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Nano Zinc Oxide Inhibits Fibrillar Growth and Suppresses Cellular Toxicity of Lysozyme Amyloid Deependra Kumar Ban, and Subhankar Paul ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11549 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 4, 2016

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ACS Applied Materials & Interfaces

Nano Zinc Oxide Inhibits Fibrillar Growth and Suppresses Cellular Toxicity of Lysozyme Amyloid Deependra Kumar Ban and Subhankar Paul*

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela

* Correspondence: Dr, Subhankar Paul, Associate Professor, Structural Biology and Nanomedicine Laboratory, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela-769008, Odisha, India, E-mail: [email protected], Phone: +91-0661-2462284 +91-0661-2463284 (R), Fax: +91-0661-2462022.

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ABSTRACT

Deposition of amyloid fibers has been a common pathological event in number of neurodegenerations such as Alzheimer’sdisease, Parkinson disease, Prion disease. Although, various therapeutic interventions have been reported; nanoparticles have recently been considered as possible inhibitor of amyloid fibrillation. Here, we reported the effect of three different forms of zinc oxide nanoparticles (ZnONP): uncapped (ZnONPuncap), starch capped (ZnONPST) and self-assembled (ZnONPassmb) (average size of 10, 30 and 163 nm, respectively) having a core size of 10-15 nm, in the amyloid growth of hen egg white lysozyme (HEWL). We monitored the amyloid growth by electron microscopy as well as Thioflavin-T (ThT) measurement. We observed that ZnONP demonstrated a dose-dependent inhibition of fibrillar amyloid growth of HEWL with highest effect exhibited by ZnONPST. Such inhibition was also associated with decrease of cross β-sheet amount, surface hydrophobicity as well as increase of stability of proteins. Further, we observed that ZnONPST prolonged the nucleation phase and shortened the elongation phase of HEWL amyloid growth. Although pure amyloid caused profound cellular toxicity in both mouse carcinoma N2a and normal cells such as human keratinocytes HaCaT cells; amyloid formed in the presence of ZnONP showed much reduced cellular toxicity. We also observed that inhibition of amyloid growth was effective when ZnONP were administered during the lag phase. When our amyloid inhibition results were compared with a well-known inhibitor curcumin, we observed that ZnONPST demonstrated a better inhibitory effect than curcumin. Overall, we reported here the inhibitory activity of three different forms of ZnONP to amyloid fibrillation of HEWL and amyloid-mediated cytotoxicity to different extent while starch capped ZnONP showed the highest fibrillation inhibitory effect.

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KEYWORDS: Amyloid; zinc oxide nanoparticles; Thioflavin-T fluorescence; Circular dichroism; cellular toxicity.

1. INTRODUCTION Accumulation of fibrillar protein aggregates (commonly known as ‘amyloid’) in the brain or other tissues is a common pathological feature of a series of neurodegenerative disorders such as Alzheimer's, disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Prion disease. 1-3

Such amyloid fibrils or plaques have been reported to be implicated with highly ordered cross

β-sheet structures.1, 4-5 A variety of molecules have been reported to inhibit the amyloid growth such as various drugs6-7, small peptides8, gene therapy9, chaperones10, and chemical osmolytes.11 However, the advent of nanotechnolgy recently have opened a new frontier and opportunity to explore the interaction of various nanoparticles with amyloidogenic proteins and find for a new therapeutic avenue. Therefore, understanding the effect of various nanoparticles particularly metal and metal-based nanomaterials on amyloid fibrillation of various amyloidogentic proteins has utter importance with regard the therapeutic scope in various neurodegeration. Nanoparticles (NPs) have intensely attracted the attention of many scientific groups due to their applications in many areas including biology and medicine. Although the inherent toxicity of many nanoparticles in the physiological condition has raised a great concern, their interaction with proteins and subsequent effect on amyloid formation should also be explored in details. Moreover, the effect of NPs on amyloid growth of proteins might change the net toxicity caused by both NPs and amyloids collectively. However, very few reports available which showed

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inhibition and/or enhancement of amyloid growth with regard to various amyloid forming proteins, NPs and disease models.

Linse et al. (2007) reported the enhancement of local concentration of β2-microglobulin on various tested NP (e.g. copolymer particle, cerium oxide, quantum dot, and carbon nanotubes) that promoted the formation of toxic cluster and amyloid.12 Moreover, titanium dioxide13 demonstrated enhanced human β2-microglobulin amyloid formation by reducing the nucleation phase of amyloids. Further it was reported that citrate capped gold nanoparticles (10-14 nm) also accelerated the amyloid growth of α-synuclein, but nucleation rate was decreased by larger AuNP.14 In contrary, Cabaleiro-Lago and group reported that copolymeric NiPAM:BAM (Nisopropylacrylamide:N-tert-butylacrylamide) nanoparticles of varying hydrophobicity retarded the fibrillation of the Alzheimer's disease-associated Aβ protein.15 Kim and his group also reported that gold nanostructures inhibited the Aβ fibrillation and cytotoxicity of Aβ in neuroblastoma cells.16 Moreover, functionalized gold nanoparticles17-18 and iron oxide nanoparticles

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demonstrated the inhibition of amyloid growth. Recently few groups

demonstrated that chemically exfoliated WS2 nanosheets and graphene oxide sheet efficiently inhibited amyloid β-peptide aggregation, however, the inherent cytotoxicity of WS2 or GO was not reported.20-21 Simulation-based finding showed that hydrophobic graphite induces the quick adsorption of Aβ peptides regardless of their initial conformations and sizes.22 Although consequences of the interaction of NP with proteins highly depends on its surface properties, core material, its assembly and type of functional molecules present and its density, no detailed study has so far been performed to assess the effect of NP with varying surface conditions on the

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amyloid growth of proteins.23-26 Moreover, effect of NPs upon administration time at various stages of amyloid growth should also be explored.

Here, we chemically synthesized three different types of zinc oxide nanoparticles (ZnONP): uncapped (ZnONPuncap), starch-capped (ZnONPST), and starch mediated self-assembly (ZnONPassemb) and assessed their effect on amyloid growth of hen egge white lysozyme (HEWL) under various conditions. ZnONP was selected for the analysis due to their broad biomedical applications such as drug delivery

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, medicine29 and bioimaging.28 Although ZnONP are

reported toxic to cells like RAW 264.7 and BEAS-2B30, starch capped ZnONP have been used due to their multi-dentate hydroxyl group, biodegradable

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, and biocompatible nature.

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In

addition, ZnONP nano-materials showed low toxicity and biodegradability at acidic as well as basic condition.

