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Reduced Aggregation and Cytotoxicity of Amyloid Peptides by Graphene Oxide/Gold Nanocomposites Prepared by Pulsed Laser Ablation in Water Jingying Li, Qiusen Han, Xinhuan Wang, Ning Yu, Lin Yang, Rong Yang,* and Chen Wang*

A novel and convenient method to synthesize the nanocomposites combining graphene oxides (GO) with gold nanoparticles (AuNPs) is reported and their applications to modulate amyloid peptide aggregation are demonstrated. The nanocomposites produced by pulsed laser ablation (PLA) in water show good biocompatibility and solubility. The reduced aggregation of amyloid peptides by the nanocomposites is confirmed by Thioflavin T fluorescence and atomic force microscopy. The cell viability experiments reveals that the presence of the nanocomposites can significantly reduce the cytotoxicity of the amyloid peptides. Furthermore, the depolymerization of peptide fibrils and inhibition of their cellular cytotoxicity by GO/AuNPs is also observed. These observations suggest that the nanocomposites combining GO and AuNPs have a great potential for designing new therapeutic agents and are promising for future treatment of amyloid-related diseases.

1. Introduction Neurodegenerative disorders have been studied for decades, including Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease and prion diseases. These fatal diseases were caused by protein misfolding and followed abnormal

J. Li, Q. Han, X. Wang, L. Yang, Prof. R. Yang, Prof. C. Wang CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology Beijing 100190, (China) E-mail: [email protected]; [email protected] X. Wang Department of Chemistry Yangzhou University Yangzhou 225009, China Dr. N. Yu Chinese PLA General Hospital Beijing 100853, China DOI: 10.1002/smll.201401121

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aggregation.[1–5] A lot of attention has been devoted to reduce even eliminate the toxicity induced by protein disorders. The most common neurodegenerative disease, Alzheimer’s disease constitutes about two thirds of cases of dementia overall.[6] One of the pathological hallmarks of AD is accumulation of insoluble amyloid “plaques” in the brain containing a 40 to 42-residue peptide called β-amyloid (Aβ). Aβ is assembled into fibrillar β-amyloid due to intracellular β-amyloid precursor protein (APP) in the neuronal membrane by the secretase complex.[3,7] The inhibitors of Aβ peptide fibrillization have been developed from peptides,[8] antibodies,[9] molecules[10] to nanomaterials.[11,12] The advantage of nanomaterials is mainly focused on the high surface ratio and facilitation of multifunctions. Developing nanoparticles as drugs against amyloid related diseases provided a possibility to overcome the limitation of blood-brain barrier (BBB). Besides, some nanoparticles have been proved excellent biocompatibility and low toxicity. Gold nanoparticles (AuNPs) were used for a long time to detect the Aβ fibrillation[13] or screen the amyloid inhibitors.[14] In recent years, AuNPs with different surface modifications were reported to be efficiently inhibit

