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Yaqin Liu, Yan Zheng, Shaoyuan Li, Jinhan Li, Xiaoyu Du, Yanyun Ma, Guofu Liao, Qing Wang, Xiaohai Yang, Kemin Wang. Contradictory effect of gold nanoparticle-decorated molybdenum sulfide nanocomposites on amyloid-β-40 aggregation[J]. Chin. Chem. Lett., 2020, 31(12): 3113-3116  

Yaqin Liu, Yan Zheng, Shaoyuan Li, Jinhan Li, Xiaoyu Du, Yanyun Ma, Guofu Liao, Qing Wang^*, Xiaohai Yang, Kemin Wang^*     

Amyloid-β (Aβ) aggregation into fibrils is a pathological hallmark of Alzheimer's disease (AD) [1]. The major isoforms of Aβ are Aβ₄₀ and Aβ₄₂, which differ only in the two extra Cterminal residues. Compared with Aβ₄₂, Aβ₄₀ has higher solubility and lower aggregation propensity, which makes it the preferred peptide for in vitro kinetic analysis [2]. According to previous reports, a variety of materials such as nanoparticles [3, 4], antibodies [5], peptides [6], and organic compounds [7], are interfere with the amyloid aggregation process. Compared with other aggregation interfering agents, the advantages of nanoparticles mainly focus on their high surface ratio and easy functionalization. In addition, nanoparticles can also provide the possibility to pass blood-brain barrier (BBB) [8, 9]. Especially, nanocomposites combine many advantages of different nanoparticles such as better photocatalytic, and optical properties [10, 11]. They have gradually attracted increasing interests in the field of biomedicine. However, only few nanocomposites (graphene oxide-iron oxide; graphene oxide/gold) have been reported to show inhibitory or accelerative effects on amyloid aggregation [12, 13].

As a typical two-dimensional (2D) nanomaterial, molybdenum sulfide nanosheet (MoS₂) has attracted wide attention in the field of electronics, biochemistry, and clinical medicine [14]. Compared with MoS₂, gold nanoparticle-decorated molybdenum sulfide (AuNP-MoS₂) nanocomposites showed superior photocatalytic, electrochemical, and biocompatibility properties than that of MoS₂ [15]. Therefore, it was more suitable for biomedical applications. In previous studies, Wang et al. have showed that MoS₂ was a promising 2D nanomaterial for modulating amyloid aggregation [16]. Yang et al. showed that MoS₂-AuNR nanocomposites could enhance the ability of disrupting Aβ fibrils under NIR irradiation [17]. However, the mechanism and influence of AuNP-MoS₂ nanocomposites on amyloid aggregation still remained to be explored. In addition, the concentration effect of nanomaterials on fibrillation was controversial. Ramshini et al. found low concentrations of Ag-NPs could inhibit the aggregation of HEWL, while high concentrations of Ag-NPs could accelerate the aggregation of HEWL [18]. However, Radic et al. used molecular simulation technology to speculate that low NP/protein ratio promoted protein aggregation, while high NP/protein ratio displayed an inhibitive effect [19]. Therefore, the concentration of nanomaterials was critical especially.

In this work, the effect of different concentrations of AuNP-MoS₂ nanocomposites on Aβ₄₀ aggregation was studied using atomic force microscopy (AFM), thioflavin T (ThT) fluorescence spectra and circular dichroism (CD) spectra. The results showed that low concentration of AuNP-MoS₂ nanocomposites accelerated Aβ₄₀ fibrils aggregation and high concentration of AuNP-MoS₂ nanocomposites inhibited Aβ₄₀ aggregation process. The contradictory effect on Aβ₄₀ aggregation may be caused by the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides, and the distinctive adsorption efficiency of AuNP-MoS₂ nanocomposites, which were certified by ultraviolet-visible (UV–vis) absorption spectra and tyrosine intrinsic fluorescence spectra. This work could provide new ideas for investigating the interactions between nanocomposites and amyloid proteins.

