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Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1–40) fibrillogenesis
Biomaterials 33 (2012) 4443e4450
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Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1e40) fibrillogenesis Ho-Man Chan a, Lehui Xiao a, Kai-Ming Yeung a, See-Lok Ho a, Dan Zhao a, Wing-Hong Chan a, Hung-Wing Li a, b, * a b
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China Centre for Surface Analysis and Research, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Accepted 6 March 2012 Available online 27 March 2012
The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1e40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Ab1e40 peptides under seedmediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Ab1e40 fibrillogenesis. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Nanoparticle Self-assembly Peptide Surface modification
1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder primarily found in elderly. The formation of self-assembling neurotoxic fibrous beta-amyloid (Ab) plaques in brain is believed as a pathological hallmark of AD [1e3]. Controlling the nucleation/ elongation-growth processes of monomeric Ab peptides into insoluble oligomers, protofibrils and fibrils are reckoned as a potential therapy to AD [4,5]. While nanomaterials are now widely designed and applied as drug delivering vehicles for therapeutics and diagnostics purposes [6], precautionary selection of nanomaterials is highly recommended as the vehicle itself maybe cytotoxic in nature otherwise aggravated the conditions. Several recent studies have provided insight that nanomaterials of various sizes, shapes, compositions and functionalities may significantly intervene with the self-assembling mechanism of amyloid peptides, drastically either promoting or inhibiting the fibrillogenesis [7e14]. For instance, Wu et al. reported that 20 nm TiO2 nanoparticles could promote the growth of Ab by shortening the nucleation process [12]. Cabaleiro-Lago et al. demonstrated that co-polymeric NIPAM:BAM nanoparticles of 40 nm with different hydrophobicities inhibited the growth of Ab fibrils by adsorbing the
* Corresponding author. Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China. Tel.: þ852 3411 7065; fax: þ852 3411 7348. E-mail address: [email protected] (H.-W. Li). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.03.024
monomers onto the particle surface and thus depleting the solution monomer concentration [9]. They have also reported that cationic amino-modified polystyrene nanoparticles of 57e180 nm induced both acceleration and retardation effects on Ab fibrillation based on the concentration and coverage of peptide monomers on the particle surfaces [8]. More recently, Yoo and coworkers have presented that functionalized quantum dots (QDs) non-specifically interacted with the Ab monomers, interrupting the nucleation process and consequently inhibiting the growth of Ab fibrils [14]. Also, a recent paper from Majzik and coworkers has drawn to our attention that the covalent interaction between the gold nanoparticles and the cysteine-modified beta-amyloid peptides hindered the formation of the polypeptide chain structure [15]. There were also numerous reports that drawn our attention describing the roles of nanomaterials on the controlled fibrillation of other self-assembling proteins and peptides, such as human serum albumin (HSA) [11], islet amyloid polypeptide (IAPP) [7], and human b2-microglobulin (b2m) [10]. In view of the reported results, it is believed that surface areas, compositions and functionalities of nanoparticles play significant regulatory role on controlling the Ab fibrillation process and worth in depth investigations. Accordingly, the behind mechanism of interactions between nanoparticles and peptide monomers were still not clearly understood. Researchers proposed that particles acted to reduce the rate of nucleation, yet the elongation phase were unaffected once critical nuclei were formed [9]. On the contrary, our previous work illustrated that N-acetyl-L-cysteine-capped quantum dots
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(NAC-QDs) with hydrodynamic diameter of ca. 3 nm could effectively quench both nucleation and elongation process of the Ab fibrillogenesis at any time points in the seed-mediated growth of beta amyloid (1e40) (Ab1e40) by blocking active sites of the seed fibrils or monomers [13]. The inhibitory effect is concentration dependent, and a remarkable inhibition towards 50 mM of Ab peptide was observed when the dosage of NAC-QDs increased from 109 to 107 M. This preliminary result showed that nanomaterials not only regulate the nucleation but also the elongation phase of beta-amyloid fibrillogenesis. Based on our previous findings that small inorganic nanoparticles may disturb Ab1e40 fibrillogenesis, we explored the effect of inorganic nanoparticles of various properties on regulating the elongation of Ab1e40 fibrils under physiological conditions. Here, nanoparticles were co-incubated with the monomeric Ab1e40 peptides for seed-mediated growth such that the elongation phase in the amyloidogenesis was dominant. Water-soluble CdTe QDs of different sizes (ca. 2e4 nm) and gold nanoparticles (AuNPs) of ca. 15 nm were adopted as the nanoparticles of interest. The QDs and AuNPs were synthesized and capped with the thiolated ligands N-acetyl-L-cysteine (NAC) and 3-mercaptopropionic acid (MPA) via the previously reported hydrothermal method [16] and modified Frens method [17] respectively. The effect of particle sizes, surface charges, functionality and compositions toward beta-amyloid fibrillogenesis was investigated. The as-formed fibrils were characterized with total internal reflection fluorescence microscopy (TIRFM), for visualizing the length of the Thioflavin T (ThT) labeled fibrils and monitoring the real-time fibril growth; and transmission electron microscopy (TEM), for studying the morphology and interaction between nanoparticles and fibrils. These imaging techniques provide advantages over conventional ThT fluorescence assay with smaller sample volume consumption; and better capability to reveal fibril length, density and general morphological structures of each single fibril instead of bulk fluorescence intensity readout [18]. Furthermore, plasmonic nanoparticles of larger size may significantly scatter incident light and absorb the emitted fluorescence from ThT due to their broad absorption wavelengths respectively. This work studied the critical regulatory roles of both the nanoparticles and surface functionalized ligands on the extent of fibrillation and the fibril morphologies of the Ab1e40 peptides in the elongation phase. Particles of different dimensions, materials and surface charges were applied to interact with the peptide monomers and seeds. The fibrillogenesis of Ab1e40 peptides controlled in the presence of nanomaterials was then monitored with spectroscopy and microscopy techniques. 2. Materials and methods 2.1. Materials Tellurium (reagent powder, 99.8%), cadmium chloride hydrate (CdCl2H2O), sodium borohydride (NaBH4), hydrogen tetrachloroaurate (III) hydrate (HAuCl4), trisodium citrate, 3-mercaptopropionic acid (MPA), N-acetyl-L-cysteine (NAC), Thioflavin T (ThT), sodium phosphate monobasic and sodium phosphate dibasic were purchased from SigmaeAldrich. All chemicals were used as received without further purification. Beta-amyloid (1e40) (Ab1e40) was purchased from Invitrogen (Lot: 25315-01S, Biosource, Camarillo CA, USA). 2.2. Synthesis and characterization of MPA- and NAC-capped quantum dots The detail procedures to synthesize high quality water soluble NAC, MPAcapped CdTe quantum dots (QD) were similar to those reported before [16]. A typical experiment procedure for synthesizing NAC capped CdTe QDs was described as follow. In brief, the ice-cold aqueous NaHTe solution was prepared by mixing NaBH4 (80 mg) with Te (127 mg) at a molar ratio of 2 : 1 in DI water (Millipore, USA) in the presence of N2. CdCl2 (12.5 mmol/L) and NAC (15 mmol/L) were mixed together in 40 mL DI water in an ice-cold bath for 30 min. The pH of this precursor solution was adjusted to pH 9.5 with NaOH. Freshly prepared NaHTe solution was
then injected to the N2 saturated precursor solution with molar ratio of Cd/NAC/Te at 1 : 1.2 : 0.2. Finally, 35 mL of the mixture solution was added into a 40 mL Teflonlined stainless steel autoclave and kept at 200 C for 30 min. To remove those unreacted reagents, cold 2-propanol was added to the reaction mixture to precipitate NAC-capped CdTe QDs and then rinsed with DI water. The UVevisible adsorption and fluorescence spectra were measured by a Cary 300 UVevisible spectrophotometer (Varian, Inc., USA) and a Perkin Elmer LS-50B luminescence spectrometer (Buckinghamshire, U.K.) respectively. The concentration of NACcapped CdTe QDs was determined according to the method reported before [19]. Zeta potential of these QDs was measured by Zetasizer Nano (Malvern Instruments Ltd., U.K.). 2.3. Preparation of citrate-, MPA- and NAC-stabilized gold nanoparticles Distilled water used in the preparation of solution and synthesis of nanoparticles and was filtered with 0.22 mm nylon membrane twice prior to use. All glasswares were cleaned with aqua regia and rinsed thoroughly with distilled water before use. Citrate stabilized gold nanoparticle (citrate-AuNP) was synthesized by the modified Frens method [17]. A 50 mL of solution containing 1 mM HAuCl4 was brought to boil. Under continuous heating and vigorous stirring, 5 mL of 38.8 mM sodium citrate was added to the vortex of the stirring solution. Boiling was continued for 10 min and the colour of the solution change from pale yellow to red wine. The heating source was removed and stirring was continued for an additional 15 min. The solution was cooled to room temperature. Gold nanoparticles functionalized with MPA and NAC were prepared by place exchange reactions. In brief, the pH of the as-prepared citrate-AuNP was tuned to approximately pH 7 with ammonia solution. The AuNP hydrosols were then added with 5 105 M MPA and NAC dissolved in absolute ethanol under vigorous stirring respectively. The solution was stirred for 2 h at room temperature. UVevis spectroscopy measurements of the AuNP solution yielded an absorbance maximum centered at ca. 520 nm (Fig. S2) and the size of the nanoparticles was characterized by TEM measurement (Fig. S3). The concentrations of all synthesized AuNP were estimated using the absorbance of the surface plasmon resonance peak [20]. 