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amyloid fibrillization.[15,16] Carbon materials were also demonstrated to have the potential to inhibit amyloid fibrillization and avoid the cytotoxic effects.[17–19] As a novel nanocarbon material, graphene[20a] has received much attention in recent years in the fields of materials science and biotechnology. Significant progress has been made for the utilization of graphene in nanoelectronics, nanocomposites, biosensors, and drug delivery.[20a–22] Compared with graphene, graphene oxide (GO) possessed higher stability and dissolubility; therefore, it was more suitable for treatment. Mahmoudi et al. have proved that GO was efficient in reducing aggregation and toxicity of Aβ(1–42).[17] Qu group utilized the near-infrared (NIR) adsorption properties of GO and combined with an Aβ fibril staining dye (thioflavin-S) then dissociated amyloid aggregation with NIR laser irradiation.[23] In previous studies, researchers reported that the hybrid materials combining GO and other nanoparticles (such as TiO2, Au) have improved photocatalytic and optical properties than GO itself.[24] However, combined GO with AuNPs to act as regulators against amyloid peptide fibrillation have not been studied. The AuNPs in the nanocomposite could play an important role for further potential investigation, such as utilizing its photo-thermal affect to depolymerize Aβ fibrils or further conjugating target peptides to disturb the aggregation. In this work, we report the greatly reduced aggregation and cytotoxicity of Aβ(1–42) peptide induced by the GO/Au nanocomposites. The nanocomposites were synthesized using pulsed laser ablation (PLA) in water. Previously we have prepared inorganic fullerene-like MoS2 nanoparticles by PLA method and shown that this technique provides a green and convenient method to synthesize novel biocompatible nanomaterials.[25] PLA in liquid is a relatively new method to produce nanocomposites. Comparing with chemical methods, PLA in water has unique properties: 1) The nanomaterials prepared have high water solubility; 2) Procedure is relatively convenient; 3) The nanomaterials have less chemical remnants from the chemical reactions. The GO/Au nanocomposites were synthesized and characterized with transmission electron microscopy (TEM) and UV–vis spectroscopy. The second structures of Aβ(1–42) in the presence and absence of GO/Au nanocomposites were studied by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). Cell viability of SH-SY5Y cells (neuroblastoma cell) was detected by MTT (3-[4,5-dimethyl-2-thiazoyl]-2,5-diphenyltetrazolium bromide) assay. Tyrosine fluorescence and ANS (8-anilino-1-naphthalenesulfonic acid) fluorescence were also carried out to verify the possible mechanism for the reduced aggregation of Aβ(1–42) peptide by the GO/Au nanocomposites in view of the hydrophobic interactions between of the nanoconposites with hydrophobic amino acids of Aβ(1–42). The results showed the enhanced effect of the GO/Au nanocomposites than GO on reducing Aβ(1–42) aggregation and cytotoxicity. Further more, the deploymerization of Aβ(1–42) fibrils and inhibition of their cellular cytotoxicity was also observed. small 2014, 10, No. 21, 4386–4394

Figure 1. Schematic illustration of synthesis of the GO/Au nanocomposites by PLA method; B) TEM image of GO/AuNPs; C) UV–vis spectrum of the GO/Au nanocomposites. (Inset of Figure 1C is a zoom-in TEM image of GO/AuNPs). C,D) XPS spectra of GO/AuNPs.

2. Results and Discussion 2.1. Synthesis of GO/Au Nanocomposites In our study, we utilized a simple and convenient laser ablation technique to prepare GO/Au nanocomposites. GO/ metal hybrid materials can be produced by chemical reduction methods. However, traditional chemical methods using a reducing agent usually result in some chemical remnants in the products.[25b] PLA in water can have a clean and wellcontrolled condition. A schematic illustration of the synthesis is shown in Figure 1A. The applied laser-based approach to nanoparticles consists in the ablation of a gold plate in GO solutions by intense laser radiation, leading to an ejection of its constituent and the formation of a GO/Au nanocomposite solution. Figure 1B is the TEM image of the sample showing the particle distribution of AuNPs on GO. Figure 1C is the UV-vis spectrum of GO/AuNPs. The peak at 230 nm was due to GO adsorption. Meanwhile, another adsorption peak at 523 nm was attributed to AuNPs. The inset of Figure 1C is zoom-in image of the TEM shown in Figure 1B. The size of AuNPs was smaller than 10 nm. The spectroscopy and morphology characterization confirmed that the quite uniform dispersion of AuNPs on GO sheets was obtained. Raman spectra of GO/AuNPs (Figure S1, Supporting Information) indicated that GO sheets were mulilayers.[26]

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2.2. GO/Au Nanocomposites Inhibit Aβ(1–42) Fibrillation The formation of Aβ(1–42) aggregates was studied with and without GO/Au nanocomposites using the thioflavin T (ThT) binding assay. ThT is a dye which can be specific binding with β-sheet structure and generate fluorescence at 485 nm while excited at 440 nm.[28] Figure 2A shows the fibrillization processes for Aβ(1–42) alone and with GO (20 mg L−1) or GO/AuNPs (20 mg L−1). When the nanomaterials were introduced, the fluorescence intensity at saturation decreased indicating the inhibition of fibrillation. Interestingly, adding the same amount of nanomaterials, the inhibition effect of GO/AuNPs was more pronounced than that of GO solution. We also compared ThT fluorescence signals of Aβ(1–42) in presence of AuNPs and GO/AuNPs at the same concentration (5 mg L−1). The results (Figure S3, Supporting Information) showed that the nanocomposites inhibit aggregation of Aβ(1–42) more effectively than AuNPs alone. The modulation effect on fibrillization was also dependent on the concentration of nanocomposites. In our experiments, three concentrations of GO/AuNPs (5, 10, 20 mg L−1) were used. As we increased the concentration, the inhibitory effect enhanced due to the larger surface areas presented in the solution (Figure 2B). The fibrillation of Aβ(1–42) follows a typical nucleatedgrowth mechanism which can be described by a lag phase, a rapid exponential growth, and a final equilibrium state. The experimental curves were fitted by a sigmoidal empirical equation: y = y0 +

