ratio mixture of FGQDs and hIAPP (20 µM) and pipetted over copper grid, left over sample preparation was same as mentioned for aggregates study. These samples were examined using Tecnai G2 F20 U-TWIN transmission electron microscope (TEM), (FEI, USA) at an acceleration voltage of 200 kV. Comparison of FGQDs with GQDs and N-GQDs. Control experiments for ThT fluorescence assay, circular dichroism and morphological characterization were also performed using GQDs without any heteroatom and N-GQDs. GQDs and N-GQDs were mixed with 20 µM hIAPP at 1:1 mass ratio and compared with results of inhibitory performance of FGQDs taken at same mass ratio. 30

ACS Paragon Plus Environment

Page 31 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

Cytotoxicity assay. Insulinoma cell line (INS-1 cells) were grown in RPMI 1640 medium (100 UI/ml penicillin, 0.1 mg/mL streptomycin, 10% FBS (fetal bovine serum), sodium pyruvate (1 nmol/L) and β-mercaptoethanol (50 µmol/L)) in humidified (95 % air, 5 % CO2) atmosphere at 37 ˚C. 6000 cells/well were plated in 96-well micro plate followed by 24 hr incubation, after that cells were exposed to peptide and FGQDs containing media and incubated at 37 ˚C for 72 hr. Cell viability was determined by adding 20 µL of Cell counting Kit 8 (CCK 8) solution followed by 2 hr incubation. Later on absorbance was measured at 450 nm (SpectraMax i3, Molecular Devices, USA), all experiments were conducted in triplicates and presented as mean ± S.D. Statistical analysis. Data was statistically analyzed by applying the analysis of variance (ANNOVA) using graph pad prism software and significance of results is expressed in terms of the p value. ASSOCIATED CONTENT Supporting information Synthesis protocol and characterization of GQD and N-GQDs, images morphological characterization and CD results for inhibitory effect of GQD and N-GQDs, specificity assay of FGQDs for hIAPP and results for control experiments are given in supporting information. AUTHOR INFORMATION Corresponding Author 31

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 32 of 39

*Yanlian Yang [email protected] Tel.: +86 10 82545559; fax: +86 10 62656765

Author Contributions Maryam Yousaf designed and done research, Dr. Huan Huang and Dr. Ping Li helped in analyzing data. Prof. Dr. Chen Wang and Prof. Dr. Yanlian Yang guided in designing research and revising manuscript. Conflict of interest There is no conflicts of interest among authors. Acknowledgment The National Natural Science Foundation of China (21273051) and the Chinese Academy of Sciences (XDA09030306) supported this work. Financial support from Beijing Municipal Natural Science Foundation (2162044) also gratefully acknowledged. References

32

ACS Paragon Plus Environment

Page 33 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

[1] Wang, H., Ridgway, Z., Cao, P., Ruzsicska, B., and Raleigh, D. P. (2015) Analysis of the ability of pramlintide to inhibit amyloid formation by human islet amyloid polypeptide reveals a balance between optimal recognition and reduced amyloidogenicity, Biochemistry. 54, 6704-6711. [2] Jean, L., Lee, C. F., Hodder, P., Hawkins, N., and Vaux, D. J. (2016) Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide, Sci. Rep. 6. [3] Hong, Y., Meng, L., Chen, S., Leung, C. W. T., Da, L.-T., Faisal, M., Silva, D.-A., Liu, J., Lam, J. W. Y., and Huang, X. (2012) Monitoring and inhibition of insulin fibrillation by a small organic fluorogen with aggregation-induced emission characteristics, J. Am. Chem. Soc. 134, 1680-1689. [4] Clark, A., Lewis, C., Willis, A., Cooper, G., Morris, J., Reid, K., and Turner, R. (1987) Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes, The Lancet. 330, 231-234. [5] Brender, J. R., Hartman, K., Nanga, R. P. R., Popovych, N., de la Salud Bea, R., Vivekanandan, S., Marsh, E. N. G., and Ramamoorthy, A. (2010) Role of Zinc in Human Islet Amyloid Polypeptide Aggregation, J. Am. Chem. Soc. 132, 8973-8983. [6] Eisenberg, D., Nelson, R., Sawaya, M. R., Balbirnie, M., Sambashivan, S., Ivanova, M. I., Madsen, A. Ø., and Riekel, C. (2006) The structural biology of protein aggregation diseases: Fundamental questions and some answers, Acc. Chem. Res. 39, 568-575. [7] Clark, A., Wells, C., Buley, I., Cruickshank, J., Vanhegan, R., Matthews, D., Cooper, G., Holman, R., and Turner, R. (1988) Islet amyloid, increased A-cells, reduced B-cells and exocrine fibrosis: quantitative changes in the pancreas in type 2 diabetes, Diabetes research (Edinburgh, Scotland) 9, 151-159. [8] Stefani, M., and Dobson, C. M. (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution, J. Mol. Med. 81, 678-699. [9] Hu, R., Zhang, M., Chen, H., Jiang, B., and Zheng, J. (2015) Cross-Seeding Interaction between β-Amyloid and Human Islet Amyloid Polypeptide, ACS Chem. Neurosci. 6, 1759-1768. [10] Sims-Robinson, C., Kim, B., Rosko, A., and Feldman, E. L. (2010) How does diabetes accelerate Alzheimer disease pathology?, Nat. Rev. Neurol. 6, 551-559. [11] Mishra, R., Bulic, B., Sellin, D., Jha, S., Waldmann, H., and Winter, R. (2008) Small Molecule Inhibitors of Islet Amyloid Polypeptide Fibril Formation, Angew. Chem. Int. Ed. 47, 4679-4682. 33