AuNP-MoS₂ nanocomposites were first prepared by hydrothermal synthesis method [20]. The synthesized AuNP-MoS₂ nanocomposites were then characterized. As shown in Fig. S1A (Supporting information), the absorption peak of MoS₂ solution was 304 nm. (curve b). After adding HAuCl₄ into the MoS₂ solution, the absorption peak of the HAuCl₄ at 290 nm (curve a) decreased obviously. At the same time, a new absorption peak at around 537 nm emerged, which suggested the consumption of Au³⁺ and the formation of AuNPs (curve c) [21]. XRD was also used to characterize the structure of AuNP-MoS₂ nanocomposites. As shown in Fig. S1B (Supporting information), strong diffraction peaks emerged at 38.2, 44.6, 64.7, 77.7, and 82.0, which could be assigned to the (111, 200, 220, 311, and 222) planes of Au respectively (JCPDS No. 04-0784) [22]. TEM was also used to characterize AuNP-MoS₂ nanocomposites. As shown in Fig. S1C (Supporting information), the AuNPs around 15 nm were modified on MoS₂ nanosheets. The synthesis of AuNP-MoS₂ nanocomposites was also investigated by scanning transmission electron microscope-energy dispersive X-ray spectroscopy (STEM-EDX) maps. As shown in Fig. S1D (Supporting information), there were three elements from the signals, Mo (image b), Au (image c), and S (image d). The above results implied the formation of AuNP-MoS₂ nanocomposites.

Given that AFM has a comparative advantage in the imaging of biological samples [23-25], the influence of AuNP-MoS₂ nanocomposites on Aβ₄₀ aggregation were evaluated by AFM. Fig. 1 showed the AFM images of Aβ₄₀ peptides with different concentrations of AuNP-MoS₂ nanocomposites from different time points. As shown in Fig. 1A, the Aβ₄₀ fibrils gradually formed until 24 h as the AuNP-MoS₂ nanocomposites were absent. As shown in Fig. 1D, short Aβ₄₀ fibrils could be observed when the incubation time was 3 h. However, without the presence of AuNP-MoS₂ nanocomposites, the Aβ₄₀ peptides were still in monomeric or oligomeric status after 3 h of incubation (Fig. 1A). The results showed that AuNP-MoS₂ nanocomposites could shorten the nucleation stage of Aβ₄₀ peptides. Furthermore, the number and length of Aβ₄₀ fibrils also increased after Aβ₄₀ peptides incubated with 3 pmol/L AuNP-MoS₂ nanocomposites for 8 h and 24 h, which showed the aggregation process was accelerated. However, the number of Aβ₄₀ fibrils decreased slightly after 8 h or 24 h as 39 pmol/L or 60 pmol/L AuNP-MoS₂ nanocomposites was present, which showed the aggregation process was inhibited at elongation stage [26].

Figure 1

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Fig. 1. AFM images of Aβ40 peptides with various concentrations of AuNP-MoS2 nanocomposites. The concentrations of AuNP-MoS2 nanocomposites were 0 pmol/L (A), 3 pmol/L (B), 39 pmol/L (C) and 60 pmol/L (D), respectively.