ymax − y0 1 + e −(t − t 1/2 )k

(1)

where y represents the fluorescence intensity value at time t, ymax, and y0 are the maximum and initial fluorescence intensity values, respectively; t1/2 is the time when fluorescence intensity reaches at half maximum value; k is the first-order aggregation constant.[29] The lag time is the time which is required to form critical nuclei that lead to the fibril formation. The lag time is described as[29] lag time = t 1/2 − 2 / k

Figure 2. Kinetics of Aβ(1–42) fibrillation. A) ThT fluorescence signal of Aβ(1–42) in absence and presence of GO and GO/AuNPs (10 mg L−1). B) ThT fluorescence signal of Aβ(1–42) with GO/AuNPs (concentrations of 0, 10, and 20 mg L−1). C) Lag time versus concentration of GO/AuNPs from the experiments shown in Figure 2B.

X-ray photon spectroscopy (XPS) was applied to investigate the chemical state of GO/Au nanocomposites. Figure 1C,D are XPS spectra of GO/AuNPs. Both show clearly Au 4f signals which correspond to the binding energy of Au and suggest that AuNPs have been effectively assembled on the surface of GO. C1s XPS spectrum of GO/ AuNP nanocomposites is shown in Figure S2 (Supporting Information).[27]

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(2)

Figure 2C shows the lag time as a function of the GO/ AuNPs concentration. One can clearly see that as the concentration of GO/AuNPs increases, the lag time for fibrillization increases as well. The lag phase was extended from 77.0 min for the peptide alone to 171.6, 255.6, and 311.8 min for GO/AuNPs concentrations of 5, 10, and 20 mg L−1, respectively. This indicated that the nucleation process of Aβ(1–42) was strongly disturbed by the presence of the GO/Au nanocomposites and the retardation of fibrillation depended on the surface area of the nanocomposites. Since the fibrillation followed the nucleation-elongation process, the increase of lag time and reduce of the plateau intensity in ThT fluorescence indicated a lower amount of fibrils formation. It could be due to the high surface areas of GO/ AuNPs which adsorbed Aβ(1–42) monomers or oligomers rapidly and strongly to prevent them from aggregating into fibrils.[12b,c]

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Figure 3. AFM images of Aβ(1–42) fibrils A) alone, B) in presence of GO, and C) GO/Au nanocomposites with concentration of 10 mg L−1.

The inhibition effect of GO/AuNPs on the Aβ(1–42) fibrillation was confirmed by AFM experiments. Figure 3 shows the typical AFM images without and with nanomaterials. AFM images showed difference in morphology and length of the fibrils. Without the presence of nanomaterials, the Aβ(1–42) aggregated into an extensive network of amyloid fibrils after 7-days’s incubation (Figure 3A). On the other hand, in the sample with GO (20 mg L−1), no mature fibrils were observed as shown in Figure 3B. With the presence of GO/Au nanocomposites (20 mg L−1), the fibrils became further shorter and less. The results indicate that the inhibition is more profound with GO/AuNPs, which is in agreement with ThT results shown in Figure 2. The modulation effect of nanocomposites with different concentrations was also studied. As the GO/AuNPs concentration increased, the inhibitory effect enhanced (Figure S4, Supporting Information).

2.3. The Interactions of GO/AuNPs with Aβ(1–42) The interactions of GO/Au nanocomposites with Aβ(1–42) and the inhibition processes were further studied by several complementary spectroscopy techniques. Tyrosine fluorescence is an intrinsic probe of Aβ(1–42).[30] We examined the tyrosine fluorescence spectra for the three samples in the absence or presence of nanomaterials. The results (Figure 4A) showed a notable quenching of the tyrosine fluorescence signal at 309 nm when Aβ(1–42) was mixed with GO or GO/ AuNPs. The quenched intensity with GO was about 50% compared to the intensity of Aβ(1–42) itself, meanwhile the fluorescence intensity with GO/AuNPs further decreased to one-forth of that of Aβ(1–42) alone. The results indicated that the GO/AuNPs could adsorb and bind the peptide to quench the tyrosine fluorescence signal. This phenomenon happened could be due to the FRET (Fluorescence resonance energy transfer) mechanism between the donor (tyrosine residue) and the acceptor (GO or GO/AuNPs). This was an indirect evidence that Aβ(1–42) adsorbed on GO or GO/AuNPs, which was also observed in the AFM image (Figure S5, Supporting Information). We further studied the influence of different concentrations of GO or GO/Au nanocomposites on Aβ(1–42) fibrillation, the signal of fluorescence declined with the increasing concentration of the nanomaterials (Figure S6, Supporting Information). small 2014, 10, No. 21, 4386–4394

