Regulation And Molecular Mechanism Of TiO₂ Nanoparticles On Amyloid Aβ₄₂ Aggregation

Nanoparticles（NPs）have been extensively applied in biomedicine,food packaging,food additives,cosmetics,coatings,etc.,due to their small size,unique surface properties,stable physical and chemical properties and good biocompatibility.Among them,metal oxide nanoparticles are increasingly used in industrial production and human living environment,and the human body and other organisms are exposed to the tiny particles more frequently.The potential risks of NPs to human body and environment have gradually attracted people’s attention.When these NPs enter the biological environment,they may pass through the blood-brain barrier and change its permeability,adsorb peptides and proteins in the brain and interact with them.For proteins,the structure or spatial conformation is changed,the normal function is adversely affected.In addition,the surface properties of NPs and the structure of the protein have an important impact on the interaction performance between them.Therefore,researching the interaction of metal oxide NPs with peptides and proteins in the brain will help us to evaluate their biosafety comprehensively and deeply.β-amyloid protein（Aβ）is generated by amyloid precursor protein（APP）unde proteolytic action ofα,β-andγ-secretase.Aβmonomers themselvs are in a disordered state,under the conditions of p H and temperature change,they interact with each other to formβ-sheet or partiallyβ-sheet states,assembleβ-sheet oligomers of different sizes at their own recognition sites and further form long fibrils.The soluble oligomers formed during the early aggregation of Aβpeptide have been hypothesized to be the major toxic substance of amyloid production.Factors that accelerate and increase the formation of the oligomers may increase the risk of AD disease.Aβ₄₀ and Aβ₄₂ are the two main components of amyloid plaques,and the extension of C-terminal for Aβ₄₂ significantly increases its hydrophobicity and aggregation speed,leading to toxicity and aggregation tendency much greater than Aβ₄₀.At present,it has been reported that metal oxide NPs affect the aggregation process mainly attribute to two competitive effects of Aβ₄₂ peptide on the surface.The two effects are mediated nucleation of metal oxide NPs surface and its influence of Aβ₄₂ monomer concentration on the surface and in solution.However,the reasons for the aggregation effect of Aβ₄₂ peptide have not been completely explained,and there are few reports on the interaction and mechanism between metal oxide NPs and Aβ₄₂ peptide.Therefore,it is essential to understand the details about the effect of metal oxide NPs on the aggregation process of Aβ₄₂ peptide.This study will help us to evaluate the application of these NPs in the biomedical field more comprehensively.In this work,titanium dioxide nanoparticles（TiO₂ NPs）were selected due to their wide range of applications,and the effects of exposed TiO₂ NPs and amino modified TiO₂-NH₂ NPs on Aβ₄₂ aggregation were compared in vitro.It was found that both TiO₂NPs and TiO₂-NH₂ NPs could accelerate the early oligomerization trend of Aβ₄₂ peptide.TiO₂-NH₂ NPs with surface charge changed could adsorb Aβ₄₂ peptide more obviously and interact with Aβ₄₂peptide.The conformation and aggregation morphology of Aβ₄₂were changed,and more toxic substances were formed.Finally,through molecular dynamics simulation analysis,it was found that TiO₂-NH₂ NPs could stabilize theβ-sheet conformation formed by Aβ₄₂ oligomer through hydrogen bonds and hydrophobic interaction,and reduce its transformation to other conformations.The effects of different TiO₂NPs on the early aggregation of Aβ₄₂ peptide and the corresponding molecular mechanism were revealed.The main research contents of this paper are as follows:PartⅠ:This section aims to explore the effects of two TiO₂ NPs with different surface properties on Aβ₄₂ aggregation and related toxicity.In this chapter,TiO₂ NPs and TiO₂-NH₂ NPs were used as nano models,Aβ₄₂ peptide was used asβ-amyloid protein model for in vitro study.It was found that TiO₂ NPs and TiO₂-NH₂NPs with different particle sizes could promote the aggregation of Aβ₄₂ peptide in different degrees by Th T fluorescence,and showed concentration dependent effect.Th T aggregation kinetics experiments showed that TiO₂ NPs and TiO₂-NH₂NPs with particle size of 25 nm could accelerate the early aggregation of Aβ₄₂ peptide and promote the formation ofβ-sheet structure.This effect was related to the surface charge.It was found that TiO₂-NH₂ NPs could adsorb and bind more Aβ₄₂ monomers on the surface to quench the tyrosine fluorescence signal by Fluorescence titration experiments.They could improve the nucleation rate of Aβ₄₂ by binding locally high concentration monomers on the surface,thus promoting the formation ofβ-sheet structure.The result further demonstrated that the interaction of TiO₂ NPs and TiO₂-NH₂ NPs with Aβ₄₂ peptide would lead to the loosening and unfolding of the