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 34 of 39

[12] Jeong, K., Chung, W. Y., Kye, Y.-S., and Kim, D. (2010) Cu (II) cyclen cleavage agent for human islet amyloid peptide, Bioorg. Med. Chem. 18, 2598-2601. [13] Cheng, B., Gong, H., Li, X., Sun, Y., Zhang, X., Chen, H., Liu, X., Zheng, L., and Huang, K. (2012) Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide, Biochem. Biophys. Res. Commun. 419, 495-499. [14] Scrocchi, L. A., Chen, Y., Waschuk, S., Wang, F., Cheung, S., Darabie, A. A., McLaurin, J., and Fraser, P. E. (2002) Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis, J. Mol. Biol. 318, 697-706. [15] Liu, P., Zhang, S., Chen, M.-s., Liu, Q., Wang, C., Wang, C., Li, Y.-M., Besenbacher, F., and Dong, M. (2012) Co-assembly of human islet amyloid polypeptide (hIAPP)/insulin, Chem. Commun. 48, 191-193. [16] Liu, Y., Xu, L.-P., Dai, W., Dong, H., Wen, Y., and Zhang, X. (2015) Graphene quantum dots for the inhibition of â amyloid aggregation, Nanoscale 7, 19060-19065. [17] Guo, J., Li, J., Zhang, Y., Jin, X., Liu, H., and Yao, X. (2013) Exploring the Influence of Carbon Nanoparticles on the Formation of â-Sheet-Rich Oligomers of IAPP(22–28) Peptide by Molecular Dynamics Simulation, PLOS ONE. 8, 65579. [18] Nedumpully-Govindan, P., Gurzov, E. N., Chen, P., Pilkington, E. H., Stanley, W. J., Litwak, S. A., Davis, T. P., Ke, P. C., and Ding, F. (2016) Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells, Phys. Chem. Chem. Phys. 18, 94-100. [19] Chan, H.-M., Xiao, L., Yeung, K.-M., Ho, S.-L., Zhao, D., Chan, W.-H., and Li, H.-W. (2012) Effect of surface-functionalized nanoparticles on the elongation phase of betaamyloid (1–40) fibrillogenesis, Biomaterials. 33, 4443-4450. [20] Wang, L., Zhu, S., Lu, T., Zhang, G., Xu, J., Song, Y., Li, Y., Wang, L., Yang, B., and Li, F. (2016) The effects of a series of carbon dots on fibrillation and cytotoxicity of human islet amyloid polypeptide, J. Mater. Chem. B 4, 4913-4921. [21] Wang, J., Cao, Y., Li, Q., Liu, L., and Dong, M. (2015) Size effect of graphene oxide on modulating amyloid peptide assembly, Chem. Eur. J. 21, 9632-9637. [22] Wang, J., Zhu, Z., Bortolini, C., Hoffmann, S., Amari, A., Zhang, H., Liu, L., and Dong, M. (2016) Dimensionality of carbon nanomaterial impacting on the modulation of amyloid peptide assembly, Nanotechnology. 27, 304001. [23] Zhang, M., Mao, X., Yu, Y., Wang, C. X., Yang, Y. L., and Wang, C. (2013) Nanomaterials for reducing amyloid cytotoxicity, Adv. Mater. 25, 3780-3801.