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Here, hen egg white lysozyme (HEWL) was studied due to its disease

causing history and its wide use as a model protein for in vitro amyloid study under laboratory condition.35-38 ZnONPs were characterized for size, shape, surface charge, and morphology. ZnONP (uncapped, capped, and self-assembled) were applied to the lysozyme solution, and amyloid growth of HEWL was monitored by electron microscopy and Thioflavin-T (ThT) measurement. The β-sheet contents of amyloid samples were observed by circular dichroism (CD). The toxicity of different amyloid samples were also assessed in mouse N2a (neuroblastoma), and normal cells such as HaCaT (human keratinocyte) cells. The cell death mechanism by amyloid and NPs were also studied.

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2. MATERIALS AND METHODS 2.1. Materials. Hen egg white lysozyme (HEWL) (EC 3.2.1.17), Zinc nitrate hexahydrate (Zn (NO3)2.6H2O), 8-anilinonapthalene-1-sulphonic acid (ANS), DMEM media, curcumin, and Fetal bovine serum (FBS) were obtained from Sigma Aldrich (Germany). Starch, Sodium hydroxide (NaOH), Hydrochloric acid (HCl), potassium di-hydrogen phosphate (KH2PO4), sodium chloride (NaCl), thioflalvin-T (ThT), and potassium chloride (KCl) were purchased from Himedia (India). Mice neuroblastoma cell line, N2a and human keratinocyte cell line, HaCaT were procured from NCCS, Pune, India. All glassware was rinsed by aqua regia and washed using double distilled water and dried in hot air oven before use. All the experiments were performed using Milli-Q water. 2.2. ZnONP synthesis.

For this study, uncapped zinc oxide nanoparticles (ZnONP) was

synthesized by wet chemical method, as described by Joshi et al. (2009), however with little modification.39 In brief, 10 mM of zinc nitrate hexahydrate [Zn(NO3)2.6H2O], the aqueous solution was prepared. NaOH was added dropwise, and the reaction was continued for 2 h with continuous stirring. The precipitate was centrifuged and calcinated for overnight at 70⁰C. For the preparation of starch capped ZnONP (ZnONPST), initially 1% (w/v) starch was added in zinc salt (10 mM), and NaOH (20 mM) and the reaction was performed for 2 h. The synthesis of self-assembly of ZnONP (ZnONPassmb) was described elsewhere

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. All three

kinds of ZnONP were characterized for shape and surface morphology by electron microscopy, hydrodynamic size by dynamic light scattering (DLS) and surface zeta potential (ζ-potential) by zeta sizer analysis. 2.3. Preparation of amyloid. Hen egg white lysozyme (HEWL) solution was prepared as described by Wang et al. with slight modification.41 Protein (70 µM) samples were prepared 6


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in hydrochloric acid (pH 2.0±0.2), which contain 2.7 mM KCl, 137 mM NaCl, and 0.01% (w/v) NaN3. The solutions (20 mL) were incubated in a shaker incubator at 58⁰C temperature and at a shaking speed of 100 rpm for 120 h (05 days). Further, amyloid samples were analyzed by various standard methods such as Thioflavin-T fluorescence assay and electron microscopy. 2.4. Electron Microscopic imaging of amyloid samples. The amyloid growth was observed by field emission scanning electron microscopy (FESEM) (Nova Nano-SEM) at 5 kV voltage, and TEM (CM 200, Phillips). For FESEM analysis, the sample was drop casted on silica wafer and dried under vacuum for overnight and gold coated for 1 min using sputtering of the sample. For TEM analysis, the sample was deposited on a copper grid of 300 meshes, negatively stained with uranyl acetate and dried in a vacuum drier for overnight before analysis. 2.5. Analyzing the effect of ZnONP. The effect of ZnONP (uncapped, starch capped, and selfassembly) on the HEWL amyloid growth was analyzed. A fixed concentration of three types of ZnONP (10 µM) was administered to 70 µM protein samples at 0th h and incubated at 58 ⁰C for 120 h, and amyloid growth kinetics was monitored by Thioflavin-T fluorescence assay at interval of 0, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h. Moreover, the effect of different concentration of ZnONP (1, 2, 5, 10, and 20 µM) on the growth and structure of amyloid was also analyzed after 72 h of incubation. Further, we also used curcumin (1-20 µM) as positive amyloid inhibitor as control for our analysis and comparison. The effect of ZnONP on different state of growth such as lag phase, elongation phase (when proto-fibrils and oligomer formed), and maturation phase (when long fiber forms) were also monitored by adding 10 µM NP sample. Moreover, the surface hydrophobicity was studied by protein7



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bound ANS fluorescence assay. The ζ-potential and hydrodynamic size distribution were analyzed by a zeta sizer (Nano-ZS, Malvern Pvt.). The structural change of amyloid was monitored by measuring Tryptophan (Trp) fluorescence spectra (Cary Eclipse, Agilent Pvt.) and secondary structural analysis was performed by Circular dichroism (CD) (Jasco-815) spectroscopy. The surface morphology and assembly of amyloid was observed by electron microscopy using TEM (Jeol) and FESEM. 2.6. Circular Dichroism (CD) analysis. CD spectra of lysozyme amyloid samples (14 µM) were analyzed in the range of 200-260 nm. The baseline correction of the sample was performed. The mean residual ellipticity was plotted, and percentage β-sheet was estimated by using CAPITO online server.42 All the data were converted to molar residue ellipticity (MRE) by using following equation:

· = − − − − − − − () · ·

Here, n is a number of amino acids, c is the concentration of protein in µM, ‘l’ is path length in mm. 2.7. Thioflavin-T (ThT) fluorescence measurement. The amyloid samples were diluted to 7 µM concentration and ThT was added such that the final concentration of ThT was 20 µM in sodium phosphate buffer, pH 7.4. The solutions were incubated for 20 min before spectrofluorometric (Cary eclipse, Agilent) analysis. The excitation was set at 440 nm and emission was recorded between 460-600 nm. The excitation and emission slit width were set at 5.0 and 10 nm, respectively. The growth of amyloid fibers was analyzed by ThT 8


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fluorescence at different time interval (0, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h), and emission was recorded. The amyloid growth curve was plotted with time (h), using the area under the curve of ThT emission spectra. The contribution of free ThT and ZnONP was subtracted before analysis. 2.8. 8-Anilinonaphthalene-1-sulfonic (ANS) assay. The protein-bound ANS fluorescence assay was performed in 3 mL sodium phosphate buffer (20 mM). The amyloid sample was diluted to make the final concentration of protein and ANS of 7µM and 150 µM, respectively. The solution was incubated for 30 min before measuring ANS fluorescence. The samples were excited at 350 nm and emission spectra were recorded between 400 and 600 nm by a Cary eclipse fluorescence spectrophotometer (Agilent Pvt. Ltd.). The excitation and emission slit width were adjusted at 5 and 10 nm, respectively. 2.9. Dynamic light scattering and ζ-potentialanalysis. The hydrodynamic size distribution of amyloid samples was analyzed by a zeta sizer (Malvern Nano-ZS series). Moreover, Aggregation index (AI) was also calculated to monitor the larger aggregate formation by protein. The size of protein aggregates was also monitored using DLS particle size analysis in dual angle mode. Aggregation index (AI) was calculated using the following equation:

=

Zaverage

forward

( !" ) − − − − − − − − ($) ( #" )

is hydrodynamic size analyzed at a forward angle, and Zaverage

hydrodynamic size at a backward angle.