The mixing of the amyloid peptides with GO or GO/ AuNPs may result in peptide conformation changes. To verify this we examined the secondary structure of the Aβ(1–42) with and without nanomaterials by circular dichroism (CD) spectroscopy (Figure 4B). Aβ(1–42) displayed a distinct spectrum with a maximum around 203 nm and a minimum at 217 nm as expected for β-sheets.[31–35] After incubated with GO, the intensity of negative peak at 217 nm became weaker, indicating the decrease of β-sheets. With the presence of GO/ Au composites, the intensity of the negative peak around 215 nm corresponding to β-sheet packing decreased and a additional negative peak at 222 nm corresponding to α-helix appeared.[34] Both of Tyrosine fluorescence and CD analysis indicated that there are strong interactions between GO/Au composites and Aβ(1–42) peptides. The fluorescent dye molecule ANS (l-anilinonaphthalene8-sulphonate) is a utilized “hydrophobic probe” that recognizes hydrophobic sites in proteins and peptides.[36] The emission spectrum of ANS is sensitive to the polarity of its environment. An increase in quantum yield and blue shift of the emission maximum is an indicative of hydrophobic environments.[37] ANS binding was used to detect changes in exposed hydrophobicity in Aβ(1–42) with or without nanocomposites (Figure 4C). Aβ(1–42) alone showed high ANS binding. Comparing with the fluorescence spectrum of ANS itself, the high emission peak of Aβ(1–42) shifted from 521 nm to 492 nm and indicated the high hydrophobicity of Aβ(1–42) fibrils. The intensities of ANS fluorescence of the Aβ(1–42) with GO or GO/ AuNPs decreased to one half or one tenth compared to that of Aβ(1–42) alone. The less blue-shift and declined fluorenscence indicated that Aβ(1–42) formed aggregates with lower hydrophobicity after regulated by GO or GO/AuNPs. On the other hand, this effect maybe also caused by GO or GO/AuNPs combined with the hydrophobic parts of Aβ (1–42). The phenomenon was also concentration dependent. The emission peak of ANS centered at 492–496 nm became weaker along with the higher concentrations of the nanocomposites (Figure S7, Supporting Information). To explain the reducing aggregation effects of the nanomaterials, we suppose that peptide adsorption on GO or GO/ AuNPs can lead to decrease concentration of the free Aβ(1–42) in solution and reduce amyloid aggregation. Moreover, the graphene oxides and AuNPs of the nanocomposites can bind to active sites of peptides separately and then block the formation of the fibrillogenesis.

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to compare the neurotoxicity of Aβ(1–42) in the absence or presence of the GO/Au nanocomposites. First, we studied cytotoxicity[39] of the GO/AuNPs to SH-SY5Y cells. The results were shown in Figure 5A. The cell viability data showed that there were about 91% cells alive after treated with 5 mg L−1 GO/AuNPs for 48 h. We also studied reactive oxygen species (ROS) level of GO/AuNPs. Figure 5B showed that the ROS level of GO/Au nanocomposites to SH-SY5Y cells was concentration-dependent. Higher the concentration of GO/AuNPs is, higher the ROS level is. In our study on modulation of Aβ(1–42) cytotoxicity to SH-SY5Y cells by GO/AuNPs, we will choose the concentration of GO/AuNPs at 5 mg L−1, in which case the ROS level of the nanocomposites is not high and the nanocomposites still have good biocompatibility to the SH-SY5Y cells. Second, we checked the genotoxicity of GO/Au nanocomposites using the comet assay. No DNA tails could be observed after exposure to 5 mg L−1 GO/AuNPs (Figure S8, Supporting Information). It indicated that GO/AuNPs exhibited little DNA damages to SH-SY5Y cells. Next we studied the cytotoxicity of Aβ(1–42) to SH-SY5Y cells with and without treatment of GO/AuNPs at concentration of 5 mg L−1.

