Interaction Dynamics in Inhibiting the Aggregation of AÎ² Peptides by

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The interaction dynamics in inhibiting the aggregation of A# peptides by SWCNTs: a combined experimental and coarse-grained molecular dynamic simulations study Dongdong Lin, Ruxi Qi, Shujie Li, Ruoyu He, Pei Li, Guanghong Wei, and Xinju Yang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00101 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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ACS Chemical Neuroscience

The interaction dynamics in inhibiting the aggregation of Aβ peptides by SWCNTs: a combined experimental and coarse-grained molecular dynamic simulation study Dongdong Lin, Ruxi Qi, Shujie Li, Ruoyu He, Pei Li, Guanghong Wei, and Xinju Yang* State Key Laboratory of Surface Physics and Key Laboratory for Computational Physical, Fudan University, Shanghai 200433, China

ABSTRACT The aggregation of amyloid-β peptides (Aβ) is considered as the main possible cause of the Alzheimer’s diseases (AD). How to suppress the formation of toxic Aβ aggregates has been intensively concerned over the past several decades. Increasing evidences show that whether carbon nano-materials can suppress or promote the aggregation depends on their physicochemical properties. However, their interaction dynamics remains elusive as amyloid fibrillation is a complex multistep process. In this paper, we utilized atomic force microscopy (AFM), electrostatic force microscopy (EFM), ThT/fluorescence spectroscopy and cell viability measurements, combined with coarse-grained molecular dynamic (MD) simulations to study the dynamic interaction of full length Aβ with single-walled carbon nanotubes (SWCNTs). At single SWCNTs scale, it is found that the presence of SWCNTs would result in rapid and spontaneous adsorption of Aβ1-40 peptides on their surface and stacking into non-fibrillar aggregates with reduced toxicity, which plays an important role in inhibiting the formation of toxic oligomers and mature fibrils. Our results provide new clues for studying the interaction in

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amyloid/SWCNTs system as well as for seeking amyloidosis inhibitors with carbon nanomaterials.

KEYWORDS Alzheimer’s disease, amyloid-β peptides, carbon nanotube, interaction, inhibit, toxic oligomers


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INTRODUCTION The formation of amyloid fibrils or plaques is considered as one of the most remarkable pathological characters of Alzheimer’s disease (AD).1-5 Numerous researches reveal that the fibrillation of amyloid-β peptides (Aβ) causes neuronal death both in Vitro and in Vivo.1, 6-8 Therefore, inhibiting the aggregation of Aβ peptides to insoluble fibrils has been considered as a potential goal in the AD therapy, and great efforts have been made to explore the inhibitory strategies in the past decades. Particularly, the effects of nano-materials on inhibiting Aβ aggregation has been drawn close attention due to their great potential applications in biomedicine and biotechnology.9-12 For example, various types of nanoparticles (NPs) including metal,13,

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magnetic,15 oxide16,

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and polymer18,

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have been attempted to investigate their

influence on the Aβ aggregation. The results reveal that NPs may either inhibit or accelerate the aggregation of Aβ. Carbon nano-materials such as fullerenes, carbon nanotubes (CNTs) and graphene, have been extensively used in biomedicine and biotechnology.9, 11, 20-22 In particular, CNTs have immense potential applications in tissue engineering, photothermal therapy, drug delivery et al.20, 23, 24 Therefore, it is fundamentally important to explore the influence of CNTs on Aβ aggregations as well as the interaction details between Aβ and CNTs. The influence of CNTs on the aggregation of different amyloid proteins have already been reported in literatures. For example, Ghule et al. reported that CNTs could prevent the trifluoroethanol induced aggregation of hFGF-1 protein,25 while Linse et al. found multiwall CNTs could enhance the aggregation of β2-microglobulin.26 Ge et al. investigated the interactions of single-walled carbon nanotubes (SWCNTs) with human serum proteins and found a competitive binding of these proteins with different adsorption