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backward

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The ζ-potential of amyloid samples was analyzed by the zeta sizer (Malvern Nano-ZS series) connected with a DLS particle size analyzer. The samples were diluted five times with distilled water, and ζ-potential of samples was estimated in a disposable cuvette. 2.10.

Cytotoxicity assay. To study the cytotoxicity of HEWL amyloid samples prepared under

different conditions, we applied them to two different cell lines mice neuroblastoma (N2a) cells, and human keratinocyte (HaCaT) cells. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The cells (2000 cells/well) were inoculated in a 96-well plate at 37 0C for 24 h. Amyloid samples prepared with ZnONPuncap, ZnONPST, and ZnONPassmb (5, 10, 20, and 50 µM) were applied in triplicates to N2a and HaCaT cells, and cytotoxicity was estimated after 24 h of administration. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was performed as described by Riss et al. using MTT kit (Himedia) to determine cell viability 43

. MTT was added to a final concentration of 0.5 mg/ml and cells were incubated for 2 h. The

excess formazan was dissolved in DMSO, and the absorbance was measured at 570 nm within 1 h, and percentage viability was calculated by dividing the absorbance of treated cells with control cells. To analyze the ROS dependent cell death, we administered 5 mM of N-acetyl cysteine (NAC) at 0 h in the cell culture, and cell viability was analyzed. Gallic acid (50 µM) was also used as positive control after 24 h post-administration. 2.11.

Statistical analysis. Statistical analysis was performed for all data by one-way ANOVA

followed by Tukey’s test using Origin Pro 8.0. The p-value was found to be statically significant (i.e. 10 and 20 µM), ZnONPST and curcumin showed nearly equal inhibitory effect (see Fig. S3 A and C in ‘SI’). However, curcumin showed higher amyloid inhibition potential than ZnONPuncap, and ZnONPassmb (see Fig. S3). Since ZnONPST showed highest anti-amyloid activity, we also analyzed the effect of pure starch on the inhibition effect by ZnONPST (Fig. S3 B). However, results (Fig. S3 B & C in ‘SI’) clearly revealed that free starch has insignificant effect on the amyloid growth and inhibition under working concentration (1-20 µM). Further, to support our results obtained so far, we estimated secondary structural components of amyloid samples using CD spectroscopy, which revealed a reduction in β-sheet of all samples 18


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(amy@ZnONPuncap amy@ZnONPST, and amy@ZnONPassmb) while amy@ZnONPST showed the maximum drop (68% to 39%) of β-sheet contents (see Fig. 5 A-D). Therefore, the secondary structural data also supports our previous results (Fig. 4) that ZnONP can inhibit the formation of high β-sheet contents, and hence the amyloid fibrillation.

Figure 5. CD spectra analysis of HEWL (native) and amyloid (amy) formed with different concentration of ZnONP (5-20 µM). (A) amy@ZnONPuncap, (B) amy@ZnONPST, (C) amy@ZnONPassmb, (D) Table showing % α-helix and β-sheet. From here, it was observed that ZnONPST with higher zeta potential might have a strong interaction with HEWL compared to another form of ZnONP. Two groups have already reported 19



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that NP and protein interaction reduced the free protein concentration in solution.46-47 Therefore, when the interaction between NP and protein is strong, NP inhibits the amyloid growth both in solution and on NP surface at all NP concentrations. However, when interaction is relatively weak, amyloid formation depends on the concentration of NP administrated in the solution. 46-47

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Figure 6. TEM image showed the effect of three different concentrations (5, 10 and 20 µM) of ZnONP on amyloid formation. (A)-(A2) amy@ZnONPuncap; (B)-(B2) amy@ZnONPST; (C)-(C2) amy@ZnONPassmb.

To support our ThT fluorescence and CD results, we also performed TEM imaging of various amyloid samples (produced after incubating with ZnONPs). To observe the amyloid fibril under three different concentration of ZnONP (5, 10 and 20 µM), we observed TEM images of different amyloid samples (Fig. 6). Amy@ ZnONPuncap and amy@ZnONPassmb showed less number of thicker and shorter fibers when NP concentration was increased from 5 to 20 µM (Fig.6 A-A2 & C-C2). Amy@ZnONPST showed thin fibers at 5 µM, however, showed predominantly non-fibriller aggregates at 10 and 20 µM (Fig. 6 B, B1 & B2) of NP concentration. All the above results collectively established that the administration of ZnONPST inhibited the formation amyloid fiber most efficiently. 3.4. Growth kinetics of amyloids. Further, we also analyzed the growth kinetics of amyloid by ThT and ANS fluorescence, ζ-potential as well as CD spectroscopy analysis. The amyloid formation in the presence of NP (10 µM) was analyzed till 120 h. The ThT fluorescence intensity @488 nm (original ThT spectra in Fig. S4, ‘SI’) was plotted with growth period up to 120 h. The results showed that amy@ZnONPST showed longer (till 16 h) lag phase and shorter elongation phase (till 60 h) compared to other samples (14 h lag and 72 h of elongation)(Fig. 7A). Moreover, the ThT fluorescence intensity also indicated that all three types ZnONP (10 µM) were able to inhibit the formation of cross β-sheet amount while highest effect was demonstrated by ZnONPST (Fig. 7A) (original ThT spectra in Fig. S4). 21



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Higher cross β-sheet is one of the important features of amyloid fibril, and ThT fluorescence intensity indicates the amount proportionately by binding with fibrils and enhancing its emission. Hence, here the decrease of ThT fluorescence intensity of amyloid sample corelates with the inhibition of amyloid fibrillation. The results also supported our previous results obtained from Fig. 5D and 6. When we analyzed the time dependent measurement of surface ζ-potential at an interval of 12 h, we found that the surface charge of all amyloid samples (except pure amyloid) reached a highest value within 6072 h, which was indeed also indicating the end of amyloid growth phase (elongation). However, the pure amyloid (incubated with none) showed a longer time to reach stationary level, perhaps due to the formation of matured fibrils.