34

ACS Paragon Plus Environment

Page 35 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

[24] Li, Q., Liu, L., Zhang, S., Xu, M., Wang, X., Wang, C., Besenbacher, F., and Dong, M. (2014) Modulating Aβ33–42 Peptide Assembly by Graphene Oxide, Chem. Eur. J. 20, 7236-7240. [25] Zhang, L., Zhang, Z.-Y., Liang, R.-P., Li, Y.-H., and Qiu, J.-D. (2014) Boron-doped graphene quantum dots for selective glucose sensing based on the “abnormal” aggregation-induced photoluminescence enhancement, Anal. Chem. 86, 4423-4430. [26] Li, X., Lau, S. P., Tang, L., Ji, R., and Yang, P. (2013) Multicolour light emission from chlorine-doped graphene quantum dots, J. Mater. Chem. C 1, 7308-7313. [27] Qu, D., Zheng, M., Du, P., Zhou, Y., Zhang, L., Li, D., Tan, H., Zhao, Z., Xie, Z., and Sun, Z. (2013) Highly luminescent S, N co-doped graphene quantum dots with broad visible absorption bands for visible light photocatalysts, Nanoscale. 5, 12272-12277. [28] Panchal, I. I., Sen, D. J., Prajapati, B., and Shah, S. K. (2013) Serendipity of fluorine in discovery and development of antidiabetic agents: a bottleneck systemic review, World. J. Pharm. Sci. 1, 168-175. [29] Ojima, I., McCarthy, J. R., and Welch, J. T. (1996) Biomedical frontiers of fluorine chemistry, ACS Publications. [30] Wang, Y., Lee, W. C., Manga, K. K., Ang, P. K., Lu, J., Liu, Y. P., Lim, C. T., and Loh, K. P. (2012) Fluorinated Graphene for Promoting Neuro‐Induction of Stem Cells, Adv. Mater. 24, 4285-4290. [31] Romero‐Aburto, R., Narayanan, T., Nagaoka, Y., Hasumura, T., Mitcham, T. M., Fukuda, T., Cox, P. J., Bouchard, R. R., Maekawa, T., and Kumar, D. S. (2013) Fluorinated graphene oxide; a new multimodal material for biological applications, Adv. Mater. 25, 5632-5637. [32] Robinson, J. T., Burgess, J. S., Junkermeier, C. E., Badescu, S. C., Reinecke, T. L., Perkins, F. K., Zalalutdniov, M. K., Baldwin, J. W., Culbertson, J. C., and Sheehan, P. E. (2010) Properties of fluorinated graphene films, Nano Lett. 10, 3001-3005. [33] Zhu, S., Zhang, J., Qiao, C., Tang, S., Li, Y., Yuan, W., Li, B., Tian, L., Liu, F., Hu, R., Gao, H., Wei, H., Zhang, H., Sun, H., and Yang, B. (2011) Strongly greenphotoluminescent graphene quantum dots for bioimaging applications, Chem. Commun. 47, 6858-6860. [34] Li, L., Wu, G., Yang, G., Peng, J., Zhao, J., and Zhu, J.-J. (2013) Focusing on luminescent graphene quantum dots: current status and future perspectives, Nanoscale 5, 4015-4039. [35] Feng, Q., Cao, Q., Li, M., Liu, F., Tang, N., and Du, Y. (2013) Synthesis and photoluminescence of fluorinated graphene quantum dots, Appl. Phys. Lett. 102, 013111.

