Study On Nanomaterials Based On Lysozyme-derived Amyloid Fibrils

Amyloid fibrillation is a unique type of protein and peptide aggregation process.The abnormal accumulation of protein and peptide amyloid fibrils in the tissues and organs in human body has been shown to be relevant to several devastating human diseases.These diseases include Alzheimer’s disease,Parkinson’s disease,type II diabetes,and mad cow disease.Due to its disease significance,research about amyloid fibrillation has attracted a lot of attention in the past several decades around the world.In addition,people have found that amyloid protein fibrils possess good material properties.Now more and more scientists begin to pay attention on amyloid-based materials and their functions.Amyloid-based novel nanomaterials have showed great potential in the biomedical field.This study aims to use lysozyme,a natural origin protein,to prepare amyloid-fibril-based nanomaterials.Using lysozyme hydrolyzed peptide fragments as a low-cost and natural origin peptide source,this study explores to prepared amyloid-based injectable hydrogel and antibacterial agent.This works should be able to provide insights into the development of novel biomaterials with amyloid as their feature structures.In the study of injectable hydrogel based on amyloid fibrils,amyloidogenic peptides were obtained through the hydrolysis of lysozyme and a novel injectable hydrogel was prepared through a proper recipe using these amyloidogenic peptides plus magnesium ion.Rheological analysis demonstrated the good performance of the as-prepared hydrogel.Factors affecting the gelation properties of the hydrogel were investigated.The extent of the hydrolysis degree extent was found to be an important factor affecting the injectability.An ANS fluorescence method was proposed to be a way to accurately control the extent of hydrolysis of lysozyme.At last,using doxorubicin as a model compound,the potential of this amyloid-based hydrogel as an injectable drug carrier was explored.Three different types of amyloid fibrils by the peptides derived from lysozyme hydrolysis with different morphologies were prepared under different temperatures and their antibacterial activities were evaluated using E.coli as a model.The amyloid fibril prepared under 65?C showed the best antibacterial performance.The interaction mechanism of amyloid fibril with bacteria was investigated using FTIR spectroscopy combined with chemometrics analysis.FTIR spectral analysis indicated that the antibacterial activities of the three types of amyloid fibrils were mainly due to their destructive effects on bacterial cell membrane.This argument was also supported by transmission electron microscopy study.Results from the chemometrics method of principle component analysis showed that the antibacterial mechanisms of the three different types of amyloid fibrils were different.Based on the results from atomic force microscopy and mass spectrometry,it was proposed that the different antibacterial mechanisms of the three different types of amyloid fibrils were due to their different morphologies and different peptide compositions.The amyloid fibril based on the peptides derived from the hydrolysis of the natural protein of lysozyme provides useful insight into the preparation and design of novel antibacterial agents.Techniques involved in this study include Fourier transform infrared(FTIR)spectroscopy,atomic force microscopy(AFM),Thioflavin T(ThT)fluorescence spectroscopy,Congo red(CR)assay,mass spectrometry(MS),transmission electron microscopy(TEM),scanning electron microscopy(SEM),rheological analysis,cytotoxicity assay,antibacterial assay,chemometrics analysis.In the perspective part of this thesis,we discussed how to efficiently utilize the natural-origin protein to prepare amyloid-fibril-based materials.We hope our exploration study described herein is beneficial for the design and preparation of novel nanomaterials based on amyloid fibril structure.