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Figure 7. Plot of (A) ThT fluorescence intensity at 488 nm. (B) ζ-potential, (C) % β-sheet contents of amyloid samples (amy@ZnONPuncap, amy@ZnONPST, amy@ZnONPassmb) with time of amyloid growth. The analysis of % β-sheet contents was performed by online CAPITO software (using CD data). The measurement was taken up to 120 h of amyloid growth. ZnONP was used @10µM.

We further analyzed the quantity of β-sheet contents using CD spectroscopy in different amyloid samples during the growth process (till 120 h). We found that the formation of β-sheet was rapid during nucleation and elongation phases; however, slowed down afterwards (Fig. 7C). 23



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Amy@ZnONPST showed shortest elongation phase (~60 h) as we observed in Fig. 7A & B along with lowest β-sheet contents (44%) compared to other amyloid samples (Fig. 7C & Fig. S5, in ‘SI’) while amyloid produced without NP showed 68% of β-sheet. Therefore, it was concluded that inhibition of amyloid formation also associated with the decrease of β-sheet formation. From Fig. 7A-C, it was clear that ZnONPST shortened the HEWL amyloid elongation phase. Normally elongation phase which is responsible for polymerization of monomer or oligomers into fibers and hence here, shortening of this phase by ZnONPST perhaps was associated with the inhibition of HEWL fibrillation. Further analysis of FESEM imaging of amyloid samples at different growth time (2, 36, 48 and 72 h) was performed. After 2 h of amyloid growth, both pure amyloid and amy@ZnONPST demonstrated smaller aggregates (see Fig. 8A & C), however, amy@ZnONPuncap and amy@ZnONPassmb results larger and more poly-dispersed aggregates (Fig. 8B & D). After 36 h of growth, we observed that all samples formed amorphous (unstructured/loose) aggregates (Fig. 8 A1, B1, C1, & D1). However, at 48 h, while pure amyloid demonstrated long fibrillar structures (Fig.8A2), other amyloid samples (except amy@ZnONPST) showed small fiber deposition (Fig. 8C2 and D2) along with amorphous (unstructured/loose) aggregates. However, amy@ZnONPST results inhigher amount of loose aggregates and almost no fibers until 48 h (Fig. 8 A2). After 72 h of amyloid growth, we found that all samples except amy@ZnONPST resulted amyloid fibers (Fig. 8 A3, B3, C3 & D3). However, amy@ZnONPuncap and amy@ZnONPassmb showed small or fragmented dense fibers (Fig. 8C3 and D3). These facts are also indicative of profound inhibitory potential of ZnONPST in amyloid growth of HEWL.

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Figure 8. FESEM image of amyloid samples with time (2, 36, 48, 72 h); (A-A3) without NP, (BB3) with ZnONPuncap (C-C3) with ZnONPST, (D-D3) ZnONPassmb. ZnONP was administered at 0th h.

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3.5. Effect of time of NP administration on amyloid structure. We further attempted to understand the effect of various ZnONP administration time on amyloid growth. We administered NP at 0, 36 and 48 h (Fig. S6 in ‘SI’) in amyloid samples and samples were analyzed after 72 h. From FESEM imaging (Fig. S6) we observed that inhibitory effect towards amyloid fibrillation of HEWL was still highest when NP was administered at 0th h. When administered at 36 and 48 h, from Fig. S6, it was found that all three ZnONP produced HEWL amyloid fibers. Therefore, we concluded that administration at 0thh,i.e., in the lag phase produced highest inhibitory effect, and this fact indeed supports our hypothesis that prolonged lag phase and shorter elongation phase perhaps the key behind the inhibitory mechanism induced by ZnONPST. Therefore, so far we observed that our ZnONP in three different forms could inhibit the HEWL amyloid fibrillation to various extents. Nanoparticle plays a multifaceted role, which depends on the properties of nanoparticle surface as well as a protein of interest. The variations of these factors also vary the effect of nanoparticle on amyloid formation. The three different form of ZnONP, which was made up of core size of 10-15 nm and having varying size due to surface capping and formation of self-assembly of ZnONP of 10-15 nm core size. Moreover, selfassembly of NP by starch altered the effect of NP on cellular toxicity and amyloid growth. In fact, Linse et al. and Cabaleiro-Lago et al. reported that the effect of nanoparticle on the amyloid growth depends on the various factors such as binding strength, NP to protein ratio on the surface, intrinsic stability and aggregation propensity of protein, surface hydrophobicity of NP.12, 15

The catalytic and inhibitory effects of NPs were most pronounced with the least hydrophobic

nanoparticles. Therefore, nanoparticles show dual role, catalyzing and inhibition, which depends on the inherent stability of protein, nanoparticle surface properties, protein to nanoparticle ratio, 26


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and strength of interaction. The variations of these factors also change the effect of nanoparticles on amyloid formation. 3.6. Cellular toxicity assay. To analyze the cellular toxicity of various amyloid samples, here we monitored the cellular toxicity in N2a (mice neural cell line) and HaCaT (human keratinocyte) cells. The cell viability assay was performed using standard MTT assay after 24 h post administration, and expressed as ‘percentage cell viability’. We observed that pure amyloid caused 78% and ~80% cell death in HaCaT and N2a cells, respectively, while amyloid (50 µM) formed with different ZnONP (amy@ZnONPuncap, amy@ZnONPST, and amy@ZnONPassmb) showed a cell death of 21%, 8%, and 29%, respectively in HaCaT cells and 24%, 12% and 22%, respectively in N2a cells (Fig. 9A & C) ), (see Fig. S8 for assay result at other concentrations in ‘SI’). Similar control assay was also performed in the same cells with 50 µM (optimized concentration) of pure three types of ZnONP, which showed fair level of toxicity in both the cells (Fig. 9A) except ZnONPST that results little (~ 9% in HaCaT and 16% in N2a cell death) cellular toxicity. The result clearly revealed that all three forms of ZnONP inhibited amyloid-mediated toxicity to different extent. Since ZnONPuncap and ZnONPassmb showed fair level of cellular toxicity (Fig. 9A & C), the toxicity produced by amy@ZnONPuncap and amy@ZnONPassmb must be from both NP as well as amyloid part. Since pure amyloid sample showed ~80 % cell death, our result clearly demonstrated the inhibitory effect by all three ZnONP towards amyloid growth while ZnONPST showed highest effect. The above results also support our earlier observations of reduced cross β-sheet amount and fibrillar bodies of protein amyloids produced with ZnONP (see Fig. 6A-C), which were perhaps the reasons of causing reduced level of toxicity. Bieschke et al. (2009) reported that mature α27