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capacity and packing modes.27 More recently, Luo et al. reported the NMR results that SWCNTs directed the Aβ peptides to form a new class of β-sheet-rich but non-amyloid fibrils.28 Meanwhile, atomistic molecular dynamic (MD) simulations have been used to investigate the interaction of Aβ monomers with SWCNTs, and the influence of SWCNTs on the aggregation of the central hydrophobic core fragments of full-length Aβ. The results of these simulations suggested that the peptides could be adsorbed on the surface of SWCNTs.29-31 However, atomistic MD simulations usually provide the interaction dynamics between Aβ monomers and SWCNT(s) in hundreds of nanosecond time scale because of the computational capability. Amyloid fibrillization is a complex multistep process, containing the formation of soluble oligomers, protofibrils, and mature fibrils. In the same way, the interaction between Aβ peptides and carbon nano-materials will be several steps in aggregation process. In addition, some evidences show that the Aβ intermediates (oligomers, protofibrils et al.) during aggregation will be more toxic than mature fibrils.7 As a result, it is essential to make a careful study in every step of interaction between Aβ peptides and CNTs, especially the interaction between Aβ intermediates and CNTs. In this paper, a method combined experimental measurements with coarse-grained MD simulations is applied to investigate the effect of SWCNTs on the aggregation process of fulllength Aβ1-40 peptides. The morphology changes of Aβ1-40 with and without SWCNTs during aggregation were investigated by atomic force microscopy (AFM) at single SWCNTs scale. It is found that the Aβ1-40 peptides will quickly bind to the surface of SWCNTs, and the Aβ1-40 wrapped SWCNTs grow large subsequently. As a result, the formation of free toxic oligomers and mature fibrils is supressed, which coincides with the improved cell viability at the presence of SWCNTs. However, it is difficult to distinguish between Aβ1-40 wrapped SWCNT complex


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and Aβ1-40 fibrils by conventional AFM topography images. Therefore, AFM phase images and electrostatic force microscopy (EFM) images were explored to get more information on the interaction details of Aβ1-40-SWCNT complex. To probe the Aβ1-40-SWCNT interaction process at the molecular level, coarse-grained MD simulations were performed on the Aβ1-40-SWCNT system. The simulation illustrates the dynamic interaction process in 1.5 µs, and suggest the different conformation of peptides and oligomers between inner and upper layers. Based on these findings, a possible mechanism of how SWCNTs affect the aggregation of Aβ1-40 is proposed.

RESULTS AND DISCUSSION SWCNTs suppress the formation of free oligomers and fibrils. Extensive topography images of Aβ1-40 aggregates and Aβ1-40-SWCNT complex were measured by ex situ AFM in tapping mode as a function of incubation time. Typical AFM images are presented in Fig. 1. The aggregation process of Aβ1-40 can be clearly observed in the upper row of Fig. 1. A mass of monomers with few small oligomers are observed on the sample prepared with fresh peptide solution. With the incubation time going on, small oligomers and monomers aggregate to filaments/protofibrils after 7 and 12 days, as shown in AFM images in Fig. 1(b) and (c). Significant aggregation is observed from the image taken after 20 days’ incubation, as shown in Fig. 1(d), in which a large number of mature fibrils were formed. In the comparative experiments of Aβ1-40 mixed with SWCNTs (Aβ1-40-SWCNT), which is shown in the bottom row of Fig. 1, the aggregation behavior are dramatically different. It can be seen that with the same incubation time, the formation of mature fibrils is greatly reduced. Less long mature fibrils are observed after 20 days (Fig. 1 (h)), indicating that the mixing of SWCNTs can reduce the fibrillation of