35

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 36 of 39

[36] Gong, P., Hou, K., Ye, X., Ma, L., Wang, J., and Yang, S. (2015) Synthesis of highly luminescent fluorinated graphene quantum dots with tunable fluorine coverage and size, Mater. Lett. 143, 112-115. [37] Liu, L., Zhang, M., Xiong, Z., Hu, D., Wu, G., and Chen, P. (2015) Ammonia borane assisted solid exfoliation of graphite fluoride for facile preparation of fluorinated graphene nanosheets, Carbon. 81, 702-709. [38] Tang, L., Ji, R., Cao, X., Lin, J., Jiang, H., Li, X., Teng, K. S., Luk, C. M., Zeng, S., and Hao, J. (2012) Deep ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots, ACS Nano. 6, 5102-5110. [39] Sun, H., Ji, H., Ju, E., Guan, Y., Ren, J., and Qu, X. (2015) Synthesis of fluorinated and nonfluorinated graphene quantum dots through a new top‐down strategy for long‐time cellular imaging, Chem. Eur. J. 21, 3791-3797. [40] Yan, S., Zhao, J., Yuan, Y., Liu, S., Huang, Z., Chen, Z., Jiang, D., and Zhao, W. (2013) Preparation and liquid-phase exfoliation of graphite fluoroxide towards graphene fluoroxide, RSC Adv. 3, 21869-21876. [41] Wang, Z., Wang, J., Li, Z., Gong, P., Liu, X., Zhang, L., Ren, J., Wang, H., and Yang, S. (2012) Synthesis of fluorinated graphene with tunable degree of fluorination, Carbon. 50, 5403-5410. [42] Liu, R., Wu, D., Feng, X., and Müllen, K. (2011) Bottom-up fabrication of photoluminescent graphene quantum dots with uniform morphology, J. Am. Chem. Soc. 133, 15221-15223. [43] Zhuo, S., Shao, M., and Lee, S.-T. (2012) Upconversion and downconversion fluorescent graphene quantum dots: ultrasonic preparation and photocatalysis, ACS Nano. 6, 10591064. [44] Yang, P., Zhou, L., Zhang, S., Wan, N., Pan, W., and Shen, W. (2014) Facile synthesis and photoluminescence mechanism of graphene quantum dots, J. Appl. Phys. 116, 244306. [45] Gong, P., Wang, J., Sun, W., Wu, D., Wang, Z., Fan, Z., Wang, H., Han, X., and Yang, S. (2014) Tunable photoluminescence and spectrum split from fluorinated to hydroxylated graphene, Nanoscale. 6, 3316-3324. [46] Babudri, F., Farinola, G. M., Naso, F., and Ragni, R. (2007) Fluorinated organic materials for electronic and optoelectronic applications: the role of the fluorine atom, Chem. Commun., 1003-1022. [47] Bao, L., Zhang, Z.-L., Tian, Z.-Q., Zhang, L., Liu, C., Lin, Y., Qi, B., and Pang, D.-W. (2011) Electrochemical Tuning of Luminescent Carbon Nanodots: From Preparation to Luminescence Mechanism, Adv. Mater. 23, 5801-5806. 36

ACS Paragon Plus Environment

Page 37 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

[48] Niu, L., Liu, L., Xi, W., Han, Q., Li, Q., Yu, Y., Huang, Q., Qu, F., Xu, M., and Li, Y. (2016) Synergistic inhibitory effect of peptide–organic coassemblies on amyloid aggregation, ACS Nano. 10, 4143-4153. [49] Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S. A., Krishna, V., Grover, R. K., Roy, R., and Singh, S. (2005) Mechanism of thioflavin T binding to amyloid fibrils, J. Struct. Biol. 151, 229-238. [50] Cabaleiro-Lago, C., Quinlan-Pluck, F., Lynch, I., Lindman, S., Minogue, A. M., Thulin, E., Walsh, D. M., Dawson, K. A., and Linse, S. (2008) Inhibition of amyloid β protein fibrillation by polymeric nanoparticles, J. Am. Chem. Soc. 130, 15437-15443. [51] Rangachari, V., Moore, B. D., Reed, D. K., Sonoda, L. K., Bridges, A. W., Conboy, E., Hartigan, D., and Rosenberry, T. L. (2007) Amyloid-β(1−42) Rapidly Forms Protofibrils and Oligomers by Distinct Pathways in Low Concentrations of Sodium Dodecylsulfate, Biochemistry. 46, 12451-12462. [52] Liu, Y., Xu, L.-P., Dai, W., Dong, H., Wen, Y., and Zhang, X. (2015) Graphene quantum dots for the inhibition of β amyloid aggregation, Nanoscale. 7, 19060-19065. [53] Huang, R., Carney, R. P., Ikuma, K., Stellacci, F., and Lau, B. L. (2014) Effects of surface compositional and structural heterogeneity on nanoparticle–protein interactions: different protein configurations, ACS Nano. 8, 5402-5412. [54] Li, S., Potana, S., Keith, D. J., Wang, C., and Leblanc, R. M. (2014) Isotope-edited FTIR in H 2 O: determination of the conformation of specific residues in a model α-helix peptide by 13 C labeled carbonyls, Chem. Commun. 50, 3931-3933. [55] Greenfield, N. J. (2006) Using circular dichroism spectra to estimate protein secondary structure, Nat. Protoc. 1, 2876-2890. [56] Bahramikia, S., and Yazdanparast, R. (2013) Inhibition of human islet amyloid polypeptide or amylin aggregation by two manganese-salen derivatives, Eur. J. Pharmacol. 707, 1725. [57] Williamson, J. A., and Miranker, A. D. (2007) Direct detection of transient α‐helical states in islet amyloid polypeptide, Protein Sci. 16, 110-117. [58] Knight, J. D., Hebda, J. A., and Miranker, A. D. (2006) Conserved and cooperative assembly of membrane-bound α-helical states of islet amyloid polypeptide, Biochemistry 45, 9496-9508. [59] Wang, L., Lei, L., Li, Y., Wang, L., and Li, F. (2014) A hIAPP‐derived all‐d‐amino‐ acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α‐helical oligomeric intermediates, FEBS Lett. 588, 884-891.