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synuclein and its fibrils are toxic to the PC12 cells while polyphenol (-)-epi-gallocatechine gallate (EGCG) treatment reduces the cellular toxicity by remodeling of fibrils and reducing βsheet formation.48 Therefore, here perhaps lack of formation of mature fibrils reduced cellular toxicity. Moreover, capping of ZnONP with starch showed a cellular toxicity to 9% and 16% for HaCaT and N2a cells, respectively. However, formation of starch mediated self-assembly by ZnONP (ZnONPassemb) could not mitigate the cellular toxicity below 26% for HaCaT and 24% for N2a. The reason was perhaps having the largest size (avg size is 163 nm) of all ZnONPs used in our study which reduces surface to volume ratio and hence, the surface activity towards amyloid inhibition as well as toxicity. Moreover, we already observed that amyloid sample possessed high surface hydrophobicity (see Fig. S7, ANS fluorescence plot in ‘SI’). The reason is the misfolding of the protein which exposes hydrophobic residues on its surface that further trigger aggregation through hydrophobic interaction. Here, in our present study, we observed that samples having lower amount of surface hydrophobicity (amy@ZnONPST, amy@ZnONPuncap, and amy@ZnONPassmb) showed a reduced ROS generation and cellular death (Fig. S8). In fact, Manniniet al. (2014) recently reported that cellular toxicity of amyloid sample varies with surface hydrophobicity of protein oligomers .49

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Figure 9. The effect of ZnONP (uncapped, starch capped, self-assembly), native lysozyme (HEWL), amyloid (Amy), amyloid formed in the presence of various ZnONP was analyzed on the cellular toxicity. Gallic acid was used as a positive control for ROS-based apoptotic death. Cell viability analysis was performed in (A) HaCaT cells (B) HaCaT cells in the presence of N-

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acetyl cysteine (5 mM), an ROS scavenger. (C) Mice neuroblastoma cell line (N2a) (D) N2a in the presence of N-acetyl cysteine. Both amyloid and ZnONP of 50 µM were used in this study. Multiple reports documented that the toxicity of amyloid to cells primarily mediated via ROS generation.50-52 Gharibyan et al. reported that lysozyme amyloid oligomers and fibers cause cellular toxicity in neuroblastoma SH-SY5Y by apoptotic or necrotic pathways. They concluded that not the single species but variety of cross β-sheet containing amyloid causes cytotoxicity.50 Further, Verma et al. concluded that the toxicity of amyloid caused by both oligomers and mature fibrils to a different extent.53 Some other reports also suggested that both oligomers and mature amyloid fibrils (highly stable) showed cellular toxicity in different cells. 54-57 Here, to estimate ROS generation by cells, we administered N-acetyl cysteine (NAC), a ROS inhibitor and gallic acid (ROS mediated apoptosis-inducing agent, a positive control) in culture.58 When, we administered NAC (5 mM) at 0 h in culture medium and cell viability assay was performed, pure amyloid demonstrated a cell death within 10% in both cells (Fig. 7B & D) while gallic acid showed only 5-6% cell death (Fig. 8B & D) after 24 h post administration. These results indicated that amyloid and gallic acid predominantly caused ROS mediated apoptotic cell death. However, when NAC was administered, all three ZnONP samples showed ignorable level of cellular toxicity in both cells indicated that the cell death by pure ZnONPs was caused by ROS generation (Fig. 8A, B, C, and D). Rest 10-15% cell death, which still occurred, was perhaps due to other kinds of cell death mechanism. Therefore, from our cell viability assay, we proved that HEWL amyloid was highly toxic to both N2a cells and HaCaT cells and such toxicity was predominantly caused due to ROS generation. However, when amyloid was produced in the presence of ZnONP, such toxicity was inhibited significantly while ZnONPST demonstrated highest inhibitory activity to cellular toxicity induced by amy@ ZnONPST (toxicity 30


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is only ~10%) (Fig. 9A & C). Such drop of toxicity was perhaps associated with the interaction of ZnONP with amyloid forming HEWL molecules, which further produced higher amount of fragmented fibers and loose aggregates rather than matured amyloid fibers. Therefore, from our collective results, we strongly anticipated that the interaction of proteins with NPs is the key behind the inhibition of HEWL amyloid fibrillation. While the surface properties of NPs are responsible for NP-protein interaction, the depletion of free proteins in solution also plays a crucial role in such inhibition process.47 Since the amyloid fibrillation process involves multiple steps such as misfolding, nucleation, oligomerization, pre-fibril formation and maturation of protein, there is a chance that during each step of such process, NPs might interact with protein species and trigger the termination of the process with regard to the protein interacted with NPs. Thus, overall probability of reaching the final matured fibril state by HEWL molecules is very less due to the continuous and random interaction of proteins with NPs. From here, we proposed a scheme to demonstrate how the interaction of three different forms of ZnONP with HEWL produced fibrillar amyloids and aggregates of different proportion (Fig. 10). Although three forms of ZnONP caused the change of amyloid structure by altering fiber thickness and length, ZnONPST was observed to reduce the β-sheet formation and toxicity in both cells (N2a, and HaCaT) maximally.

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Figure 10. The schematic diagram represents the mechanism of amyloid fibrillation process of HEWL in two different conditions, without and with NP: Scheme-I: HEWL amyloid was formed when protein sample was incubated at pH 2.2 and 580C. The fibrillation process results the formation of long amyloid fibers. Scheme-II: Here ZnONPST was added in the HEWL solution and incubated at pH 2.2 and 580C. During amyloid growth, protein present in two states: one is free protein (unbound) and second is NP-bound. In each step of the growth process, protein molecules bind with NPs and thus deplete their concentration in the solution. For ZnONPST, no matured fibrils as such were formed because critical amounts of unbound 32


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oligomers were not available in the solution due to exhaustive interaction of all proteins with NPs. 4. CONCLUSION Here, we synthesized ZnONP of three different forms and sizes, however with a core size of 1015 nm. We demonstrated that all three different forms of ZnO nanoparticles (ZnONPuncap, ZnONPassmb,

ZnONPST)

inhibited

HEWL

amyloid

fibrillation

by

producing

small

fibers/aggregates of varying size while starch capped ZnONP (ZnONPST) demonstrated the highest inhibitory effect.