Figure 4. A) Tyrosine fluorescence signal of Aβ(1–42) was quenched in the presence of GO or GO/AuNPs. B) CD spectra of Aβ(1–42) alone, with GO or GO/AuNPs. C) ANS fluorescence data for Aβ(1–42) alone and with GO or GO/AuNPs.

2.4. Reduced Aβ(1–42) Cytotoxicity to SH-SY5Y Cells by GO/AuNPs It is reported that the cytotoxicity of amyloid peptides could be reduced with the decrease of peptide aggregates.[38] Here we used neuroblastoma SH-SY5Y cells as a model system

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Figure 5. A) Cell viability of SH-SY5Y cells after incubating with GO/AuNPs at different concentrations; B) ROS level of GO/Au nanocomposites to SH-SY5Y cells.

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Figure 6. Cell viability of SH-SY5Y cells in the present of Aβ(1–42) alone, and in the present of Aβ(1–42) with GO (5 mg L−1) or GO/ AuNPs (5 mg L−1). Results are mean ± SD (n = 3). Statistical differences compared with the controls are given as *, P < 0.05 and **, P < 0.01.

From Figure 6 one can see that Aβ(1–42) is toxic to SH-SY5Y cells. The cell viability treated with Aβ(1–42) is markedly decreased with increasing peptide concentration from 2 to 40 µm. When 5 mg L−1 of GO or GO/AuNPs were added to Aβ peptides, the cytotoxicity of the peptides was greatly reduced. At higher concentration of Aβ(1–42), the GO/AuNPs show more significant cell protecting effect than GO. For example, the cell viability was about 45% with 40 µm of peptides, which was increased to 65% by GO and to nearly 90% by GO/AuNPs. The results further proved that those small peptide aggregates produced after addition of nanomaterials possess lower damage to neural cells.

2.5. Deploymerization of Aβ (1–42) Fibrils and Reduced Cytotoxicity by GO/AuNPs For the practical use, it is also important to know whether the modulator can deploymerize the amyloid fibrils. So, next we investigated the effects of GO and GO/AuNPs on the deploymerization of Aβ(1–42) fibrils by ThT assay (Figure 7). The fibrils were first prepared by incubating 20 µm of Aβ monomers in room temperature with continuous shaking for 48 h. The peptide fibrils were then mixed with GO or GO/ Au nanocomposites at 20 mg L−1 for 8 h. The lowering of ThT fluorescence intensity in Figure 7 indicated a decrease of Aβ(1–42) aggregates. The results indicated that GO or GO/ Au nanocomposites also affect the properties of Aβ(1–42) fibrils. The presence of GO or GO/AuNPs leads to depolymerization of amyloid aggregates and also yields a concentration-dependent decrease of ThT fluorescence intensity (Figure S9, Supporting Information). The ability of GO and GO/Au nanocomposites to reduce the peptide fibrils could be caused by the interactions between GO or GO/AuNPs and hydrophobic residues at the surface of the fibrils and then interrupt the interface between beta-sheet structures of the peptides. This needs to be further investigated. small 2014, 10, No. 21, 4386–4394

Figure 7. Depolymerization of Aβ(1–42) fibrils induced by 20 mg L−1 GO or GO/AuNPs detected by ThT assay.

Since GO/AuNPs can cause depolymerization of amyloid aggregates, the GO nanocomposites may be used to inhibit the cellular cytotoxicity mediated by Aβ(1–42) fibrils. We used SH-SY5Y cells to study the effect of GO/AuNPs on the cell toxicity of Aβ(1–42) fibrils. Figure 8 shows that Aβ(1–42) fibrils lead to a decrease of 50% in cell numbers. Aβ(1–42) fibrils treated with GO (8 mg L−1) for 48 h increased the cell viability to about 63%. While, treatment of the cells with Aβ(1–42) fibrils in the presence of GO/AuNPs (8 mg L−1) for 48 h increased the survival of the cells to about 84%. The results show that GO/Au nanocomposites are effective to depolymerize the existing Aβ(1–42) fibrils and reduced their cell toxicity.