protein skeleton,and the interaction between TiO₂-NH₂NPs and Aβ₄₂peptide could change the proportion of secondary structure and increase theβ-sheet conformation significantly through CD spectroscopy experiments.It was also observed that TiO₂ NPs and TiO₂-NH₂ NPs could change the aggregation pathway of Aβ₄₂,influence the size and morphology of Aβ₄₂aggregates by AFM and DLS experiments.TiO₂ NPs and TiO₂-NH₂ NPs were co-cultured with Aβ₄₂peptide at certain concentration to produce oligomers,respectively.Both cell viability and LDH release assays showed that the cytotoxicity of oligomers was enhanced.It was confirmed that the two kinds of NPs formed aggregates with different structures or increased toxic aggregates after interaction with Aβ₄₂peptide.In summary,it was found that TiO₂ NPs had an important effect on the aggregation process of Aβ₄₂peptide,and their surface properties played a key role in determining their interaction with Aβ₄₂ peptide.Furthermore,we predicted that TiO₂ NPs would be very dangerous to interact with Aβ₄₂peptide in the brain,and long-term exposure in the environment would accelerate and increase the formation of this oligomer,thereby increasing the risk of AD disease.Therefore,it was very important to understand the details about the effect cause of TiO₂ NPs on the aggregation process of Aβ₄₂peptide,and it was necessary to further explore the interaction mechanism at the molecular level.PartⅡ:This section aims to explore the possibility of interaction between two kinds of TiO₂ NPs with different surface properties and Aβ₄₂ peptide,and predict their binding affinity and interaction modes.Refer to the experimental results in the previous chapter,TiO₂ and TiO₂-NH₂ nano surface,Aβ₄₂ monomer and Aβ₄₂ trimer molecular models were constructed by Visualizer module of MS 7.0 software,PDB protein database and molecular simulation calculation.The molecular docking method was used to simulate the docking of two structural forms of Aβ₄₂ protein on the nano surface.The results showed that the interaction sides between nano surfaces and Aβ₄₂ monomer with helix form were mainly at the central helical part of the Aβ₄₂ monomer peptide chain and the local hydrophilic N-terminus,while the interactions between nano surfaces and Aβ₄₂trimer withβ-sheet form were mainly located in the middle segment of the three peptide chains.From the perspective of binding energy,whether bound to Aβ₄₂ monomer or Aβ₄₂trimer,the binding affinity of TiO₂-NH₂ surface with amino modification was stronger than that of TiO₂surface with hydroxyl group,and the number of amino acids involved in the interaction was more than the latter.It could be preliminarily judged that TiO₂-NH₂surface was more prone to interact with Aβ₄₂protein.In addition,the binding affinity of TiO₂ with two different surface modifications to Aβ₄₂ trimer was generally higher than that of Aβ₄₂ monomer,which might be related to the secondary structure form of Aβ₄₂trimer itself.To summarize,the above docking experimental results could preliminarily predict that TiO₂ and TiO₂-NH₂ surfaces were prone to adsorb and combine Aβ₄₂ trimer,and interact with it.It had been reported that Aβ₄₂ oligomer inβ-sheet form was the main characteristic of amyloid fiber precipitation.In addition,compared with TiO₂ surface,TiO₂-NH₂ surface was easier to interact with Aβ₄₂ protein.PartⅢ:This section aims to explore the mechanism of interaction between TiO₂NPs with different surface properties and Aβ₄₂ monomer.Based on the molecular models and docking results in Chapter 3,the adsorption models of TiO₂ NPs with different surface properties and Aβ₄₂ monomers were constructed by all-atom molecular dynamics simulation method.In the molecular dynamics simulation,vmd was used to visually show the molecular simulation trajectories of the two kinds of TiO₂ NPs surfaces and Aβ₄₂monomers system.It was found that TiO₂ NPs and TiO₂-NH₂ NPs surfaces could quickly adsorb Aβ₄₂ monomers in the binding solution and increase the monomer concentration locally on their surfaces due to their special properties.RMSD and RMSF were calculated to evaluate the kinetic equilibrium state of each simulation system and the stability of Aβ₄₂ peptide.The results showed that the binding of TiO₂ NPs surface to Aβ₄₂ monomers reduced the deformation of Aβ₄₂ monomers,and the binding of TiO₂-NH₂ NPs surface to Aβ₄₂ monomers was more stable.Then,the changes of the secondary structure and protein conformation of Aβ₄₂ monomers which were adsorbed on the two kinds of TiO₂ NPs surfaces were analyzed by DSSP program,Rg,SASA,FEL and clusters,etc.It was found that the local concentration and stability of Aβ₄₂ monomers were increased by rapid adsorption on the two kinds of TiO₂ NPs surfaces when they exposed to Aβ₄₂ monomers solution,and tended to promote them further aggregate.The results of binding sites and molecular potential energy analysis displayed that the interaction between TiO₂ NPs and Aβ₄₂ monomers was mainly characterized by electrostatic