We also concluded that such inhibition of amyloid growth was

associated with reduced β-sheet contents (Fig. 4A-C and Fig.5D). We further observed that HEWL amyloid was highly toxic to both mouse neuroblastoma N2a and human keratinocyte HaCaT cells and such toxicity was caused predominantly by ROS (reactive oxygen species) generation (Fig.9B & D). However, the incubation of HEWL with ZnONP demonstrated substantial reduction of amyloid toxicity to both kinds of cells with ZnONPST showed the highest inhibitory effect. We further concluded that such toxicity reduction was implicated with the inhibition of amyloid fibrillation. Moreover, we also concluded that administration of NPs in lag phase produced better inhibitory result than administration in elongation or maturation phases. To establish our conclusion that our synthesized ZnONP could inhibit HEWL amyloid fibrillar growth, we compared our results with a standard amyloid inhibitor curcumin (positive control) which demonstrated that ZnONPST was as effective amyloid inhibitor as curcumin (see Fig. S3 in SI). However, at lower concentration our synthesized ZnONPST (1-5 µM) exhibited higher inhibitory activity than curcumin.

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Since ZnONP (particularly starch-capped form) revealed inhibition to amyloid fibrillation of HEWL as well as amyloid-mediated cellular toxicity potential, it can be used for the design of nanoparticle-based future therapeutic strategy against multiple amyloid-related diseases. Although our anti-amyloid findings were observed in HEWL protein, our synthesized ZnONP might also be effective in the inhibition of other protein-mediated amyloid systems such as Aβ, insulin as well as α-synuclein.

ACKNOWLEDGEMENT The authors deeply thank the Department of Biotechnology, Government of India, for financial support (Grant No. BT/ PR13853/NNT/28/481/2010) and National Institute of Technology, Government of India, providing the research facility for carrying out this work. SUPPORTING INFORMATION FESEM and TEM image of ZnONPST and ZnONPassmb (Figure S1); The DLS analysis of amyloid samples (Figure S2); Amyloid growth analysis in presence of curcumin (Figure S3); Thioflavin-T analysis of amyloid samples (Figure S4); Circular dichroism spectral analysis of different amyloid samples (Figure S5); FESEM analysis of various amyloid samples and morphological changes due to variation in administration time (Figure S6); ANS fluorescence of various amyloid samples (Figure S7); The effect of different concentration of amyloid on N2a cells (Figure S8).

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REFERENCE 1.

Chaudhuri, T. K.; Paul, S., Protein‐Misfolding Diseases And Chaperone‐Based

Therapeutic Approaches. FEBS J.2006, 273 (7), 1331-1349. 2.

Glenner, G. G., Amyloid Deposits and Amyloidosis: (First of Two Parts). N. Engl. J. Med

1980,302 (23), 1283-1292. 3.

Stefani, M., Protein Misfolding and Aggregation: New Examples in Medicine and

Biology of the Dark Side of the Protein World. Biochim. Biophys. Acta, Mol. Basis Dis.2004,1739 (1), 5-25. 4.

Jahn, T. R.; Makin, O. S.; Morris, K. L.; Marshall, K. E.; Tian, P.; Sikorski, P.; Serpell,

L. C., The Common Architecture of Cross-β Amyloid. J. Mol. Biol. 2010, 395 (4), 717-727. 5.

Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C.,

Common Core Structure of Amyloid Fibrils by Synchrotron X-Ray Diffraction. J. Mol. Biol. 1997, 273 (3), 729-739. 6.

Gilgun-Sherki, Y.; Melamed, E.; Offen, D., Anti-Inflammatory Drugs in the Treatment of

Neurodegenerative Diseases: Current State. Curr. Pharm. Des. 2006, 12 (27), 3509-3519. 7.

Nowacek, A.; Kosloski, L. M.; Gendelman, H. E., Neurodegenerative Disorders and

Nanoformulated Drug Development. Nanomedicine (London, U.K.) 2009,4 (5), 541-555. 8.

Sciarretta, K. L.; Gordon, D. J.; Meredith, S. C., Peptide‐Based Inhibitors of Amyloid

Assembly. Methods Enzymol.2006,413, 273-312. 9.

Murphy, S. R.; Chang, C. C.; Dogbevia, G.; Bryleva, E. Y.; Bowen, Z.; Hasan, M. T.;

Chang, T.-Y., Acat1 Knockdown Gene Therapy Decreases Amyloid-β in a Mouse Model of Alzheimer’s Disease. Mol. Ther. 2013, 21 (8), 1497-1506.

35



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

10.

Page 36 of 43

Muchowski, P. J.; Schaffar, G.; Sittler, A.; Wanker, E. E.; Hayer-Hartl, M. K.; Hartl, F.

U., Hsp70 and Hsp40 Chaperones can Inhibit Self-Assembly of Polyglutamine Proteins into Amyloid-like Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (14), 7841-7846. 11.

Choudhary, S.; Kishore, N.; Hosur, R. V., Inhibition of Insulin Fibrillation by Osmolytes:

Mechanistic Insights. Sci. Rep. 2015, 5. 12.

Linse, S.; Cabaleiro-Lago, C.; Xue, W.-F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford,

S. E.; Dawson, K. A., Nucleation of Protein Fibrillation by Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (21), 8691-8696. 13.

Wu, W.-h.; Sun, X.; Yu, Y.-p.; Hu, J.; Zhao, L.; Liu, Q.; Zhao, Y.-f.; Li, Y.-m., Tio2

Nanoparticles Promote β-Amyloid Fibrillation in Vitro. Biochem. Biophys. Res. Commun. 2008, 373 (2), 315-318. 14.

Álvarez, Y. D.; Fauerbach, J. A.; Pellegrotti, J. s. V.; Jovin, T. M.; Jares-Erijman, E. A.;

Stefani, F. D., Influence of Gold Nanoparticles on the Kinetics of Α-Synuclein Aggregation. Nano Lett. 2013, 13 (12), 6156-6163. 15.

Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin,

E.; Walsh, D. M.; Dawson, K. A.; Linse, S., Inhibition of Amyloid β Protein Fibrillation by Polymeric Nanoparticles. J. Am. Chem. Soc. 2008, 130 (46), 15437-15443. 16.

Liao, Y. H.; Chang, Y. J.; Yoshiike, Y.; Chang, Y. C.; Chen, Y. R., Negatively Charged

Gold Nanoparticles Inhibit Alzheimer's Amyloid‐β Fibrillization, Induce Fibril Dissociation, and Mitigate Neurotoxicity. Small 2012, 8 (23), 3631-3639. 17.

Hsieh, S.; Chang, C.-w.; Chou, H.-h., Gold Nanoparticles as Amyloid-like Fibrillogenesis

Inhibitors. Colloids Surf., B 2013, 112, 525-529.