2.4. Preparation of beta-amyloid (1e40) fibrils The beta-amyloid (1e40) (Ab1e40) fibrils were prepared as reported previously [13,21]. Stock Ab1e40 solution was prepared by dissolving in 400 mL of ice-cold 0.02% ammonia solution and stored at 80 C before use. To prepare the seed fibrils, the stock solution was diluted with to 57.7 mM with 50 mM phosphate buffer (pH 7.4, with 100 mM NaCl). After a brief sonication (about 5e10 s), the reaction solution was incubated at 37 C water bath for 24 h. The reaction solution was centrifuged at 4 C for 1 h at 1.6 104g. The supernatant solution was discarded and the precipitate was resuspended in phosphate buffer with 0.05% NaN3 and stored at 18 C. Prior to each experiment, sample of the seed fibrils was sonicated thrice for 5 s and was then added to the phosphate buffer solution containing monomer peptide (57.7 mM) with a final concentration of ca. 10 mg/mL. The reaction solution was incubated in a water bath at 37 C for 1 h. The fibril was diluted with buffer solution to appropriate concentration and labeled with the thioflavin T (ThT) dye. In general, 5 mL of diluted and labeled fibril solution was sandwiched between a pair of cover glasses (No.1, 22 22 sq. mm, Menzel-Gläser, Germany, pre-cleaned with NaOH and distilled water) for imaging under fluorescence microscope. In the QDs and AuNPs modulation experiments, different size and ligand protected CdTe QDs and AuNPs of 10 nM was added to the seed-mediated beta-amyloid peptide reaction mixture solution and incubated at 37 C. For the TIRFM assay, sample solution of 1 mL was aliqouted, diluted with buffer solution and labeled with ThT in the time-lapse experiment at different time points (t ¼ 0, 15, 30, 45, 60, 120 min) for kinetics monitoring. For the ThT fluorescence measurement assay, the Ab peptide (50 mM), Ab seeds and gold nanoparticles (10 nM) were suspended in ThT solution instead of PB buffer, and incubated in the quartz cuvette at 37 C with gentle agitation. The fluorescence signals from the as-formed ThT labeled fibrils were measured in the luminescence lifetime scanning spectrometer (PTI Time Master Model C-720) every 10 min up to 2 h. 2.5. Fluorescence imaging and data analysis The prism-type total internal reflection fluorescence microscopic imaging system was similar to the setup described before [13,21]. In brief, a fused silica Isosceles Brewster Prism (CVI Laser, USA) was mounted on an Olympus IX71 inverted microscope. An evanescent field was generated on the interface between the sample solution and the surface of a cover glass. A 445 nm diode laser (Newport, USA) was used to excite the fluorescent labeling dye ThT binding to the amyloid fibrils. A band pass filter (HQ 480/40, Chroma Technology Corp., USA) and an oilimmersion 60 (PlanApo, N.A. 1.42) objective were used. Fluorescence images were captured by an electron-multiplying charge-coupled device (EMCCD) camera (PhotonMax 512, Princeton Instrument, USA) with 100 ms exposure time. The pixel size of the EMCCD is 16 mm. The length of the fibrils was measured with the freedomain software Image J (http://rsbweb.nih.gov/ij/). A hundred fibrils were measured with the Freehand line and Measure function of the software in each
H.-M. Chan et al. / Biomaterials 33 (2012) 4443e4450 condition. The distribution of real-time fibril length of Ab was fitted with the software OriginPro 8 and Igor Pro. 2.6. Transmission electron microscopy (TEM) Fibril sample and AuNP solutions of 5 mL was dropped on a carbon-coated copper grid (T200H-Cu, Electron Microscopy Sciences, Washington, USA) and dried for 5 min. The grid with amyloid fibrils was further negatively stained with 2% uranyl acetate for another 5 min. The dried sample was examined by a Technai G2 Transmission Electron Microscope (FEI, USA) with an acceleration voltage of 200 kV, and a JEM 2100 TEM (JEOL, Japan) with an acceleration voltage of 210 kV for high solution TEM images respectively.
3. Results and discussions 3.1. Regulation of beta-amyloid growth by functionalized quantum dots of various sizes CdTe QDs of various sizes were synthesized to investigate the size effect of nanoparticles on amyloid fibrillogenesis. MPA and NAC were functionalized onto the surface of the QDs as stabilizers. With respect to the pKa value of the eCOOH group of the surfacebounded MPA (pKa z 6.6) and NAC (pKa z 4.1) [22,23], the nanoparticles were anionic at physiological pH. The negatively charged carboxylate end groups of the ligands allowed well dispersion and better stabilization of QDs by electrostatic repulsion. Characterization of the MPA- and NAC-QDs was shown in Table 1. The sizes and concentrations of the QDs were estimated from the UVevis absorption spectra [19], and the zeta-potential measurements confirmed that all QDs were stable in aqueous solution with negative charges. As according to our previous study, functionalized QDs (ca. 3 nm) showed threshold response to the fibrillation of Ab1e40 ranging from 109 to 107 M. In this work, the concentration of nanoparticles was kept constant at 108 M to investigate the effect of nanoparticle sizes and surface charges to the regulation of Ab growth. As demonstrated in our previous work that elongation process of Ab1e40 fibrils was completed in an hour under seedmediated growth [13,21], the incubation time of the mixture of Ab1e40 peptides and nanoparticles was kept at 1 h throughout unless otherwise specified. Fig. 1 showed the fluorescence and TEM images of Ab1e40 fibrils grew in the presence of MPA- and NAC-QDs of three sizes respectively. It revealed that the amyloid fibrillation was inhibited by 10 nM of 2.8 nm NAC-QDs, however, not by 2.2 nm MPA-QDs (Fig. 1A and D). This effect could be explained by the same argument as proposed in our previous work that NAC, as compared with MPA, offers more hydrogen bonding (H-bond) sites to interact with Ab1e40 peptides [13]. The small NAC-QDs blocked the elongation site of the Ab1e40 peptides and seeds, and prohibited the self-assembling fibrillation. The 2.2 nm MPA-QDs, albeit owning similar charges as the 2.8 nm NAC-QDs, did not observably affect the growth of Ab1e40 fibrils. Nevertheless, the inhibitory effect was Table 1 Absorption maximum, calculated diameter and zeta potential of MPA and NACcapped CdTe quantum dots in aqueous solution.
MPA-QD 1 MPA-QD 2 MPA-QD 3 NAC-QD 1 NAC-QD 2 NAC-QD 3
lmax/nm
Diameter/nma
z/mV
494 542 577 519 536 589
2.2 3.1 3.5 2.8 3.1 3.8
25.2 36.6 29.8 29.6 39.9 28.7
a Theoretical diameter of the stock QDs were calculated according to the equation reported previously, in which d ¼ (9.8127 107)l3max (1.7147 103) 2 lmax þ (1.0064)lmax 194.84 [18], in which lmax is the maximum absorption wavelength.
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noticed when the concentration of MPA-QDs increased to 107 M or higher (data not shown). We presumed that MPA possess a weaker interaction with the Ab1e40 peptide than NAC, and hence the MPAQDs were relatively less effective in inhibiting the fibrillogenesis although bearing similar surface charges and sizes as the NAC-QDs. Seemingly, surface ligands possessed more H-bond sites that may interact with the peptides. There was no inhibition observed on the growth of Ab1e40 peptides in the presence of medium size MPA- and NAC-QDs. The fibrillation patterns of Ab1e40 fibrils, regarding for the fibril length and fibril number, grew in the presence of medium size 3.1 nm NAC-QDs and 3.1 nm MPA-QDs (Fig. 1B and E) were very similar to the control fibrils. Both NAC- and MPA-QDs of 3.1 nm were concluded neither fibrillation inhibitor nor promoter. In principle, the number of ligands conjugated on the particle surfaces, and thus the available H-bond sites for interaction with peptides, should increase with the extended size and surface area of the AuNPs. As evidenced from the zeta-potential measurements, the medium size MPA- and NAC-QDs carried more negative surface charges than the small QDs, confirming that more ligands were bound onto the surface of the particles. However, it is revealed the relatively high density of negative charges on the surface of the particles may repel the Ab1e40 peptides with net charges of 3 and thus retarded the interactions between QDs and Ab1e40. The above results proved that despite the increase in sites for H-bond interactions should theoretically further facilitate the blockage of amyloid elongation sites, the high density of surface charges generated also by the elevated number of surface ligands may counteract the conceivable hydrogen bonds between particles and peptides, and accordingly offset the inhibitory effect of the NAC- and MPA- QDs to the fibrillogenesis. Unexpectedly, TIRFM imaging observed that Ab1e40 fibrillogenesis was promoted in the batches of Ab1e40 peptides incubated with larger size of NAC-QDs (3.8 nm) and MPA-QDs (3.5 nm) respectively. The measured zeta potentials of the large QDs were higher than those of medium size. We presumed that the charge to surface area ratio of the conjugated QD was reduced in the presence of limited amount of ligands but enlarged particle sizes, and thus, the electrostatic repulsions of large QDs to peptides were attenuated. Here, we found the peptides spontaneously assembled into large and high-density aggregates with large QDs (Fig. 1C and F). In order to validate for the formation of the amyloid clusters in the presence of large QDs, the real-time seed-mediated fibrillation of Ab1e40 peptides was monitored. Fluorescence images were taken at t ¼ 0, 15, 30, 45 and 60 min respectively as shown in Fig. 2. Large fibrillar aggregates appeared notably earlier in the presence of QDs, meaning that the interactions between the QDs and fibrils were highly favorable. To review if the QDs interrupted the fibrillation rate of Ab1e40, the length of a hundred control and QDsinteracted fibrils were measured from the TIRFM images respectively. The number of pixels occupied by the fibrils allowed estimation of the length of each Ab1e40 fibril. It was worth noting that the individual cluster-forming fibrils cannot be well resolved under fluorescence microscopy as they appeared as a single large bright cluster, and thus the length of the fibrils inside the cluster cannot be analyzed while only the length of the free non-clustered fibrils were measured and compared in this work. Hereby, the lengths of fibrils in the presence of MPA-QDs (6.7 mm 0.04 mm, mean standard error of mean, n ¼ 100, hereinafter) and NAC-QDs (8.0 mm 0.1 mm) were slightly shorter than the control fibrils (10.4 mm 0.3 mm) after 1 h of incubation. We expected that the QDs, which have similar sizes as the Ab seeds, maybe large enough to serve as additional nucleation sites. Monomeric Ab1e40 interacted through hydrogen bonds and adsorbed onto the functionalized surface of the QDs. The concentration of monomeric
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Fig. 1. Fluorescence (left) and TEM (right) images of Ab1e40 fibrils formed after incubating (A) 2.8 nm, (B) 3.1 nm, (C) 3.8 nm NAC-capped QDs; and (D) 2.2 nm, (E) 3.1 nm, (F) 3.5 nm MPA-capped QDs with Ab1e40 seed mediated growth solution for 1 h at 37 C. Scale bar in fluorescence and TEM images are 16 mm and 0.2 mm respectively.