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Aβ1-40 efficiently. Instead of numerous mature fibrils formed in control sample, some Aβ1-40 wrapped rod-like structures (simple termed as wraps afterwards) can be observed on Aβ1-40SWCNT samples. The wraps even emerged on the sample prepared immediately after the mixing of SWCNTs with Aβ1-40 (Fig. 1(e)), certifying that they should not be Aβ fibrils. In addition, the suppression of oligomers can be observed from the zoomed AFM images of Fig. 1(b, c) and (f, g), as shown in Fig. S3. The quantity of oligomers on Aβ1-40-SWCNT sample (Fig. S3 (f’)) is much less than that on Aβ1-40 sample (Fig. S3 (b’)) for same incubation time of 2 days, indicating the presence of SWCNTs suppresses the formation of free toxic oligomers. Fig. S3 (g’) also gives evidence that some short protofibrils (~ 100 nm) are formed at the presence of SWCNTs. This phenomenon may be attributed to the large surface of Aβ1-40-SWCNTs wraps provides a platform for quick adsorption of Aβ1-40 peptides and increase the local concentration for aggregation. Meanwhile, the adsorption of peptides reduces the formation of free oligomers/protofibrils in solution. As a result, the presence of SWCNTs promotes the aggregation, however, the number of promoted fibrils is small (Fig. 1(h)), as the majority of peptides/oligomers are restricted on SWCNTs. Aβ1-40-SWCNT binding analysis at single SWCNT scale. To investigate the combination details of Aβ1-40-SWCNT complex, magnified AFM images are presented in Fig. 2. It clearly shows that the height of formed wraps is about 3 ~ 4 nm after being mixed immediately, which is higher than that of bare SWCNT (less than 2 nm as shown in Fig. S4) but lower than mature fibrils (about 8 nm as shown in Fig. 2(d)), indicating that these wraps should be SWCNTs adsorbed with peptides. Additionally, the wraps exhibit the character of a string of beads along the SWCNT, we attribute this feature to the adsorption of few Aβ1-40 monomers or oligomers on the surface of SWCNT. The size of some wraps becomes larger with the continuous adsorption


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of peptides (Fig. 2(b) (3 days)). In addition, the morphology of wraps is amorphous, and the height of the largest wraps even reaches about 30 nm, suggesting that the absorbed peptides on SWCNTs are much random. However, the height of the large wrap reaches a uniform height of ~ 8 nm after 20 day’s incubation (Fig. 2 (c)), which is lower than that after less incubation time (212 days). In this case, the migration and rearrangement of peptides should have occurred and the loose compounds turn into much compact and ordered structures, because of the hydrophobic and aromatic character from peptides-peptides and peptides-SWCNT.32 More evidences can be achieved from the AFM phase images since they can produce more information about the material contrast by comparing the different mechanical properties of SWCNTs and peptides.33, 34 From the phase images of Aβ1-40-SWCNT presented in Fig. 2(a’) to (d’), it can be observed that for the freshly mixed sample, the phase image exhibits the feature like scattered dots along the tube, consisting with the adsorption of oligomers on the SWCNT as observed in Fig. 2(a). The phase shift profile along the wrap shows a fluctuated ups and downs, which is attributed to the height and material change of oligomers and SWCNT. There is a dramatic phase contrast along the wrap of Aβ1-40-SWCNT (Fig. 2(b’)) after being incubated for 3 days, where a large phase shift fluctuation up to 10° is measured along the wrap, indicating that the Aβ1-40 peptides are adsorbed heavily and randomly on the surface of SWCNTs. In addition, a core-shell feature is observed for both large and small wraps in Fig. 2(b’), presenting that the core has a lower phase shift compared to the shell. This feature indicates the mechanical difference between the core and shell. After 20 days’ incubation, the core-shell structure is no longer observed. Instead, a large but relatively uniform phase shift is observed along the wrap (Fig. 2(c’)). It is consistent with the results obtained from the height images. We speculate the absorbed peptides may rearrange and form much tight and ordered structures around the


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SWCNT, which reduce the influence of SWCNT on the measured phase shift. For comparison, the phase image of Aβ1-40 fibrils is also measured (Fig. 2(d’)). The image exhibits a long-period twist and a small phase shift variation, which is significantly different compared with Aβ1-40SWCNT wraps and bare SWCNTs (1~2° as shown in Fig.S4). Here, it should be mentioned the measured phase shift would be influenced by the topographic factor. To get more accurate information about the location of SWCNT inside Aβ-SWCNT aggregates, EFM measurement was carried out. As SWCNTs and Aβ fibrils have different electrical properties, the influence of the topography factor on the phase shift can be excluded at enough lift height. In a previous literature, EFM has been applied to distinguish SWCNT and polyimide by utilizing the higher dielectric constant of SWCNT than that of the polyimide.35 The topography and electrical phase shift images on Aβ1-40-SWCNT incubated after 2 days at a sample bias of +3 V and a lift height of 20 nm are presented in Fig. 3, together with their line profiles. From the EFM phase shift image (Fig. 3(b)), a thin groove can be observed at the top centre of the Aβ1-40-SWCNT wrap. Clear feature can be obtained from the line profiles as shown in Fig. 3(c). It exhibits that there is a break on the peak of the EFM phase shift, resulting as a concave shape located in the centre of the wrap. This feature can be obtained repeatedly on most of the Aβ1-40-SWCNT wraps but cannot be observed in pristine SWCNT and bare Aβ1-40 fibrils. The phase image presented in Fig. S5 also displays clear difference compared with Fig. 3(a). Similar concave EFM phase shift has been observed by Clausen et al. on hollow and silver-filled peptide nanotubes, and it was attributed to the different permittivity of the air and silver located in center.36 In our case, we hold the same viewpoint as SWCNTs and Aβ1-40 peptides have different dielectric coefficients, and the concave EFM phase shift should be attributed to the location of the SWCNT in the centre of the wrap. The EFM result is well consistent with the