36


Page 37 of 43

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


18.

Palmal, S.; Maity, A. R.; Singh, B. K.; Basu, S.; Jana, N. R.; Jana, N. R., Inhibition of

Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles. Chem. - Eur. J. 2014, 20 (20), 6184-6191. 19.

Bellova, A.; Bystrenova, E.; Koneracka, M.; Kopcansky, P.; Valle, F.; Tomasovicova,

N.; Timko, M.; Bagelova, J.; Biscarini, F.; Gazova, Z., Effect of Fe3O4 Magnetic Nanoparticles on Lysozyme Amyloid Aggregation. Nanotechnology 2010, 21 (6), 065103. 20.

Ghavami, M.; Rezaei, M.; Ejtehadi, R.; Lotfi, M.; Shokrgozar, M. A.; Abd Emamy, B.;

Raush, J.; Mahmoudi, M., Physiological Temperature has a Crucial Role in Amyloid Beta in the Absence and Presence of Hydrophobic and Hydrophilic Nanoparticles. ACS Chem. Neurosci. 2012, 4 (3), 375-378. 21.

Li, M.; Zhao, A.; Dong, K.; Li, W.; Ren, J.; Qu, X., Chemically Exfoliated WS2

Nanosheets Efficiently Inhibit Amyloid β-Peptide Aggregation and can be Used for Photothermal Treatment of Alzheimer’s Disease. Nano Res. 2015, 8 (10), 3216-3227. 22.

Yu, X.; Wang, Q.; Lin, Y.; Zhao, J.; Zhao, C.; Zheng, J., Structure, Orientation, and

Surface Interaction of Alzheimer Amyloid-β Peptides on the Graphite. Langmuir 2012, 28 (16), 6595-6605. 23.

Anderson, D. S.; Patchin, E. S.; Silva, R. M.; Uyeminami, D. L.; Sharmah, A.; Guo, T.;

Das, G. K.; Brown, J. M.; Shannahan, J.; Gordon, T., Influence of Particle Size on Persistence and Clearance of Aerosolized Silver Nanoparticles in the Rat Lung. Toxicol. Sci. 2015, 144 (2), 366-381. 24.

Osmond-McLeod, M. J.; Osmond, R. I.; Oytam, Y.; McCall, M. J.; Feltis, B.; Mackay-

Sim, A.; Wood, S. A.; Cook, A. L., Surface Coatings of Zno Nanoparticles Mitigate

37



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 43

Differentially a Host of Transcriptional, Protein and Signalling Responses in Primary Human Olfactory Cells. Part. Fibre Toxicol. 2013, 10 (1), 1-18. 25.

Podila, R.; Brown, J. M., Toxicity of Engineered Nanomaterials: A Physicochemical

Perspective. J. Biochem. Mol. Toxicol. 2013, 27 (1), 50-55. 26.

Podila, R.; Chen, R.; Ke, P. C.; Brown, J.; Rao, A. M., Effects of Surface Functional

Groups on the Formation of Nanoparticle-Protein Corona. Appl. Phys. Lett. 2012, 101 (26), 263701. 27.

Fakhar-e-Alam, M.; Rahim, S.; Atif, M.; Aziz, M. H.; Malick, M. I.; Zaidi, S.; Suleman,

R.; Majid, A., ZnO Nanoparticles as Drug Delivery Agent for Photodynamic Therapy. Laser Phys. Lett. 2013, 11 (2), 025601. 28.

Xiong, H. M., ZnO Nanoparticles Applied to Bioimaging and Drug Delivery. Adv. Mater.

(Weinheim, Ger.) 2013, 25 (37), 5329-5335. 29.

Rasmussen, J. W.; Martinez, E.; Louka, P.; Wingett, D. G., Zinc Oxide Nanoparticles for

Selective Destruction of Tumor Cells and Potential for Drug Delivery Applications. Expert Opin. Drug Delivery2010, 7 (9), 1063-1077. 30.

Xia, T.; Kovochich, M.; Liong, M.; Mädler, L.; Gilbert, B.; Shi, H.; Yeh, J. I.; Zink, J. I.;

Nel, A. E., Comparison of the Mechanism of Toxicity of Zinc Oxide And Cerium Oxide Nanoparticles Based on Dissolution ond Oxidative Stress Properties. ACS Nano 2008, 2 (10), 2121-2134. 31.

Zamiri, R.; Zakaria, A.; Ahangar, H. A.; Darroudi, M.; Zak, A. K.; Drummen, G. P.,

Aqueous Starch as a Stabilizer in Zinc Oxide Nanoparticle Synthesis via Laser Ablation. J. Alloys Compd.2012, 516, 41-48.

38


Page 39 of 43

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


32.

Gomes, M.; Salgado, A.; Reis, R., Bone Tissue Engineering using Starch Based Scaffolds

Obtained by Different Methods. In Polymer Based Systems on Tissue Engineering, Replacement and Regeneration, Springer: 2002, 86, 221-249. 33.

Zhang, Y.; R Nayak, T.; Hong, H.; Cai, W., Biomedical Applications of Zinc Oxide

Nanomaterials. Curr. Mol. Med. 2013, 13 (10), 1633-1645. 34.

Zhou, J.; Xu, N. S.; Wang, Z. L., Dissolving Behavior and Stability of ZnO Wires in

Biofluids: A Study on Biodegradability and Biocompatibility Of ZnO Nanostructures. Adv. Mater. (Weinheim, Ger.) 2006, 18 (18), 2432-2435. 35.

Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E.;

Hawkins, P. N.; Dobson, C. M.; Radford, S. E.; Blake, C. C. F.; Pepys, M. B., Instability, Unfolding and Aggregation of Human Lysozyme Variants Underlying Amyloid Fibrillogenesis. Nature 1997, 385 (6619), 787-793. 36.

Granel, B.; Valleix, S.; Serratrice, J.; Chérin, P.; Texeira, A.; Disdier, P.; Weiller, P.-J.;

Grateau, G., Lysozyme Amyloidosis: Report of 4 Cases and A Review Of The Literature. Medicine 2006, 85 (1), 66-73. 37.

Pepys, M.; Hawkins, P.; Booth, D.; Vigushin, D.; Tennent, G.; Soutar, A.; Totty, N.;

Nguyen, O.; Blake, C.; Terry, C., Human Lysozyme Gene Mutations cause Hereditary Systemic Amyloidosis. Nature 1993, 362 (6420), 553-557. 38.