Fig. 2. Real-time monitoring of the seed-mediated fibrillation of Ab1e40 with and without the addition of QDs. Occurrence of large Ab1e40 aggregates was earlier in the presence of both MPA- and NAC-capped QDs compared to the control. Scale bar is 20 mm.
H.-M. Chan et al. / Biomaterials 33 (2012) 4443e4450
Ab1e40 in free solution was reduced [8], and thus relatively shorter fibrils were generated in the presence of large QDs.
3.2. Regulation of beta-amyloid growth by functionalized gold nanoparticles To further explore the factors controlling the Ab1e40 growth and the role of nanoparticles played in the regulation of fibrillogenesis, we extended the size scale of the nanoparticles from QDs smaller than 5 nm to gold nanoparticles of 15 nm in the study. The surface charges and functional ligands on the surface of the particles were found to be comparable to those used in the study of QDs-Ab1e40 interaction. Recent studies on the effect of particles towards the growth of protein fibril limited in mostly polymeric or semiconducting metallic nanoparticles of drastic sizes (either below 5 nm or above 40 nm) [8e11]. However, the interaction between amyloid peptide and metallic nanoparticles ranging from 10 to 40 nm, which were commonly used in bioassays, were not reported. AuNP is chosen to interact with amyloid peptide for its wellknown biocompatibility, high monodispersity and simply fabrication method compared to other nanoparticles [24,25]. Furthermore, the derivatization of AuNP with different ligands such as aliphatic thiols and cysteine residues through AueS bond were well established and hence the surface charges and functionality of the AuNP could be easily regulated [26,27]. Previous report revealed that protein such as bovine serum albumin (BSA) appeared to bind spontaneously to the surface of citrate-stabilized gold nanoparticles (citrate-AuNP) even though both BSA and citrate-AuNP were negatively charged and should repel from each other at physiological pH [28]. It was proposed that the positive lysine residues of BSA interact strongly with the negative charge from the citrate ion by salt-bridge interaction and facilitated strong binding. Similarly, monomeric Ab1e40 peptides, although with a net charge of 3e, bearing three positive and 18 hydrophobic residues should have high potential to bind to the AuNP surface through hydrogen bondings or other electrostatic interactions and hence intervened with the fibrillation mechanism of beta-amyloid. Herein, monomeric Ab1e40 peptides were co-incubated with 15 nm AuNPs stabilized by citrate, MPA and NAC into amyloid fibrils using the same conditions as described in the former section respectively. Since the acidity of the stock AuNP solution may disturb the overall pH of the Ab1e40 growth solution and so do AuNP has a high flocculation rate at low pH [29], all AuNP solutions used were therefore adjusted to approximately pH 7 prior to experiments. Table 2 showed the characterization of the functionalized AuNPs used in this study. The conjugation of MPA and NAC was achieved by ligand displacement method, in which citrate ion electrostatically stabilized on the AuNP surface was displaced by
Table 2 Absorption maximum, calculated diameter, measured diameter and zeta potential of citrate, MPA and NAC-stabilized gold nanoparticles in aqueous solution at pH 7.
Citrate-AuNP MPA-AuNP NAC-AuNP
lmax/nm
Diameter/nma
Diameter/nmb
z/mV
519 520 520
16.90 16.10 16.44
14.05 1.2 14.49 1.2 14.40 1.1
38.7 48.7 25.0
a Theoretical diameter of the AuNP were calculated according to the equation reported previously, in which d ¼ [Aspr 5.89 106/(Cau e4.75)](1/0.314) [19], in which Aspr is the absorbance of the surface plasmon resonance peak, Cau is the initial concentration of the gold ion used in the synthesis. b TEM measured diameter of the AuNP was verified by fitting the size distribution of 100 AuNPs with the Gaussian distribution and hence obtained the average diameter. The TEM images and the size distributions of citrate-, MPA- and NAC-AuNP were shown in the supporting information.
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the thiolated ligands respectively. The ligands covalently bound to the surface of the AuNP through strong AueS bonds and resulted in a charged monolayer surface of AuNPs as proven by the decline in absorbance in the UVevis absorption spectra (Fig. S2 in supporting information). The particles were remained spherical and monodisperse after the surface modifications as evaluated by the TEM imaging. As shown in Fig. S3 in the supporting information, the MPA- and NAC-AuNPs shared comparable diameter as the stock citrate-AuNP, displaying that the ligand exchange reaction did not reduce the stability of the nanoparticles. Neither flocculation was observed from TEM nor positive zeta-potentials (which implying to the instability of the colloids) were measured in all batches of samples confirming that the AuNPs have both successfully functionalized with the thiolated ligands and possessed with high monodispersity to interact with monomeric Ab1e40 peptides. Preliminarily, the fibrillation kinetics of the seed-mediated growth of 50 mM Ab1e40 peptides in the absence and presence of 108 M citrate-, MPA- and NAC-AuNPs were studied with the typical ThT fluorescence assays. Fig. S4 showed the corresponding realtime fluorescence spectra of the amyloid fibrils grew in the presence of ThT and the corresponding colloids. Fluorescence enhancement at lem ¼ 480 nm was observed over 2 h of incubation in all cases (Fig. S5) except in the presence of NAC-AuNP (Fig. S4D), indicating that citrate and MPA functionalized AuNPs did not inhibit the fibrillation. However, it is worth noting that the peak shapes for all nanoparticles-interacted Ab1e40 fibrils (Fig. S4BeD) was significantly different from that of the control (Fig. S4A), implicating that AuNPs may interfere with the spectral characteristics of the ThT fluorescence spectra by scattering the excitation light source or absorbing the emitted ThT fluorescence (quenching) as mentioned. Therefore, we reckoned that one should neither (i) serve ThT fluorescence assay as the sole explanatory experiment to manifest the regulatory role of AuNPs on Ab1e40 fibrillation, nor (ii) conclude that NAC-AuNPs limited the growth of Ab1e40 fibrils based on the unexpected differences in ThT emission spectrum. For extended verification on the fibril-particles interaction, fluorescence and electron microscopic imaging were exercised for authentic examination of the resultant fibril lengths and morphologies. Fig. 3 showed the length distributions, fluorescence and TEM images of the Ab1e40 fibrils grew for 1 h in the absence and presence of nanoparticles respectively. As similar to the case of QDs, fluorescence images showed that Ab has a tendency to form cluster-like aggregates in the presence of nanoparticles. The appearance frequency and density of the amyloid cluster was in the following order: NAC-AuNP > Citrate-AuNP > MPAAuNP > Control. The cluster formation was induced by the introduction of the functionalized nanoparticles rather than the ligand compounds, as proven by the control experiments incubating ligand and Ab1e40 peptides only that no cluster-like aggregates were observed in the fibrils grew from the Ab1e40 peptides with sodium citrate, MPA and NAC respectively (Fig. S6). The real-time growth of amyloid fibrils in the absence and presence of functionalized AuNPs were shown in Fig. 4. The fibril length measured in fluorescence images showed no significant deviation among control and the existence of three different nanoparticles. The 1-hincubated fibril lengths were measured to be 11.1 mm 0.2 mm (control), 11.0 mm 0.2 mm (citrate-AuNPs interacted fibrils), 8.6 mm 0.1 mm (MPA-AuNPs interacted fibrils) and 10.5 mm 0.2 mm (NAC-AuNPs interacted fibrils) respectively. The overall distribution of the length of 100 fibrils surveyed in each of the concerned conditions did not significantly deviated from that of control (Fig. S7e10). Although the surface charges of the AuNPs were similar to that of the QDs, the variation in length was not as obvious as the case of QDs since the size of the AuNPs are larger
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Fig. 3. Fluorescence (left panel), TEM (middle panel) and amyloid fibril length distribution (right panel) of Ab1e40 fibrils formed (A) in the absence of nanoparticles; in the presence of 108 M of (B) citrate-AuNP; (C) MPA-AuNP; (D) NAC-AuNP with Ab1e40 seed mediated growth solution for 1 h at 37 C. Scale bar in fluorescence and TEM images are 20 mm and 0.2 mm respectively.