The effect of AuNP-MoS₂ nanocomposites on Aβ₄₀ aggregation was also evaluated by ThT fluorescence spectra [25, 27]. According to the previous research [17], the aggregation of Aβ₄₀ is a two-step reaction. The initial lag period reflected the thermodynamic barrier to form a nucleation "seed", then a rapid fibril propagation and aggregation stage were followed. As shown in Fig. 2A, the addition of AuNP-MoS₂ nanocomposites significantly decreased the lag time of Aβ₄₀, implying that the AuNP-MoS₂ nanocomposites enhanced the nucleus formation. Interestingly, 3 pmol/L AuNP-MoS₂ nanocomposites increased ThT fluorescence intensity of Aβ₄₀ by about 25%, while 39 pmol/L and 60 pmol/L AuNP-MoS₂ nanocomposites decreased ThT fluorescence intensity of Aβ₄₀ by almost 30% and 60%, respectively. It indicated that the Aβ₄₀ aggregation process was inhibited. The results of ThT fluorescence were consistent with that of AFM imaging. To further study the effects of AuNP-MoS₂ nanocomposites on Aβ₄₀ fibrillation, different concentrations of AuNP-MoS₂ nanocomposites (3, 39 and 60 pmol/L) were added to Aβ₄₀ solution at nucleation stage. As shown in Fig. 2B, the Aβ₄₀ peptides were incubated for 1 h to grow to oligomers. Then different concentrations of AuNP-MoS₂ nanocomposites were incubated with oligomers solution for 23 h afterwards. The results of kinetic curves showed that there was no significant difference with or without AuNP-MoS₂ nanocomposites (Fig. 2B). The results showed that the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides was at monomeric status. In addition, the effect of MoS₂ or AuNPs on Aβ₄₀ aggregation was also discussed. As shown in Fig. S2A (Supporting information), low or high concentration of MoS₂ only showed inhibition effect on Aβ₄₀ aggregation. The concentration of MoS₂ was consistent with MoS₂ in AuNP-MoS₂ nanocomposites here, i.e., 0.9 nmol/L MoS₂ was consistent with MoS₂ in 3 pmol/L AuNP-MoS₂ nanocomposites, and 12 nmol/L MoS₂ was consistent with MoS₂ in 39 pmol/L AuNP-MoS₂ nanocomposites. Fig. S2B (Supporting information) showed 3 pmol/L AuNPs had no effect on Aβ₄₀ aggregation, while 39 pmol/L AuNPs showed inhibition effect on Aβ₄₀ aggregation. The results were consistent with the previous researches [16, 28]. Comparing with MoS₂ and AuNPs, AuNP-MoS₂ nanocomposites showed concentration dependent roles for Aβ₄₀ aggregation.

Figure 2

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Fig. 2. ThT fluorescence spectra and CD spectra of Aβ40 peptides with different concentrations of AuNP-MoS2 nanocomposites. The concentrations of AuNP-MoS2 nanocomposites were 0, 3, 39, and 60 pmol/L, respectively. (A) ThT fluorescence spectra of Aβ40 peptides with different concentrations of AuNP-MoS2 nanocomposites. The addition time was 0 h. (B) ThT fluorescence spectra of Aβ40 peptides with different concentrations of AuNP-MoS2 nanocomposites. The addition time was 1 h. (C) CD spectra of Aβ40 peptides with different concentrations of AuNP-MoS2 nanocomposites.

CD spectra were also used to investigate the transition of Aβ₄₀ peptides structure with AuNP-MoS₂ (Fig. 2C). After Aβ₄₀ peptides were incubated for 24 h, CD spectrum of Aβ₄₀ fibrils showed a negative valley at around 218 nm, which indicated the existence of β-sheet structure [29]. In the presence of AuNP-MoS₂ nanocomposites, different changes appeared in the CD spectra after Aβ₄₀ peptides were incubated for 24 h. When the concentration of AuNP-MoS₂ nanocomposites was 3 pmol/L, the intensity of negative peak at 218 nm increased, indicating the increase in content of Aβ₄₀ peptides β-sheet. However, when the concentration of AuNP-MoS₂ nanocomposites was 39 or 60 pmol/L, the negative peak at 218 nm became weaker, indicating the decrease content of Aβ₄₀ peptides β-sheet. The results showed low concentration of AuNP-MoS₂ nanocomposites increased the content of Aβ₄₀ peptides β-sheet, while high concentration of AuNP-MoS₂ nanocomposites decreased the content of Aβ₄₀ peptides β-sheet. The CD experiment also supported the results of AFM and ThT fluorescence experiments.