37

ACS Paragon Plus Environment

ACS Chemical Neuroscience

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 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 57 58 59 60

Page 38 of 39

[60] Haataja, L., Gurlo, T., Huang, C. J., and Butler, P. C. (2008) Islet amyloid in type 2 diabetes, and the toxic oligomer hypothesis, Endocr. Rev. 29, 303-316. [61] Soong, R., Brender, J. R., Macdonald, P. M., and Ramamoorthy, A. (2009) Association of highly compact type II diabetes related islet amyloid polypeptide intermediate species at physiological temperature revealed by diffusion NMR spectroscopy, J. Am. Chem. Soc. 131, 7079-7085. [62] Saraiva, A. M., Cardoso, I., Pereira, M. C., Coelho, M. A., Saraiva, M. J., Möhwald, H., and Brezesinski, G. (2010) Controlling Amyloid‐β Peptide (1–42) Oligomerization and Toxicity by Fluorinated Nanoparticles, Chem.Bio.Chem. 11, 1905-1913. [63] Rocha, S., Thünemann, A. F., Pereira, M. d. C., Coelho, M., Möhwald, H., and Brezesinski, G. (2008) Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide, Biophys. Chem. 137, 35-42. [64] Okazoe, T. (2009) Overview on the history of organofluorine chemistry from the viewpoint of material industry, Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 85, 276-289. [65] Barbier, O., Arreola-Mendoza, L., and Del Razo, L. M. (2010) Molecular mechanisms of fluoride toxicity, Chem.-Biol. Interact. 188, 319-333. [66] Clayton Jr, J. W. (1966) The mammalian toxicology of organic compounds containing fluorine, In Pharmacology of fluorides, pp 459-500, Springer. [67] Teo, W. Z., Chua, C. K., Sofer, Z., and Pumera, M. (2015) Fluorinated nanocarbons cytotoxicity, Chem. Eur. J. 21, 13020-13026. [68] Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63. [69] Teo, W. Z., Sofer, Z., Sembera, F., Janousek, Z., and Pumera, M. (2015) Cytotoxicity of fluorographene, RSC Adv. 5, 107158-107165. [70] Jaikaran, E. T., Higham, C. E., Serpell, L. C., Zurdo, J., Gross, M., Clark, A., and Fraser, P. E. (2001) Identification of a novel human islet amyloid polypeptide β-sheet domain and factors influencing fibrillogenesis, J. Mol. Biol. 308, 515-525. [71] Kajava, A. V., Aebi, U., and Steven, A. C. (2005) The Parallel Superpleated Beta-structure as a Model for Amyloid Fibrils of Human Amylin, J. Mol. Biol. 348, 247-252. [72] Guo, J., Li, J., Zhang, Y., Jin, X., Liu, H., and Yao, X. (2013) Exploring the influence of carbon nanoparticles on the formation of β-sheet-rich oligomers of IAPP 22–28 peptide by molecular dynamics simulation, PLoS One 8, e65579. [73] Vieira, E. P., Hermel, H., and Möhwald, H. (2003) Change and stabilization of the amyloidβ (1–40) secondary structure by fluorocompounds, Biochimica et Biophysica ActaProteins and Proteomics 1645, 6-14. 38

ACS Paragon Plus Environment

Page 39 of 39

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 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 57 58 59 60

ACS Chemical Neuroscience

[74] Gurzov, E. N., Wang, B., Pilkington, E. H., Chen, P., Kakinen, A., Stanley, W. J., Litwak, S. A., Hanssen, E. G., Davis, T. P., and Ding, F. (2016) Inhibition of hIAPP Amyloid Aggregation and Pancreatic β‐Cell Toxicity by OH‐Terminated PAMAM Dendrimer, Small 12, 1615–1626. [75] Cui, W., Ma, J. w., Lei, P., Wu, W. h., Yu, Y. p., Xiang, Y., Tong, A. j., Zhao, Y. f., and Li, Y. m. (2009) Insulin is a kinetic but not a thermodynamic inhibitor of amylin aggregation, FEBS J. 276, 3365-3371.

Inhibition of amyloid aggregation by FGQDs

39

ACS Paragon Plus Environment