and the charge-to-surface area ratio of the particles are much reduced. On the other hand, TEM images displayed that NAC-AuNPs adhered densely on the backbone of the amyloid fibrils while citrate-AuNPs and MPA-AuNPs were much dispersedly attached to
the fibrils. It seemed like certain interactions were presented in between the NAC-AuNP and Ab1e40 peptides. While in our previous work [13] and the former section of this article hypothesized that NAC-QDs strongly interacted with the Ab1e40 peptides through H-
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Fig. 4. Fibrillation kinetics of Ab1e40 was monitored by measuring the fibril length at different time points by TIRFM under the addition of functionalized AuNPs at 108 M: (B) no particles, (,) Citrate-AuNP, (C) MPA-AuNP, and (-) NAC-AuNP. The fibrillation kinetics follows the pseudo first order kinetics.
bond, we herein testified if the proposed mechanism was also valid for NAC-AuNPs. The occurrence of hydrogen bonding interactions between functionalized AuNPs and monomeric Ab1e40 peptides were proved by UVevis spectroscopy. Fig. S11 showed the UVevis spectra of functionalized AuNPs dispersed in (i) DMSO, (ii) DMSO with Ab1e40 peptides, (iii) ethanol (EtOH), and (iv) EtOH with Ab1e40 peptides respectively. Here, no notable differences in the spectral shape and the lmax were displayed when functionalized AuNPs were dispersed in either DMSO or DMSO with monomeric Ab1e40 peptides. We deduced that DMSO, as a strong polar aprotic solvent, interacted with the ligands and the Ab1e40 peptides individually. The solvent environment obstructed and shielded the interactions between nanoparticles and peptides and hence, even with the addition of Ab1e40 monomers, no obvious change in lmax of the AuNP spectrum was achieved. Interestingly, the situation was totally different when the particles were suspended in EtOH. Nanoparticles were found aggregated in EtOH since the surface charges of the functionalized AuNPs were neutralized. The particles were agglomerated and resulted in broad UVevis spectra with lmax > 600 nm for both MPA-AuNP and NAC-AuNP. However, with the addition of Ab1e40 monomers, the spectra maxima were well preserved at w530 nm. Comparing with the absorption maxima of nanoparticles in EtOH only, a significant blue shift of 128 nm and 96 nm in lmax was observed for MPA-AuNP and NAC-AuNP respectively after exposing the colloids to monomeric Ab1e40 peptides. It was expected that the peptide molecules adsorbed on the surface of the nanoparticles provided extra stabilization to the particles and avoided aggregation. While EtOH diluted the electrostatic charges of the particles, the potential interactions between the functionalized AuNPs and the Ab peptides were therefore likely to be hydrogen bondings. The above arguments provided evidences that the interactions between Ab1e40 peptides and AuNPs were highly correlated to the ligand functionality and surface charges of the particles, in which the cluster-like aggregates were found in the batches of functionalized AuNP with more H-bond sites and less negative surface charges (e.g. NAC-AuNP). We inferred that the NAC-AuNP, which offers more hydrogen bonding sites and less negatively charged surface, favored a stronger interaction with the amyloid fibrils; on
the other hand, the citrate-AuNP and MPA-AuNP with highly negative surface charges discouraged the interaction between the particles and the Ab1e40 peptides. This hypothesis also explained the dissimilarity in spectral characteristics of the ThT fluorescence spectra of Ab1e40 peptides interacted with NAC-AuNPs (Fig. S4D). Since NAC conjugated strongly with the Ab1e40 peptides, the intermolecular distances between the ThT molecules and the NACAuNPs were expected to be close. The fluorescence emitted from the fibril-bound ThT maybe absorbed and quenched by the AuNPs and thus the fluorescence enhancement at lem ¼ 480 nm was not projected as expected. It also supports that TIRFM imaging is more suitable than conventional ThT fluorescence assays for the study of amyloid growth involving other fluorescent dyes or nanoparticles. Our results showed there is no significant influence on the length and kinetics of Ab1e40 fibrillogenesis in the presence of functionalized AuNPs. The phenomenon also agreed well with the mechanism proposed in the preceding reports from CabaleiroeLago and coworkers [8]. Under a high peptide to nanoparticle surface area ratio (5000 peptide against 1 nanoparticle), a remarkably large fraction of the Ab1e40 monomer was present in the solution instead of on the particle surface. Thus, the fibrillation of Ab1e40 in solution, instead of that on particle surface, dominated the fibrillation pathway and hence the fibrillation patterns of Ab1e40 were not eminently disturbed in the presence of 15 nm AuNPs.
4. Conclusions Nanoparticles with various sizes, surface charges and potential hydrogen-bonding sites act differently in the elongation phase of the Ab1e40 fibrillogenesis. Slight change in sizes and charges of small nanoparticles may induce contrary effect on amyloid fibrillation. It has been shown that many nanoparticles are small enough to pass through different barriers in the body and are regarded as valuable therapeutic materials, for example, in the treatment of Alzheimer’s disease [30]. Knowing that nanoparticles of various functionalities play critical regulatory role on the fibrillation of Ab1e40 peptide, our findings provide insight that well-controlling the surface functionality, size and concentration of nanoparticles
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is very crucial and essential in the development of nanomaterials for future biological and biomedical applications. Acknowledgements This work was fully supported by grants from the University Grant Council of Hong Kong Special Administrative Region, China (HKBU 201208) and Faculty Research Grant from Hong Kong Baptist University (FRG2/08-09/073) and (FRG2/09-10/037). The FEI-Technai G2 TEM used in this work was supported by the Center for Surface Analysis and Research (CSAR) with funding from the Special Equipment Grant from the University Grant Committee of the Hong Kong Special Administrative Region, China (SEG_HKBU06). We thank Mr. Benson Leung (HKBU) and Mr. Roy Ho (HKUST) for TEM measurement. We also thank Dr. Kai-Chung Lau and Miss Queeny Lo of the City University of Hong Kong for zeta-potential measurement. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2012. 03.024. References [1] Caughey B, Lansbury PT. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 2003;26:267e98. [2] Pepys MB. Amyloidosis. Annu Rev Med 2006;57:223e41. [3] Rauk A. The chemistry of alzheimer’s disease. Chem Soc Rev 2009;38: 2698e715. [4] Hamley IW. Peptide fibrillization. Angew Chem-Int Edit 2007;46:8128e47. [5] Selkoe DJ. Normal and abnormal biology of the beta-amyloid precursor protein. Annu Rev Neurosci 1994;17:489e517. [6] Stark WJ. Nanoparticles in biological systems. Angew Chem Int Edit 2011;50: 1242e58. [7] Cabaleiro-Lago C, Lynch I, Dawson KA, Linse S. Inhibition of iapp and iapp (20e29) fibrillation by polymeric nanoparticles. Langmuir 2010;26:3453e61. [8] Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Dawson KA, Linse S. Dual effect of amino modified polystyrene nanoparticles on amyloid beta protein fibrillation. ACS Chem Neurosci 2010;1:279e87. [9] Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Lindman S, Minogue AM, Thulin E, et al. Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles. J Am Chem Soc 2008;130:15437e43. [10] Linse S, Cabaleiro-Lago C, Xue WF, Lynch I, Lindman S, Thulin E, et al. Nucleation of protein fibrillation by nanoparticles. Proc Natl Acad Sci U S A 2007;104:8691e6.