Next, the influence of AuNP-MoS₂ nanocomposites on preformed Aβ₄₀ fibrils was evaluated by AFM, and ThT fluorescence spectra. Fig. S3 (Supporting information) showed the AFM images of Aβ₄₀ fibrils in the presence of different concentrations of AuNP-MoS₂ nanocomposites from different time points. The results showed that the Aβ₄₀ fibrils dissociated gradually with the incubation of 60 pmol/L AuNP-MoS₂ nanocomposites, implying that AuNP-MoS₂ nanocomposites could affect the properties of preformed Aβ₄₀ fibrils. In addition, the ThT experiment also verified the results of AFM experiments. As shown in Fig. S4 (Supporting information), as 60 pmol/L of AuNP-MoS₂ nanocomposites was present, the ThT fluorescence intensity of Aβ₄₀ fibrils decreased gradually with increasing incubation time, implying the depolymerization of Aβ fibrils. It could be due to fibril sedimentation as described in the previous literature [28]. In addition, 30 pmol/L AuNP-MoS₂ nanocomposites were also used to explore the depolymerization of AuNP-MoS₂ nanocomposites on preformed Aβ₄₀ fibrils (Fig. S5 in Supporting information). It showed that the effect of 30 pmol/L AuNP-MoS₂ nanocomposites was not obvious than that of 60 pmol/L AuNP-MoS₂ nanocomposites. The results showed that high concentration of AuNP-MoS₂ nanocomposites may depolymerize the Aβ₄₀ fibrils.

Taken the SH-SY5Y cell as a model, the effects of AuNP-MoS₂ nanocomposites on Aβ₄₀ peptides mediated cytotoxicity of cells was then studied [30]. As shown in Fig. 3A, the cell viability was higher than 80% after the cell was treated with concentration of < 39 pmol/L AuNP-MoS₂ for 48 h, implying the low cytotoxicity of AuNP-MoS₂. The cytotoxicity of Aβ₄₀ peptides with different concentrations of AuNP-MoS₂ nanocomposites was then investigated. As shown in Fig. 3B, the cell viability decreased gradually with the treatment of increasing Aβ₄₀ peptide concentration when the AuNP-MoS₂ nanocomposites were absent. When the AuNP-MoS₂ nanocomposites were present, the effects of different concentrations of AuNP-MoS₂ nanocomposites on Aβ₄₀ peptides mediated cytotoxicity of cells were different. Taken 15 μmol/L Aβ₄₀ peptides as an example, when 3 pmol/L AuNP-MoS₂ was added to Aβ₄₀ peptides, the cytotoxicity of the peptides increased, and the cell viability decreased from 51.6%–34.1%. On the contrary, the 39 pmol/L AuNP-MoS₂ decreased the cytotoxicity of the peptides apparently, and the cell viability increased from 51.6% to about 73.5%. The results showed that different concentrations of AuNP-MoS₂ nanocomposites could regulate the neurotoxicity of amyloid peptides. In addition, although 60 pmol/L of AuNP-MoS₂ could depolymerize the Aβ₄₀ fibrils (Fig. S4), it is slightly toxic to cells (Fig. 3A). Therefore, it implied the importance of appropriate concentration of AuNP-MoS₂ on Aβ₄₀ aggregation.

Figure 3

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Fig. 3. (A) Cell viability of SH-SY5Y cells with different concentrations of AuNP-MoS2. The concentrations of AuNP-MoS2 nanocomposites were 0, 1, 3, 6, 13, 26, 39 and 60 pmol/L, respectively. (B) Effects of AuNP-MoS2 nanocomposites with different concentrations (0, 3, 39 pmo/L) on Aβ40-mediated cytotoxicity of SH-SY5Y cells.

According to the results, it was shown that low concentration of AuNP-MoS₂ nanocomposites could not only enhance the nucleus formation of Aβ₄₀ peptides, but also promote Aβ₄₀ fibrils aggregation. However, although high concentration of AuNP-MoS₂ nanocomposites could enhance the nucleus formation of Aβ₄₀ peptides, it eventually inhibited Aβ₄₀ aggregation process. The possible mechanism may be explained as shown in Fig. 4. Fig. 4A showed the normal pathway of Aβ₄₀ aggregation without AuNP-MoS₂ nanocomposites. In the presence of low concentration of AuNP-MoS₂ nanocomposites, it acted as nuclei as demonstrated in the previous works [31], and then Aβ₄₀ peptides aggregated around the AuNP-MoS₂ nanocomposites, resulting in the acceleration of the nucleation process (Fig. 4B). With increasing concentration of AuNP-MoS₂ nanocomposites, the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides may limit the structural flexibility of Aβ₄₀ peptides for conformational conversion, resulting in the inhibition of Aβ₄₀ fibrils (Fig. 4C).