3. Conclusion In conclusion, we have accomplished a novel and convenient method to synthesize the GO/Au nanocomposites and demonstrated their applications to modulate amyloid peptide aggregation. The nanocomposites produced by

Figure 8. Effect of GO (8 mg L−1) or GO/AuNPs (8 mg L−1) on the cell toxicity of Aβ(1–42) fibrils.

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PLA in water show good biocompatibility and solubility. We believe that this green and convenient technique of PLA in water can be readily extended to the synthesis of other biocompatible nanocomposites which can be used in various biomedical applications. The ThT fluorescence and AFM experiments demonstrated the efficient inhibition of Aβ(1–42) fibrillation by GO/AuNPs. Moreover, quenching of tyrosine signal and ANS fluorescence was observed in the presence of GO or GO/AuNPs, which indicated an interaction of the nanocomposites with Aβ(1–42). The cell viability experiments revealed that the presence of the nanocomposites could significantly reduce the cytotoxicity of Aβ(1–42). Compared with GO alone, the treatment with the nanocomposites demonstrated a higher inhibition efficacy for Aβ(1–42) aggregation and cytotoxicity. Furthermore, the depolymerization of Aβ(1–42) fibrils and inhibition of their cellular cytotoxicity by GO/AuNPs was also observed. Taken together, our results demonstrate the viability of utilizing GO/Au nanocomposites as the modulator for regulating amyloid-β aggregation and depolymerization, which may have potential applications in future treatment of amyloidrelated diseases.

4. Experimental Section Materials: Aβ(1–42) (purity 98%) was purchased from Shanghai Science Peptide Biological Technology Co. Ltd (China). Hexafluorisopropanol was obtained from J&K Scientific Ltd (China). Trifluoroethanol was purchased from Acros (Belgium). Dimethyl sulfoxide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Amresco (USA). DMEM medium and fetal bovine serums for SH-SY5Y cells culture were purchased from HyClone (USA) and Gibico (USA), respectively. Preparation of GO/Au Composites: GO was synthesized from natural graphite by a modified Hummers method. The GO/Au nanocomposites were prepared by a pulsed laser ablation (PLA) in water. A gold plate (>99.99%) was placed at the bottom of a centrifuge tube filled with 14 mL GO solution at concentration of 40 mg L−1. A Nd:YAG pulsed laser (532 nm, 10 Hz, pulse duration between 6–9 ns and pulse energy of 100 mJ), was focused on the plate for ablation. The ablation was typically done for duration of 10 min. During the course of the ablation, the colloidal solution was continuously stirred by an ultrasonicator. Raman spectra of GO and GO/AuNPs were collected with 534 nm excitation wavelength (Renishaw). The chemical states of GO/AuNPs were examined by X-ray photoelectron spectroscopy (XPS) using XPS spectrometer (ESCALAB 250Xi). Peptide Solution: Aβ(1–42) was dissolved at a concentration of 1 mg mL–1 in hexafluorisopropanol (HFIP) and incubated at room temperature overnight and stored at –20 °C. The HFIP was evaporated under vacuum before using and the peptide was resuspended with or without GO/Au composite in 50% trifluoroethanol for CD spectroscopy or 1% DMSO for other experiments. Amyloid fibril formation of Aβ(1–42) in the presence and absence of GO/Au composites was investigated by incubation of Aβ at 37 °C for 7 days in a shaker (THZ-D, Huamei). The Aβ (1–42) concentration was 20 µM. ThT Fluorescence Assay: To investigate the effects of GO/Au composite on fibrillation, Thioflavin T (ThT) was used to study the