[11] Vannoy CH, Leblanc RM. Effects of dhla-capped cdse/zns quantum dots on the fibrillation of human serum albumin. J Phys Chem B 2010;114:10881e8. [12] Wu WH, Sun X, Yu YP, Hu J, Zhao L, Liu Q, et al. Tio2 nanoparticles promote beta-amyloid fibrillation in vitro. Biochem Biophys Res Commun 2008;373: 315e8. [13] Xiao LH, Zhao D, Chan WH, Choi MMF, Li HW. Inhibition of beta 1-40 amyloid fibrillation with n-acetyl-l-cysteine capped quantum dots. Biomaterials 2010; 31:91e8. [14] Yoo SI, Yang M, Brender JR, Subramanian V, Sun K, Joo NE, et al. Inhibition of amyloid peptide fibrillation by inorganic nanoparticles: functional similarities with proteins. Angew Chem Int Edit 2011;50:5110e5. [15] Majzik A, Fulop L, Csapo E, Bogar F, Martinek T, Penke B, et al. Functionalization of gold nanoparticles with amino acid, beta-amyloid peptides and fragment. Colloid Surf B-Biointerfaces 2010;81:235e41. [16] Zhao D, He ZK, Chan WH, Choi MMF. Synthesis and characterization of highquality water-soluble near-infrared-emitting cdte/cds quantum dots capped by N-acetyl-L-cysteine via hydrothermal method. J Phys Chem C 2009;113: 1293e300. [17] Grabar KC, Freeman RG, Hommer MB, Natan MJ. Preparation and characteriation of au colloid monolayers. Anal Chem 1995;67:735e43. [18] Ban T, Morigaki K, Yagi H, Kawasaki T, Kobayashi A, Yuba S, et al. Real-time and single fibril observation of the formation of amyloid beta spherulitic structures. J Biol Chem 2006;281:33677e83. [19] Yu WW, Qu LH, Guo WZ, Peng XG. Experimental determination of the extinction coefficient of cdte, cdse, and cds nanocrystals. Chem Mat 2003;15: 2854e60. [20] Haiss W, Thanh NTK, Aveyard J, Fernig DG. Determination of size and concentration of gold nanoparticles from UV-Vis spectra. Anal Chem 2007;79: 4215e21. [21] Man BYW, Chan HM, Leung CH, Chan DSH, Bai LP, Jiang ZH, et al. Group 9 metal-based inhibitors of beta-amyloid (1e40) fibrillation as potential therapeutic agents for alzheimer’s disease. Chem Sci 2011;2:917e21. [22] Choi MMF, Douglas AD, Murray RW. Ion-pair chromatographic separation of water-soluble gold monolayer-protected clusters. Anal Chem 2006;78: 2779e85. [23] Park JJ, Weiger MC, Lacerda S, Pristinski D, Becker ML, Douglas JF, et al. Characterization of non-equilibrium nanoparticle adsorption on a model biological substrate. Langmuir 2010;26:4822e30. [24] Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 2004;104:293e346. [25] Murphy CJ, Gole AM, Stone JW, Sisco PN, Alkilany AM, Goldsmith EC, et al. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc Chem Res 2008;41:1721e30. [26] Love JC, Estroff LA, Kriebel JK, Nuzzo RG, Whitesides GM. Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem Rev 2005;105:1103e69. [27] Weisbecker CS, Merritt MV, Whitesides GM. Molecular self-assembly of aliphatic thiols on gold colloids. Langmuir 1996;12:3763e72. [28] Brewer SH, Glomm WR, Johnson MC, Knag MK, Franzen S. Probing bsa binding to citrate-coated gold nanoparticles and surfaces. Langmuir 2005;21:9303e7. [29] Mayya KS, Patil V, Sastry M. On the stability of carboxylic acid derivatized gold colloidal particles: the role of colloidal solution ph studied by optical absorption spectroscopy. Langmuir 1997;13:3944e7. [30] Liu G, Garrett MR, Men P, Zhu XW, Perry G, Smith MA. Nanoparticle and other metal chelation therapeutics in alzheimer disease. Bba-Mol Basis Dis 2005; 1741:246e52.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1e40) fibrillogenesis Ho-Man Chan a, Lehui Xiao a, Kai-Ming Yeung a, See-Lok Ho a, Dan Zhao a, Wing-Hong Chan a, Hung-Wing Li a, b, * a b
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China Centre for Surface Analysis and Research, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Accepted 6 March 2012 Available online 27 March 2012
The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1e40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Ab1e40 peptides under seedmediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Ab1e40 fibrillogenesis. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Nanoparticle Self-assembly Peptide Surface modification
1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder primarily found in elderly. The formation of self-assembling neurotoxic fibrous beta-amyloid (Ab) plaques in brain is believed as a pathological hallmark of AD [1e3]. Controlling the nucleation/ elongation-growth processes of monomeric Ab peptides into insoluble oligomers, protofibrils and fibrils are reckoned as a potential therapy to AD [4,5]. While nanomaterials are now widely designed and applied as drug delivering vehicles for therapeutics and diagnostics purposes [6], precautionary selection of nanomaterials is highly recommended as the vehicle itself maybe cytotoxic in nature otherwise aggravated the conditions. Several recent studies have provided insight that nanomaterials of various sizes, shapes, compositions and functionalities may significantly intervene with the self-assembling mechanism of amyloid peptides, drastically either promoting or inhibiting the fibrillogenesis [7e14]. For instance, Wu et al. reported that 20 nm TiO2 nanoparticles could promote the growth of Ab by shortening the nucleation process [12]. Cabaleiro-Lago et al. demonstrated that co-polymeric NIPAM:BAM nanoparticles of 40 nm with different hydrophobicities inhibited the growth of Ab fibrils by adsorbing the
* Corresponding author. Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China. Tel.: þ852 3411 7065; fax: þ852 3411 7348. E-mail address: [email protected] (H.-W. Li). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.03.024
monomers onto the particle surface and thus depleting the solution monomer concentration [9]. They have also reported that cationic amino-modified polystyrene nanoparticles of 57e180 nm induced both acceleration and retardation effects on Ab fibrillation based on the concentration and coverage of peptide monomers on the particle surfaces [8]. More recently, Yoo and coworkers have presented that functionalized quantum dots (QDs) non-specifically interacted with the Ab monomers, interrupting the nucleation process and consequently inhibiting the growth of Ab fibrils [14]. Also, a recent paper from Majzik and coworkers has drawn to our attention that the covalent interaction between the gold nanoparticles and the cysteine-modified beta-amyloid peptides hindered the formation of the polypeptide chain structure [15]. There were also numerous reports that drawn our attention describing the roles of nanomaterials on the controlled fibrillation of other self-assembling proteins and peptides, such as human serum albumin (HSA) [11], islet amyloid polypeptide (IAPP) [7], and human b2-microglobulin (b2m) [10]. In view of the reported results, it is believed that surface areas, compositions and functionalities of nanoparticles play significant regulatory role on controlling the Ab fibrillation process and worth in depth investigations. Accordingly, the behind mechanism of interactions between nanoparticles and peptide monomers were still not clearly understood. Researchers proposed that particles acted to reduce the rate of nucleation, yet the elongation phase were unaffected once critical nuclei were formed [9]. On the contrary, our previous work illustrated that N-acetyl-L-cysteine-capped quantum dots
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(NAC-QDs) with hydrodynamic diameter of ca. 3 nm could effectively quench both nucleation and elongation process of the Ab fibrillogenesis at any time points in the seed-mediated growth of beta amyloid (1e40) (Ab1e40) by blocking active sites of the seed fibrils or monomers [13]. The inhibitory effect is concentration dependent, and a remarkable inhibition towards 50 mM of Ab peptide was observed when the dosage of NAC-QDs increased from 109 to 107 M. This preliminary result showed that nanomaterials not only regulate the nucleation but also the elongation phase of beta-amyloid fibrillogenesis. Based on our previous findings that small inorganic nanoparticles may disturb Ab1e40 fibrillogenesis, we explored the effect of inorganic nanoparticles of various properties on regulating the elongation of Ab1e40 fibrils under physiological conditions. Here, nanoparticles were co-incubated with the monomeric Ab1e40 peptides for seed-mediated growth such that the elongation phase in the amyloidogenesis was dominant. Water-soluble CdTe QDs of different sizes (ca. 2e4 nm) and gold nanoparticles (AuNPs) of ca. 15 nm were adopted as the nanoparticles of interest. The QDs and AuNPs were synthesized and capped with the thiolated ligands N-acetyl-L-cysteine (NAC) and 3-mercaptopropionic acid (MPA) via the previously reported hydrothermal method [16] and modified Frens method [17] respectively. The effect of particle sizes, surface charges, functionality and compositions toward beta-amyloid fibrillogenesis was investigated. The as-formed fibrils were characterized with total internal reflection fluorescence microscopy (TIRFM), for visualizing the length of the Thioflavin T (ThT) labeled fibrils and monitoring the real-time fibril growth; and transmission electron microscopy (TEM), for studying the morphology and interaction between nanoparticles and fibrils. These imaging techniques provide advantages over conventional ThT fluorescence assay with smaller sample volume consumption; and better capability to reveal fibril length, density and general morphological structures of each single fibril instead of bulk fluorescence intensity readout [18]. Furthermore, plasmonic nanoparticles of larger size may significantly scatter incident light and absorb the emitted fluorescence from ThT due to their broad absorption wavelengths respectively. This work studied the critical regulatory roles of both the nanoparticles and surface functionalized ligands on the extent of fibrillation and the fibril morphologies of the Ab1e40 peptides in the elongation phase. Particles of different dimensions, materials and surface charges were applied to interact with the peptide monomers and seeds. The fibrillogenesis of Ab1e40 peptides controlled in the presence of nanomaterials was then monitored with spectroscopy and microscopy techniques. 2. Materials and methods 2.1. Materials Tellurium (reagent powder, 99.8%), cadmium chloride hydrate (CdCl2H2O), sodium borohydride (NaBH4), hydrogen tetrachloroaurate (III) hydrate (HAuCl4), trisodium citrate, 3-mercaptopropionic acid (MPA), N-acetyl-L-cysteine (NAC), Thioflavin T (ThT), sodium phosphate monobasic and sodium phosphate dibasic were purchased from SigmaeAldrich. All chemicals were used as received without further purification. Beta-amyloid (1e40) (Ab1e40) was purchased from Invitrogen (Lot: 25315-01S, Biosource, Camarillo CA, USA). 2.2. Synthesis and characterization of MPA- and NAC-capped quantum dots The detail procedures to synthesize high quality water soluble NAC, MPAcapped CdTe quantum dots (QD) were similar to those reported before [16]. A typical experiment procedure for synthesizing NAC capped CdTe QDs was described as follow. In brief, the ice-cold aqueous NaHTe solution was prepared by mixing NaBH4 (80 mg) with Te (127 mg) at a molar ratio of 2 : 1 in DI water (Millipore, USA) in the presence of N2. CdCl2 (12.5 mmol/L) and NAC (15 mmol/L) were mixed together in 40 mL DI water in an ice-cold bath for 30 min. The pH of this precursor solution was adjusted to pH 9.5 with NaOH. Freshly prepared NaHTe solution was