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results obtained by phase images, indicating that both the phase and EFM images can be applied to confirm the location of SWCNT by using their different mechanical and electrical properties at a single SWCNT scale. Aβ1-40-SWCNT prevent fibrillation and reduce cytotoxicity. Thioflavin T (ThT) fluorescence were measured to obtain the secondary structure information of those Aβ1-40SWCNT wraps. ThT fluorescence intensity of 2.3 µM Aβ1-40 with and without SWCNTs (< 0.005 mg/mL) was compared as a function of incubation time under same conditions (Fig. 4 (a)). The results show that the ThT fluorescence intensity of Aβ1-40 solution with SWCNTs is a little higher than that without SWCNTs in the first 3 days (lag phase), then grows quickly in the following several days. As ThT fluorescence intensity is proportional to the amount of the βsheet structures,37, 38 by combining with our AFM results shown in Fig. 1 and Fig. S3, it is inferred that the wraps formed in lag phase contain β-sheet structures. In addition, the lag phase becomes short compared with pure Aβ1-40 sample, which is consistent with the results reported that SWCNTs promoted the process of Aβ nucleation.28 With the increase of incubation time, the ThT fluorescence intensity of the Aβ1-40-SWCNT solution grows more slowly than that of Aβ1-40 solution. After 25 days’ incubation, the ThT intensity of Aβ1-40-SWCNT reduced about 40 percent compared with Aβ1-40 sample. The results suggest that the presence of SWCNTs in Aβ140

solution can promote the surface adsorbed peptides forming into non-fibrillar aggregates but

reduce the formation of fibrils finally, which is in line well with the large reduction of mature fibrils in the AFM observation. In previous simulation researches, CNTs could reduce the formation of β-sheet structures dramatically by absorbing all of the peptides in a several-peptides system (eight peptides and one CNT).32 however, the results were different in this work. when in an open system (about 40:1 of


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peptides : SWCNTs), some of the peptides and preformed oligomers were adsorbed on SWCNTs directly, while the rest would form oligomers in the aqueous solution (Fig. 2(a)). Those free oligomers will also tend to be adsorbed on peptides-wrapped-SWCNT and make a locally high concentration to promote the aggregation (Fig. 2(b)), which shorten the lag phase of Aβ1-40 peptides. However, the number of final formed fibrils is limited. Furthermore, the dosedependent inhibitory effect was explored by ThT fluorescence. Typical ThT fluorescence spectra of Aβ1-40-SWCNTs after 20-day-incubation with different mass ratio of Aβ1-40: SWCNTs = 2:1, 5:1, 10:1 and 20:1 (equivalent molar ratio equal to 31:1, 76:1, 155:1 and 310:1 respectively) were presented in Fig. 4(b), together with the control spectra of pure fibrils and free ThT solution. Their normalized ThT fluorescence intensities (Fig. 4(c)) show that the inhibitory effects by SWCNTs becomes stronger with the increasing percentage of SWCNTs. This is because more Aβ1-40 peptides can be adsorbed on the surface of SWCNTs with the increasing number of SWCNTs, while less peptides are left in solution to form fibrils finally. In addition, SH-SY5Y cell viability (CV) was applied to investigate the cytotoxicity of Aβ1-40SWCNT aggregates. The measured samples have a final concentration of

Interaction Dynamics in Inhibiting the Aggregation of AÎ² Peptides by

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