Swaminathan, R.; Ravi, V. K.; Kumar, S.; Kumar, M. V. S.; Chandra, N., Lysozyme: A

Model Protein for Amyloid Research. Adv. Protein. Chem Struct. Biol.2011, 84 (1), 63-111. 39.

Joshi, P.; Chakraborti, S.; Chakrabarti, P.; Haranath, D.; Shanker, V.; Ansari, Z.; Singh,

S. P.; Gupta, V., Role of Surface Adsorbed Anionic Species in Antibacterial Activity of ZnO Quantum Dots Against Escherichia coli. J. Nanosci. Nanotechnol. 2009, 9 (11), 6427-6433. 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

40.

Page 40 of 43

Ban, D. K.; Pratihar, S. K.; Paul, S., An Investigation of Optical Properties of Zinc Oxide

Nanoparticle Synthesized by Starch Mediated Assembly and Its Application in Photocatalytic Bleaching of Methyl Green and Rhodamine-B. Mater. Sci. Semicond. Process. 2015, 39, 691701. 41.

Wang, S. S.-S.; Liu, K.-N.; Wang, B.-W., Effects of Dithiothreitol on the Amyloid

Fibrillogenesis of Hen Egg-White Lysozyme. Eur. Biophys. J. 2010, 39 (8), 1229-1242. 42.

Wiedemann, C.; Bellstedt, P.; Görlach, M., CAPITO-A Web Server Based Analysis and

Plotting Tool for Circular Dichroism Data. Bioinformatics 2013, btt278. 43.

Riss, T. L.; Moravec, R. A.; Niles, A. L.; Benink, H. A.; Worzella, T. J.; Minor, L., Cell

Viability Assays. 2015. 44.

Biancalana, M.; Koide, S., Molecular Mechanism of Thioflavin-T Binding to Amyloid

Fibrils. Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804 (7), 1405-1412. 45.

Münch, C.; Bertolotti, A., Exposure of Hydrophobic Surfaces Initiates Aggregation of

Diverse ALS-Causing Superoxide Dismutase-1 Mutants. J. Mol. Biol. 2010, 399 (3-3), 512-525. 46.

Galvagnion, C.; Buell, A. K.; Meisl, G.; Michaels, T. C.; Vendruscolo, M.; Knowles, T.

P.; Dobson, C. M., Lipid Vesicles Trigger α-Synuclein Aggregation by Stimulating Primary Nucleation. Nat. Chem. Biol. 2015, 11 (3), 229-234. 47.

Radic, S.; Davis, T. P.; Ke, P. C.; Ding, F., Contrasting Effects of Nanoparticle–Protein

Attraction on Amyloid Aggregation. RSC Adv. 2015, 5 (127), 105489-105498. 48.

Bieschke, J.; Russ, J.; Friedrich, R. P.; Ehrnhoefer, D. E.; Wobst, H.; Neugebauer, K.;

Wanker, E. E., EGCG Remodels Mature α-Synuclein and Amyloid-β Fibrils and Reduces Cellular Toxicity. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (17), 7710-7715.

40


Page 41 of 43

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


49.

Mannini, B.; Mulvihill, E.; Sgromo, C.; Cascella, R.; Khodarahmi, R.; Ramazzotti, M.;

Dobson, C. M.; Cecchi, C.; Chiti, F., Toxicity of Protein Oligomers is Rationalized by a Function Combining Size and Surface Hydrophobicity. ACS Chem. Biol. 2014, 9 (10), 23092317. 50.

Gharibyan, A. L.; Zamotin, V.; Yanamandra, K.; Moskaleva, O. S.; Margulis, B. A.;

Kostanyan, I. A.; Morozova-Roche, L. A., Lysozyme Amyloid Oligomers and Fibrils Induce Cellular Death via Different Apoptotic/Necrotic Pathways. J. Mol. Biol. 2007, 365 (5), 13371349. 51.

Kadowaki, H.; Nishitoh, H.; Urano, F.; Sadamitsu, C.; Matsuzawa, A.; Takeda, K.;

Masutani, H.; Yodoi, J.; Urano, Y.; Nagano, T.; Ichijo, H., Amyloid [beta] induces Neuronal Cell Death through ROS-mediated ASK1 Activation. Cell Death Differ. 2005, 12 (1), 19-24. 52.

Mossuto, M. F.; Dhulesia, A.; Devlin, G.; Frare, E.; Kumita, J. R.; de Laureto, P. P.;

Dumoulin, M.; Fontana, A.; Dobson, C. M.; Salvatella, X., The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity. J. Mol. Biol. 2010, 402 (5), 783-796. 53.

Verma, M.; Vats, A.; Taneja, V., Toxic Species in Amyloid Disorders: Oligomers or

Mature Fibrils. Annals of Indian Academy of Neurology 2015, 18 (2), 138-145. 54.

Bucciantini, M.; Nosi, D.; Forzan, M.; Russo, E.; Calamai, M.; Pieri, L.; Formigli, L.;

Quercioli, F.; Soria, S.; Pavone, F.; Savistchenko, J.; Melki, R.; Stefani, M., Toxic Effects of Amyloid Fibrils on Cell Membranes: The Importance of Ganglioside GM1. The FASEB J.l 2012, 26 (2), 818-831. 55.

Dirix, C.; Meersman, F.; MacPhee, C. E.; Dobson, C. M.; Heremans, K., High

Hydrostatic Pressure Dissociates Early Aggregates of TTR105–115, but not the Mature Amyloid Fibrils. J. of Mol. Biol. 2005, 347 (5), 903-909. 41



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

56.

Page 42 of 43

Harte, N. P.; Klyubin, I.; McCarthy, E. K.; Min, S.; Garrahy, S. A.; Xie, Y.; Davey, G.

P.; Boland, J. J.; Rowan, M. J.; Mok, K. H., Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms. J. of Biol. Chem. 2015, 290 (47), 28343-28352. 57.

Meersman, F.; Dobson, C. M., Probing the Pressure–temperature Stability of Amyloid

Fibrils Provides New Insights into their Molecular Properties. Biochim. Biophys. Acta, Proteins and Proteomics 2006, 1764 (3), 452-460. 58.

Russell, L. H.; Mazzio, E.; Badisa, R. B.; Zhu, Z.-P.; Agharahimi, M.; Oriaku, E. T.;

Goodman, C. B., Autoxidation of Gallic Acid Induces ROS-Dependent Death in Human Prostate Cancer Lncap Cells. Anticancer Res. 2012, 32 (5), 1595-1602.

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TOC Graphic 910x474mm (96 x 96 DPI)


Nano Zinc Oxide Inhibits Fibrillar Growth and ... - ACS Publications

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