Figure 4

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Fig. 4. Mechanism diagram of concentration dependent effect of AuNP-MoS2 nanocomposites on Aβ40 aggregation.

In the hypothesis, the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides was mentioned. The UV–vis absorption spectra and tyrosine intrinsic fluorescence spectra were used for investigating the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides. As shown in Fig. S6 (Supporting information), the absorption peak of AuNP-MoS₂ nanocomposites broadened gradually with increasing concentration of Aβ₄₀ peptides, which indicated Aβ₄₀ peptides could trigger the aggregation of AuNP-MoS₂ nanocomposites. The interactions between AuNP-MoS₂ and Aβ₄₀ peptides could also be proved by tyrosine intrinsic fluorescence. As shown in Fig. S7 (Supporting information), a quenching of the tyrosine fluorescence signal at 315 nm was observed when Aβ₄₀ peptides were incubated with AuNP-MoS₂ nanocomposites, which indicated that AuNP-MoS₂ could absorb Aβ₄₀ peptides. As a contrast, the tyrosine fluorescence of Aβ₄₀ peptides incubated with AuNPs, or MoS₂ was also evaluated (Figs. S8 and S9 in Supporting information). Moreover, the binding constant and the number of binding sites could be got from the double logarithm regression curve (Eq. 1) [32].

(1)

where F and F₀ are the fluorescence intensities with and without AuNP-MoS₂ nanocomposites, respectively. K is the binding constant for quencher-protein interaction; n is the number of binding sites of Aβ₄₀ peptides. [Q] is the concentration of AuNP-MoS₂ nanocomposites. The values of K and n could be calculated from the slope and intercept by plotting log(F₀-F)/F against log[AuNP-MoS₂]. The binding constant K and binding sites n of AuNP-MoS₂ to Aβ₄₀ peptides were 2.82 × 10¹⁵ and 1.36, respectively. The K and n of AuNPs or MoS₂ to Aβ₄₀ peptides could be calculated as shown in Table S1 (Supporting information). The results showed that the binding sites of AuNP-MoS₂, AuNPs, or MoS₂ to Aβ₄₀ peptides were essentially identical. However, the binding constant of AuNP-MoS₂ to Aβ₄₀ peptides was much higher than that of AuNPs and MoS₂, indicating that AuNP-MoS₂ was easier to combine with Aβ₄₀ than AuNPs and MoS₂. From the results, it was also speculated that the binding constant for AuNP-MoS₂/protein interaction may increase with the increasing surface density of AuNP on MoS₂. The contradictory effect of Aβ₄₀ fibrillation was caused by the ratio of peptides adsorbed on AuNP-MoS₂ nanocomposites against free peptides in solution. Presumably, higher binding constant may increase the ratio of peptides adsorbed on AuNP-MoS₂ nanocomposites, which will reduce the aggregation lag phase of Aβ₄₀, and the concentration border line between the two phenomena.

In summary, we explored the effect of AuNP-MoS₂ on Aβ₄₀ aggregation. Compared with AuNPs and MoS₂, AuNP-MoS₂ nanocomposites played different roles on Aβ₄₀ aggregation. It was concluded that low concentration of AuNP-MoS₂ nanocomposites accelerated the nucleation process and elongation process. High concentration of AuNP-MoS₂ nanocomposites accelerated the nucleation process, but it eventually inhibited Aβ₄₀ aggregation process. The phenomenon might be caused by the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides, and the ratio of peptides adsorbed on AuNP-MoS₂ nanocomposites against free peptides in solution. The results demonstrated the possibility to regulate amyloid peptide aggregation and depolymerization, which might have potential applications to provide an effective way of treating related diseases.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by the National Natural Science Foundation of China (Nos. 21375034, 21675047 and 21735002) and Natural Science Foundation for Distinguished Young Scholars of Hunan Province (No. 2016JJ1008).

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2020.04.052.