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process of fibrillation. 20 µL aliquots from each sample were taken at different time points and added to a black 96-well plate, and then 180 µL ThT (10 µM) was added. The ThT fluorescence was measured at 485 nm with excitation at 440 nm using a microplate reader. FTIR and Circular Dichroism (CD) Spectra: FTIR and CD were used to characterize the secondary structure of Aβ (1–42). The solutions were dropped onto the CaF2 surface, followed by air drying prior to FTIR measurements. FTIR spectra were recorded on a PerkinElmer Fourier transform infrared spectrometer at a resolution of 4 cm−1. FTIR spectroscopic measurements were performed in the transmission mode with CaF2 windows. Peptides used for CD experiment were dissolved in 1:1 (v/v) mixture of trifluoroethanol and deionized water. CD spectra of the peptides were recorded on a Jasco-J810 sepctropolarimeter (Jasco, Japan) and collected spectra between 190 nm and 260 nm. Fluorescence Emission Spectra: Fluorescence spectra were collected using a Hitachi FP-4500 fluorescence spectrophotometer. An excitation wavelength of 280 nm (slit width = 5 nm) was used and data were collected over 290–350 nm (slit width = 5 nm). Samples were placed in a four-sided quartz fluorescence cell and data were recorded at room temperature. Besides, ANS-Na fluorescent probe was used to assess changes in hydrophobicity upon interaction of GO/Au composites with Aβ(1–42). An excitation wavelength of 400 nm (slit width = 5 nm) was used and data were collected over 450–650nm (slit width = 10 nm). The fluorescence emission intensity was measured in 200 µL samples titrated with 3 µL aliquots of a 10 mM ANS solution with 1 min of stirring after each addition. Atomic Force Microscopy: Morphology of the peptide aggregation on mica surfaces were characterized by AFM. AFM sample was applied to freshly cleaved mica and dried by N2. AFM experiments were performed under ambient condition using silicon cantilevers in tapping mode on a Dimension 3100 system (Bruker Nano, USA). MTT Cytotoxicity Assay: SH-SY5Y cells were cultured in 1640 supplemented with 15% fetal bovine serum, 100 U mL–1 penicillin and 100 mg L-1 streptomycin. Cells were kept at 37 °C in an incubator with 5% CO2. The cell viability was tested using MTT assay, which is based on the mitochondrial conversion of tetrazolium salt. SH-SY5Y cells at a density of 104 cells/well were seeded in a 96-well microplate (Costar, Corning, NY) in 100 µL 1640 medium containing 15% FBS. After attachment for 24 h, the cells were incubated with Aβ(1–42) or with Aβ(1–42) fibrils in the presence or absence of GO/Au nanocomposites for another 48 h in a final volume containing 150 µL medium. After that, 10 µL of MTT (5 mg mL−1 in PBS) buffer were added to each well, and the culture plates were incubated at 37 °C and 5% CO2 for 4 h. After removal of the medium, 150 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the dye. The absorbance at 492 nm was measured using a microplate reader. Each data point was derived from three parallel samples. Reactive Oxygen Species (ROS) Test: Cells were seeded into 48-well plates and cultured overnight, then loaded 100 µM ROS probe (DCFH-DA) for 2 h. Then, they were washed by PBS three times and cultured with the different concentrations of the GO/Au nanocomposites for an additional 8 h. The fluorescence intensity of the cells was measured. Comet Assay: The comet assay was reported to detect DNA migration resulted in DNA fragmentation. At first, the microscope

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slides were dipped in a molten regular agarose and allowed to curing at 4 °C. Then, cells were suspended in low-melting point (LMP) agarose and added to the first layer and cured at the same condition. The prepared slides were placed in a lysis solution containing 2.5 M NaCl, 100 mM EDTA, at pH 10.0 for 1.5 h. In addition, 1% triton X-100 was added just prior to use. Before the electrophoresis, the slides were incubated in an alkaline (pH > 13) electrophoresis buffer for 20 min to produce single-stranded DNA (ss-DNA). The alkaline solution contained 1 mM EDTA and 300 mM NaOH. Then, the ss-DNA in the gels was electrophoresed at 1.0 V cm−1 for 20 min under the same alkaline condition to produce comets. After the electrophoresis, the alkali in the gels was neutralized for 5 min. The DAPI utilized for comet visualization and fluorescence image was also captured by confocal microscope (Carl Zeiss LSM710). Statistical Analysis: Unless otherwise indicated, results are represented as the mean ± SD of five replicates, and at least three separate experiments was performed. Student’s t-test was used for two groups. P < 0.05 was accepted as statistically significant.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements J.L. and Q.H. contributed equally to this work. This work was supported by National Natural Science Foundation of China (21261130090, 21073047, and 91127043). Financial supports from Chinese Academy of Sciences (XDA09030303) and CAS Key Laboratory of Nano Bioeffect and Biosafety are also gratefully acknowledged.

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Received: April 22, 2014 Revised: June 18, 2014 Published online: July 24, 2014

small 2014, 10, No. 21, 4386–4394

gold nanocomposites prepared by pulsed laser ablation in water.

A novel and convenient method to synthesize the nanocomposites combining graphene oxides (GO) with gold nanoparticles (AuNPs) is reported and their ap...
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