then injected to the N2 saturated precursor solution with molar ratio of Cd/NAC/Te at 1 : 1.2 : 0.2. Finally, 35 mL of the mixture solution was added into a 40 mL Teflonlined stainless steel autoclave and kept at 200 C for 30 min. To remove those unreacted reagents, cold 2-propanol was added to the reaction mixture to precipitate NAC-capped CdTe QDs and then rinsed with DI water. The UVevisible adsorption and fluorescence spectra were measured by a Cary 300 UVevisible spectrophotometer (Varian, Inc., USA) and a Perkin Elmer LS-50B luminescence spectrometer (Buckinghamshire, U.K.) respectively. The concentration of NACcapped CdTe QDs was determined according to the method reported before [19]. Zeta potential of these QDs was measured by Zetasizer Nano (Malvern Instruments Ltd., U.K.). 2.3. Preparation of citrate-, MPA- and NAC-stabilized gold nanoparticles Distilled water used in the preparation of solution and synthesis of nanoparticles and was filtered with 0.22 mm nylon membrane twice prior to use. All glasswares were cleaned with aqua regia and rinsed thoroughly with distilled water before use. Citrate stabilized gold nanoparticle (citrate-AuNP) was synthesized by the modified Frens method [17]. A 50 mL of solution containing 1 mM HAuCl4 was brought to boil. Under continuous heating and vigorous stirring, 5 mL of 38.8 mM sodium citrate was added to the vortex of the stirring solution. Boiling was continued for 10 min and the colour of the solution change from pale yellow to red wine. The heating source was removed and stirring was continued for an additional 15 min. The solution was cooled to room temperature. Gold nanoparticles functionalized with MPA and NAC were prepared by place exchange reactions. In brief, the pH of the as-prepared citrate-AuNP was tuned to approximately pH 7 with ammonia solution. The AuNP hydrosols were then added with 5 105 M MPA and NAC dissolved in absolute ethanol under vigorous stirring respectively. The solution was stirred for 2 h at room temperature. UVevis spectroscopy measurements of the AuNP solution yielded an absorbance maximum centered at ca. 520 nm (Fig. S2) and the size of the nanoparticles was characterized by TEM measurement (Fig. S3). The concentrations of all synthesized AuNP were estimated using the absorbance of the surface plasmon resonance peak [20]. 2.4. Preparation of beta-amyloid (1e40) fibrils The beta-amyloid (1e40) (Ab1e40) fibrils were prepared as reported previously [13,21]. Stock Ab1e40 solution was prepared by dissolving in 400 mL of ice-cold 0.02% ammonia solution and stored at 80 C before use. To prepare the seed fibrils, the stock solution was diluted with to 57.7 mM with 50 mM phosphate buffer (pH 7.4, with 100 mM NaCl). After a brief sonication (about 5e10 s), the reaction solution was incubated at 37 C water bath for 24 h. The reaction solution was centrifuged at 4 C for 1 h at 1.6 104g. The supernatant solution was discarded and the precipitate was resuspended in phosphate buffer with 0.05% NaN3 and stored at 18 C. Prior to each experiment, sample of the seed fibrils was sonicated thrice for 5 s and was then added to the phosphate buffer solution containing monomer peptide (57.7 mM) with a final concentration of ca. 10 mg/mL. The reaction solution was incubated in a water bath at 37 C for 1 h. The fibril was diluted with buffer solution to appropriate concentration and labeled with the thioflavin T (ThT) dye. In general, 5 mL of diluted and labeled fibril solution was sandwiched between a pair of cover glasses (No.1, 22 22 sq. mm, Menzel-Gläser, Germany, pre-cleaned with NaOH and distilled water) for imaging under fluorescence microscope. In the QDs and AuNPs modulation experiments, different size and ligand protected CdTe QDs and AuNPs of 10 nM was added to the seed-mediated beta-amyloid peptide reaction mixture solution and incubated at 37 C. For the TIRFM assay, sample solution of 1 mL was aliqouted, diluted with buffer solution and labeled with ThT in the time-lapse experiment at different time points (t ¼ 0, 15, 30, 45, 60, 120 min) for kinetics monitoring. For the ThT fluorescence measurement assay, the Ab peptide (50 mM), Ab seeds and gold nanoparticles (10 nM) were suspended in ThT solution instead of PB buffer, and incubated in the quartz cuvette at 37 C with gentle agitation. The fluorescence signals from the as-formed ThT labeled fibrils were measured in the luminescence lifetime scanning spectrometer (PTI Time Master Model C-720) every 10 min up to 2 h. 2.5. Fluorescence imaging and data analysis The prism-type total internal reflection fluorescence microscopic imaging system was similar to the setup described before [13,21]. In brief, a fused silica Isosceles Brewster Prism (CVI Laser, USA) was mounted on an Olympus IX71 inverted microscope. An evanescent field was generated on the interface between the sample solution and the surface of a cover glass. A 445 nm diode laser (Newport, USA) was used to excite the fluorescent labeling dye ThT binding to the amyloid fibrils. A band pass filter (HQ 480/40, Chroma Technology Corp., USA) and an oilimmersion 60 (PlanApo, N.A. 1.42) objective were used. Fluorescence images were captured by an electron-multiplying charge-coupled device (EMCCD) camera (PhotonMax 512, Princeton Instrument, USA) with 100 ms exposure time. The pixel size of the EMCCD is 16 mm. The length of the fibrils was measured with the freedomain software Image J (http://rsbweb.nih.gov/ij/). A hundred fibrils were measured with the Freehand line and Measure function of the software in each
H.-M. Chan et al. / Biomaterials 33 (2012) 4443e4450 condition. The distribution of real-time fibril length of Ab was fitted with the software OriginPro 8 and Igor Pro. 2.6. Transmission electron microscopy (TEM) Fibril sample and AuNP solutions of 5 mL was dropped on a carbon-coated copper grid (T200H-Cu, Electron Microscopy Sciences, Washington, USA) and dried for 5 min. The grid with amyloid fibrils was further negatively stained with 2% uranyl acetate for another 5 min. The dried sample was examined by a Technai G2 Transmission Electron Microscope (FEI, USA) with an acceleration voltage of 200 kV, and a JEM 2100 TEM (JEOL, Japan) with an acceleration voltage of 210 kV for high solution TEM images respectively.
3. Results and discussions 3.1. Regulation of beta-amyloid growth by functionalized quantum dots of various sizes CdTe QDs of various sizes were synthesized to investigate the size effect of nanoparticles on amyloid fibrillogenesis. MPA and NAC were functionalized onto the surface of the QDs as stabilizers. With respect to the pKa value of the eCOOH group of the surfacebounded MPA (pKa z 6.6) and NAC (pKa z 4.1) [22,23], the nanoparticles were anionic at physiological pH. The negatively charged carboxylate end groups of the ligands allowed well dispersion and better stabilization of QDs by electrostatic repulsion. Characterization of the MPA- and NAC-QDs was shown in Table 1. The sizes and concentrations of the QDs were estimated from the UVevis absorption spectra [19], and the zeta-potential measurements confirmed that all QDs were stable in aqueous solution with negative charges. As according to our previous study, functionalized QDs (ca. 3 nm) showed threshold response to the fibrillation of Ab1e40 ranging from 109 to 107 M. In this work, the concentration of nanoparticles was kept constant at 108 M to investigate the effect of nanoparticle sizes and surface charges to the regulation of Ab growth. As demonstrated in our previous work that elongation process of Ab1e40 fibrils was completed in an hour under seedmediated growth [13,21], the incubation time of the mixture of Ab1e40 peptides and nanoparticles was kept at 1 h throughout unless otherwise specified. Fig. 1 showed the fluorescence and TEM images of Ab1e40 fibrils grew in the presence of MPA- and NAC-QDs of three sizes respectively. It revealed that the amyloid fibrillation was inhibited by 10 nM of 2.8 nm NAC-QDs, however, not by 2.2 nm MPA-QDs (Fig. 1A and D). This effect could be explained by the same argument as proposed in our previous work that NAC, as compared with MPA, offers more hydrogen bonding (H-bond) sites to interact with Ab1e40 peptides [13]. The small NAC-QDs blocked the elongation site of the Ab1e40 peptides and seeds, and prohibited the self-assembling fibrillation. The 2.2 nm MPA-QDs, albeit owning similar charges as the 2.8 nm NAC-QDs, did not observably affect the growth of Ab1e40 fibrils. Nevertheless, the inhibitory effect was Table 1 Absorption maximum, calculated diameter and zeta potential of MPA and NACcapped CdTe quantum dots in aqueous solution.
MPA-QD 1 MPA-QD 2 MPA-QD 3 NAC-QD 1 NAC-QD 2 NAC-QD 3
lmax/nm
Diameter/nma
z/mV
494 542 577 519 536 589
2.2 3.1 3.5 2.8 3.1 3.8
25.2 36.6 29.8 29.6 39.9 28.7
a Theoretical diameter of the stock QDs were calculated according to the equation reported previously, in which d ¼ (9.8127 107)l3max (1.7147 103) 2 lmax þ (1.0064)lmax 194.84 [18], in which lmax is the maximum absorption wavelength.