interaction such as hydrogen bond and ionic bond（include salt bridge）,and the electrostatic interaction was mainly distributed in the N-terminal binding region and turn binding region of Aβ₄₂ peptide.When these monomers contacted fully with TiO₂ NPs surface,the helical structures of Aβ₄₂ monomers were maintained and stabilized through hydrogen bond.However,the internal interaction of the polypeptides would be affected to some extent,resulting in the transformation of partial random coil and turn structures to other structures,but could not convert helix structure intoβ-sheet structure.TiO₂-NH₂ NPs modified by amino groups were equipped with alkyl chains.TiO₂-NH₂ surface interacted with Aβ₄₂ monomers after adsorbing them,formed stable intermolecular hydrogen bond and hydrophobic interaction in many parts of the peptide chain to further enhance the adsorption.To conclude,the calculation results in this chapter showed that the surfaces of both TiO₂ NPs and TiO₂-NH₂ NPs could increase the generation of oligomer by adsorbing and binding of locally high concentration of Aβ₄₂ monomers,which was consistent with the previous experimental results.According to the results of interactions,it was found that more amino acid residues interacting on its surface were generated and the adsorption of TiO₂-NH₂ surface was enhanced to stabilize the structure of polypeptides and maintain more helix structures which were attributed to more hydrogen bonds and obvious hydrophobicity when Aβ₄₂ monomers contacted fully with TiO₂-NH₂ surface.However,the internal interaction of the polypeptides would be affected to some extent,and the helix structure could not be converted toβ-sheet.PartⅣ:This section aims to explore the mechanism of interaction between two kinds of TiO₂ NPs and Aβ₄₂ oligomers.In this chapter,Aβ₄₂ trimer was used as the oligomer model,the molecular models of TiO₂ NPs with different surface properties and Aβ₄₂trimer were optimized by all-atom molecular dynamics simulation.In the molecular dynamics simulation,the vmd results reflected that the two kinds of TiO₂NPs could make the orientation of Aβ₄₂ trimer rotate towards the surface substrate in different directions by adsorbing and binding Aβ₄₂trimer on their surfaces.At the same time,a fewβ-sheet structures formed in advance were destroyed by TiO₂ NPs surface.The structure of Aβ₄₂trimer could be stabilized after having been adsorbed on the surface of TiO₂-NH₂ NPs.The kinetic equilibrium state of each simulation system and the stability of Aβ₄₂ trimer were proved by RMSD and RMSF.DSSP results showed that the secondary structure of Aβ₄₂ trimer could change significantly in the presence of TiO₂-NH₂ NPs.Compared with TiO₂ NPs,TiO₂-NH₂ NPs could make Aβ₄₂ protein keep moreβ-sheet structures and less random coil structures.Then,we analyzed the effects of the two kinds of TiO₂ NPs on the conformation of Aβ₄₂ trimer through Rg,SASA,FEL and clusters analysis.The strong absorption characteristics of TiO₂-NH₂ NPs to Aβ₄₂ trimer were proved directly at the molecular level.Aβ₄₂ trimer peptide chain could be partially or completely adsorbed on the surface of TiO₂-NH₂ NPs to stabilize or increase theβ-sheet structure.The results of binding sites and molecular potential energy analysis showed that the interaction between TiO₂ NPs and Aβ₄₂ trimer was also mainly characterized by electrostatic interaction and partial hydrophobic interaction,the secondary structures of Aβ₄₂ trimer were maintained and stabilized.While interacting with TiO₂-NH₂ NPs whose surface were modified with amino groups,in addition to hydrogen bond,the interaction was mainly characterized by hydrophobic interaction.Hydrogen bond and hydrophobic interaction could further enhance the adsorption of nano surface,the amino modified surface could better stabilize theβ-sheet conformation formed spontaneously by Aβ₄₂ oligomers and reduce the transformation to other conformations.In conclusion,the research of this chapter demonstrated that TiO₂-NH₂ NPs could promote the early aggregation of Aβ₄₂ and accelerate the formation of oligomers.The oligomers formed by Aβ₄₂ peptides were mainly rich inβ-sheet structure in the early aggregation process.It had been confirmed that amyloid polypeptides with low tendency to form aggregates could be accelerated by interacting with NPs which had strong attraction,and oligomers could be promoted to form fibrils by increasing the local concentration of Aβ₄₂ on the surface.Meanwhile,the surface charge and hydrophobicity of inorganic nanoparticles determine the structures of Aβaggregates and their formation kinetics at the interface.Therefore,in a sense,the research results of this paper can explain the effects and the corresponding molecular mechanisms of TiO₂ NPs on Aβ₄₂aggregation,and provide a new understanding of the different pathways of Aβ₄₂aggregation in the presence of nanoparticles.It is of great significance for studing the aggregation theory of Aβ₄₂ peptide and the further application of TiO₂ NPs in biomedicine.