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noticed when the concentration of MPA-QDs increased to 107 M or higher (data not shown). We presumed that MPA possess a weaker interaction with the Ab1e40 peptide than NAC, and hence the MPAQDs were relatively less effective in inhibiting the fibrillogenesis although bearing similar surface charges and sizes as the NAC-QDs. Seemingly, surface ligands possessed more H-bond sites that may interact with the peptides. There was no inhibition observed on the growth of Ab1e40 peptides in the presence of medium size MPA- and NAC-QDs. The fibrillation patterns of Ab1e40 fibrils, regarding for the fibril length and fibril number, grew in the presence of medium size 3.1 nm NAC-QDs and 3.1 nm MPA-QDs (Fig. 1B and E) were very similar to the control fibrils. Both NAC- and MPA-QDs of 3.1 nm were concluded neither fibrillation inhibitor nor promoter. In principle, the number of ligands conjugated on the particle surfaces, and thus the available H-bond sites for interaction with peptides, should increase with the extended size and surface area of the AuNPs. As evidenced from the zeta-potential measurements, the medium size MPA- and NAC-QDs carried more negative surface charges than the small QDs, confirming that more ligands were bound onto the surface of the particles. However, it is revealed the relatively high density of negative charges on the surface of the particles may repel the Ab1e40 peptides with net charges of 3 and thus retarded the interactions between QDs and Ab1e40. The above results proved that despite the increase in sites for H-bond interactions should theoretically further facilitate the blockage of amyloid elongation sites, the high density of surface charges generated also by the elevated number of surface ligands may counteract the conceivable hydrogen bonds between particles and peptides, and accordingly offset the inhibitory effect of the NAC- and MPA- QDs to the fibrillogenesis. Unexpectedly, TIRFM imaging observed that Ab1e40 fibrillogenesis was promoted in the batches of Ab1e40 peptides incubated with larger size of NAC-QDs (3.8 nm) and MPA-QDs (3.5 nm) respectively. The measured zeta potentials of the large QDs were higher than those of medium size. We presumed that the charge to surface area ratio of the conjugated QD was reduced in the presence of limited amount of ligands but enlarged particle sizes, and thus, the electrostatic repulsions of large QDs to peptides were attenuated. Here, we found the peptides spontaneously assembled into large and high-density aggregates with large QDs (Fig. 1C and F). In order to validate for the formation of the amyloid clusters in the presence of large QDs, the real-time seed-mediated fibrillation of Ab1e40 peptides was monitored. Fluorescence images were taken at t ¼ 0, 15, 30, 45 and 60 min respectively as shown in Fig. 2. Large fibrillar aggregates appeared notably earlier in the presence of QDs, meaning that the interactions between the QDs and fibrils were highly favorable. To review if the QDs interrupted the fibrillation rate of Ab1e40, the length of a hundred control and QDsinteracted fibrils were measured from the TIRFM images respectively. The number of pixels occupied by the fibrils allowed estimation of the length of each Ab1e40 fibril. It was worth noting that the individual cluster-forming fibrils cannot be well resolved under fluorescence microscopy as they appeared as a single large bright cluster, and thus the length of the fibrils inside the cluster cannot be analyzed while only the length of the free non-clustered fibrils were measured and compared in this work. Hereby, the lengths of fibrils in the presence of MPA-QDs (6.7 mm 0.04 mm, mean standard error of mean, n ¼ 100, hereinafter) and NAC-QDs (8.0 mm 0.1 mm) were slightly shorter than the control fibrils (10.4 mm 0.3 mm) after 1 h of incubation. We expected that the QDs, which have similar sizes as the Ab seeds, maybe large enough to serve as additional nucleation sites. Monomeric Ab1e40 interacted through hydrogen bonds and adsorbed onto the functionalized surface of the QDs. The concentration of monomeric
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Fig. 1. Fluorescence (left) and TEM (right) images of Ab1e40 fibrils formed after incubating (A) 2.8 nm, (B) 3.1 nm, (C) 3.8 nm NAC-capped QDs; and (D) 2.2 nm, (E) 3.1 nm, (F) 3.5 nm MPA-capped QDs with Ab1e40 seed mediated growth solution for 1 h at 37 C. Scale bar in fluorescence and TEM images are 16 mm and 0.2 mm respectively.
Fig. 2. Real-time monitoring of the seed-mediated fibrillation of Ab1e40 with and without the addition of QDs. Occurrence of large Ab1e40 aggregates was earlier in the presence of both MPA- and NAC-capped QDs compared to the control. Scale bar is 20 mm.
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Ab1e40 in free solution was reduced [8], and thus relatively shorter fibrils were generated in the presence of large QDs.
3.2. Regulation of beta-amyloid growth by functionalized gold nanoparticles To further explore the factors controlling the Ab1e40 growth and the role of nanoparticles played in the regulation of fibrillogenesis, we extended the size scale of the nanoparticles from QDs smaller than 5 nm to gold nanoparticles of 15 nm in the study. The surface charges and functional ligands on the surface of the particles were found to be comparable to those used in the study of QDs-Ab1e40 interaction. Recent studies on the effect of particles towards the growth of protein fibril limited in mostly polymeric or semiconducting metallic nanoparticles of drastic sizes (either below 5 nm or above 40 nm) [8e11]. However, the interaction between amyloid peptide and metallic nanoparticles ranging from 10 to 40 nm, which were commonly used in bioassays, were not reported. AuNP is chosen to interact with amyloid peptide for its wellknown biocompatibility, high monodispersity and simply fabrication method compared to other nanoparticles [24,25]. Furthermore, the derivatization of AuNP with different ligands such as aliphatic thiols and cysteine residues through AueS bond were well established and hence the surface charges and functionality of the AuNP could be easily regulated [26,27]. Previous report revealed that protein such as bovine serum albumin (BSA) appeared to bind spontaneously to the surface of citrate-stabilized gold nanoparticles (citrate-AuNP) even though both BSA and citrate-AuNP were negatively charged and should repel from each other at physiological pH [28]. It was proposed that the positive lysine residues of BSA interact strongly with the negative charge from the citrate ion by salt-bridge interaction and facilitated strong binding. Similarly, monomeric Ab1e40 peptides, although with a net charge of 3e, bearing three positive and 18 hydrophobic residues should have high potential to bind to the AuNP surface through hydrogen bondings or other electrostatic interactions and hence intervened with the fibrillation mechanism of beta-amyloid. Herein, monomeric Ab1e40 peptides were co-incubated with 15 nm AuNPs stabilized by citrate, MPA and NAC into amyloid fibrils using the same conditions as described in the former section respectively. Since the acidity of the stock AuNP solution may disturb the overall pH of the Ab1e40 growth solution and so do AuNP has a high flocculation rate at low pH [29], all AuNP solutions used were therefore adjusted to approximately pH 7 prior to experiments. Table 2 showed the characterization of the functionalized AuNPs used in this study. The conjugation of MPA and NAC was achieved by ligand displacement method, in which citrate ion electrostatically stabilized on the AuNP surface was displaced by
Table 2 Absorption maximum, calculated diameter, measured diameter and zeta potential of citrate, MPA and NAC-stabilized gold nanoparticles in aqueous solution at pH 7.
Citrate-AuNP MPA-AuNP NAC-AuNP
lmax/nm
Diameter/nma
Diameter/nmb
z/mV
519 520 520
16.90 16.10 16.44
14.05 1.2 14.49 1.2 14.40 1.1
38.7 48.7 25.0
a Theoretical diameter of the AuNP were calculated according to the equation reported previously, in which d ¼ [Aspr 5.89 106/(Cau e4.75)](1/0.314) [19], in which Aspr is the absorbance of the surface plasmon resonance peak, Cau is the initial concentration of the gold ion used in the synthesis. b TEM measured diameter of the AuNP was verified by fitting the size distribution of 100 AuNPs with the Gaussian distribution and hence obtained the average diameter. The TEM images and the size distributions of citrate-, MPA- and NAC-AuNP were shown in the supporting information.
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the thiolated ligands respectively. The ligands covalently bound to the surface of the AuNP through strong AueS bonds and resulted in a charged monolayer surface of AuNPs as proven by the decline in absorbance in the UVevis absorption spectra (Fig. S2 in supporting information). The particles were remained spherical and monodisperse after the surface modifications as evaluated by the TEM imaging. As shown in Fig. S3 in the supporting information, the MPA- and NAC-AuNPs shared comparable diameter as the stock citrate-AuNP, displaying that the ligand exchange reaction did not reduce the stability of the nanoparticles. Neither flocculation was observed from TEM nor positive zeta-potentials (which implying to the instability of the colloids) were measured in all batches of samples confirming that the AuNPs have both successfully functionalized with the thiolated ligands and possessed with high monodispersity to interact with monomeric Ab1e40 peptides. Preliminarily, the fibrillation kinetics of the seed-mediated growth of 50 mM Ab1e40 peptides in the absence and presence of 108 M citrate-, MPA- and NAC-AuNPs were studied with the typical ThT fluorescence assays. Fig. S4 showed the corresponding realtime fluorescence spectra of the amyloid fibrils grew in the presence of ThT and the corresponding colloids. Fluorescence enhancement at lem ¼ 480 nm was observed over 2 h of incubation in all cases (Fig. S5) except in the presence of NAC-AuNP (Fig. S4D), indicating that citrate and MPA functionalized AuNPs did not inhibit the fibrillation. However, it is worth noting that the peak shapes for all nanoparticles-interacted Ab1e40 fibrils (Fig. S4BeD) was significantly different from that of the control (Fig. S4A), implicating that AuNPs may interfere with the spectral characteristics of the ThT fluorescence spectra by scattering the excitation light source or absorbing the emitted ThT fluorescence (quenching) as mentioned. Therefore, we reckoned that one should neither (i) serve ThT fluorescence assay as the sole explanatory experiment to manifest the regulatory role of AuNPs on Ab1e40 fibrillation, nor (ii) conclude that NAC-AuNPs limited the growth of Ab1e40 fibrils based on the unexpected differences in ThT emission spectrum. For extended verification on the fibril-particles interaction, fluorescence and electron microscopic imaging were exercised for authentic examination of the resultant fibril lengths and morphologies. Fig. 3 showed the length distributions, fluorescence and TEM images of the Ab1e40 fibrils grew for 1 h in the absence and presence of nanoparticles respectively. As similar to the case of QDs, fluorescence images showed that Ab has a tendency to form cluster-like aggregates in the presence of nanoparticles. The appearance frequency and density of the amyloid cluster was in the following order: NAC-AuNP > Citrate-AuNP > MPAAuNP > Control. The cluster formation was induced by the introduction of the functionalized nanoparticles rather than the ligand compounds, as proven by the control experiments incubating ligand and Ab1e40 peptides only that no cluster-like aggregates were observed in the fibrils grew from the Ab1e40 peptides with sodium citrate, MPA and NAC respectively (Fig. S6). The real-time growth of amyloid fibrils in the absence and presence of functionalized AuNPs were shown in Fig. 4. The fibril length measured in fluorescence images showed no significant deviation among control and the existence of three different nanoparticles. The 1-hincubated fibril lengths were measured to be 11.1 mm 0.2 mm (control), 11.0 mm 0.2 mm (citrate-AuNPs interacted fibrils), 8.6 mm 0.1 mm (MPA-AuNPs interacted fibrils) and 10.5 mm 0.2 mm (NAC-AuNPs interacted fibrils) respectively. The overall distribution of the length of 100 fibrils surveyed in each of the concerned conditions did not significantly deviated from that of control (Fig. S7e10). Although the surface charges of the AuNPs were similar to that of the QDs, the variation in length was not as obvious as the case of QDs since the size of the AuNPs are larger
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Fig. 3. Fluorescence (left panel), TEM (middle panel) and amyloid fibril length distribution (right panel) of Ab1e40 fibrils formed (A) in the absence of nanoparticles; in the presence of 108 M of (B) citrate-AuNP; (C) MPA-AuNP; (D) NAC-AuNP with Ab1e40 seed mediated growth solution for 1 h at 37 C. Scale bar in fluorescence and TEM images are 20 mm and 0.2 mm respectively.
and the charge-to-surface area ratio of the particles are much reduced. On the other hand, TEM images displayed that NAC-AuNPs adhered densely on the backbone of the amyloid fibrils while citrate-AuNPs and MPA-AuNPs were much dispersedly attached to
the fibrils. It seemed like certain interactions were presented in between the NAC-AuNP and Ab1e40 peptides. While in our previous work [13] and the former section of this article hypothesized that NAC-QDs strongly interacted with the Ab1e40 peptides through H-
H.-M. Chan et al. / Biomaterials 33 (2012) 4443e4450
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Fig. 4. Fibrillation kinetics of Ab1e40 was monitored by measuring the fibril length at different time points by TIRFM under the addition of functionalized AuNPs at 108 M: (B) no particles, (,) Citrate-AuNP, (C) MPA-AuNP, and (-) NAC-AuNP. The fibrillation kinetics follows the pseudo first order kinetics.
bond, we herein testified if the proposed mechanism was also valid for NAC-AuNPs. The occurrence of hydrogen bonding interactions between functionalized AuNPs and monomeric Ab1e40 peptides were proved by UVevis spectroscopy. Fig. S11 showed the UVevis spectra of functionalized AuNPs dispersed in (i) DMSO, (ii) DMSO with Ab1e40 peptides, (iii) ethanol (EtOH), and (iv) EtOH with Ab1e40 peptides respectively. Here, no notable differences in the spectral shape and the lmax were displayed when functionalized AuNPs were dispersed in either DMSO or DMSO with monomeric Ab1e40 peptides. We deduced that DMSO, as a strong polar aprotic solvent, interacted with the ligands and the Ab1e40 peptides individually. The solvent environment obstructed and shielded the interactions between nanoparticles and peptides and hence, even with the addition of Ab1e40 monomers, no obvious change in lmax of the AuNP spectrum was achieved. Interestingly, the situation was totally different when the particles were suspended in EtOH. Nanoparticles were found aggregated in EtOH since the surface charges of the functionalized AuNPs were neutralized. The particles were agglomerated and resulted in broad UVevis spectra with lmax > 600 nm for both MPA-AuNP and NAC-AuNP. However, with the addition of Ab1e40 monomers, the spectra maxima were well preserved at w530 nm. Comparing with the absorption maxima of nanoparticles in EtOH only, a significant blue shift of 128 nm and 96 nm in lmax was observed for MPA-AuNP and NAC-AuNP respectively after exposing the colloids to monomeric Ab1e40 peptides. It was expected that the peptide molecules adsorbed on the surface of the nanoparticles provided extra stabilization to the particles and avoided aggregation. While EtOH diluted the electrostatic charges of the particles, the potential interactions between the functionalized AuNPs and the Ab peptides were therefore likely to be hydrogen bondings. The above arguments provided evidences that the interactions between Ab1e40 peptides and AuNPs were highly correlated to the ligand functionality and surface charges of the particles, in which the cluster-like aggregates were found in the batches of functionalized AuNP with more H-bond sites and less negative surface charges (e.g. NAC-AuNP). We inferred that the NAC-AuNP, which offers more hydrogen bonding sites and less negatively charged surface, favored a stronger interaction with the amyloid fibrils; on
the other hand, the citrate-AuNP and MPA-AuNP with highly negative surface charges discouraged the interaction between the particles and the Ab1e40 peptides. This hypothesis also explained the dissimilarity in spectral characteristics of the ThT fluorescence spectra of Ab1e40 peptides interacted with NAC-AuNPs (Fig. S4D). Since NAC conjugated strongly with the Ab1e40 peptides, the intermolecular distances between the ThT molecules and the NACAuNPs were expected to be close. The fluorescence emitted from the fibril-bound ThT maybe absorbed and quenched by the AuNPs and thus the fluorescence enhancement at lem ¼ 480 nm was not projected as expected. It also supports that TIRFM imaging is more suitable than conventional ThT fluorescence assays for the study of amyloid growth involving other fluorescent dyes or nanoparticles. Our results showed there is no significant influence on the length and kinetics of Ab1e40 fibrillogenesis in the presence of functionalized AuNPs. The phenomenon also agreed well with the mechanism proposed in the preceding reports from CabaleiroeLago and coworkers [8]. Under a high peptide to nanoparticle surface area ratio (5000 peptide against 1 nanoparticle), a remarkably large fraction of the Ab1e40 monomer was present in the solution instead of on the particle surface. Thus, the fibrillation of Ab1e40 in solution, instead of that on particle surface, dominated the fibrillation pathway and hence the fibrillation patterns of Ab1e40 were not eminently disturbed in the presence of 15 nm AuNPs.
4. Conclusions Nanoparticles with various sizes, surface charges and potential hydrogen-bonding sites act differently in the elongation phase of the Ab1e40 fibrillogenesis. Slight change in sizes and charges of small nanoparticles may induce contrary effect on amyloid fibrillation. It has been shown that many nanoparticles are small enough to pass through different barriers in the body and are regarded as valuable therapeutic materials, for example, in the treatment of Alzheimer’s disease [30]. Knowing that nanoparticles of various functionalities play critical regulatory role on the fibrillation of Ab1e40 peptide, our findings provide insight that well-controlling the surface functionality, size and concentration of nanoparticles
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is very crucial and essential in the development of nanomaterials for future biological and biomedical applications. Acknowledgements This work was fully supported by grants from the University Grant Council of Hong Kong Special Administrative Region, China (HKBU 201208) and Faculty Research Grant from Hong Kong Baptist University (FRG2/08-09/073) and (FRG2/09-10/037). The FEI-Technai G2 TEM used in this work was supported by the Center for Surface Analysis and Research (CSAR) with funding from the Special Equipment Grant from the University Grant Committee of the Hong Kong Special Administrative Region, China (SEG_HKBU06). We thank Mr. Benson Leung (HKBU) and Mr. Roy Ho (HKUST) for TEM measurement. We also thank Dr. Kai-Chung Lau and Miss Queeny Lo of the City University of Hong Kong for zeta-potential measurement. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2012. 03.024. References [1] Caughey B, Lansbury PT. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 2003;26:267e98. [2] Pepys MB. Amyloidosis. Annu Rev Med 2006;57:223e41. [3] Rauk A. The chemistry of alzheimer’s disease. Chem Soc Rev 2009;38: 2698e715. [4] Hamley IW. Peptide fibrillization. Angew Chem-Int Edit 2007;46:8128e47. [5] Selkoe DJ. Normal and abnormal biology of the beta-amyloid precursor protein. Annu Rev Neurosci 1994;17:489e517. [6] Stark WJ. Nanoparticles in biological systems. Angew Chem Int Edit 2011;50: 1242e58. [7] Cabaleiro-Lago C, Lynch I, Dawson KA, Linse S. Inhibition of iapp and iapp (20e29) fibrillation by polymeric nanoparticles. Langmuir 2010;26:3453e61. [8] Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Dawson KA, Linse S. Dual effect of amino modified polystyrene nanoparticles on amyloid beta protein fibrillation. ACS Chem Neurosci 2010;1:279e87. [9] Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Lindman S, Minogue AM, Thulin E, et al. Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles. J Am Chem Soc 2008;130:15437e43. [10] Linse S, Cabaleiro-Lago C, Xue WF, Lynch I, Lindman S, Thulin E, et al. Nucleation of protein fibrillation by nanoparticles. Proc Natl Acad Sci U S A 2007;104:8691e6.
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