Construction And Bioanalysis Application Of Peptide Based Nanomaterials

Short peptides have attracted much attention in the field of bionanotechnology due to their simple structure,adjustable function,low cost.The nanomaterials based on self-assembly of short peptides have become a hot research object in the fields of biocatalysis,drug/gene controlled release,functional materials and clinical therapy due to its advantages of simple preparation process,good biocompatibility,large specific surface area,easy modification and high bioactivity.By rationally designing the molecular structure of short peptides and giving specific environmental stimulation,short peptides can spontaneously organize or aggregate through multiple noncovalent interactions to form nanostructures with special morphology and function.The superior biochemical properties of short peptides are conducive to the construction of intelligent,bionic,multivalent,functional nanomaterials.As an innovative biomaterial of biological homology,short peptide self-assembled nanomaterials are useful for the construction of multifunctional fluorescent nanoprobes.These short peptide fluorescent nanoprobes have the characteristics of specific targeting,high selectivity,high stability,high sensitivity,accuracy and rapidity,ratio response,high biosafety and biodegradation in the application of bioanalysis,which show great application prospects in complex biological systems.Alzheimer's disease?AD?and malignant tumor are difficult to prevent,cure and even slow down,which have become the killer of human life and health.Studies have shown that the pathological mechanism of these diseases is closely related to a variety of active substances.As a tumor marker,biomolecular sialic acid is often overexpressed in the malignant transformation and metastasis of tumor.In addition,sialic acid in the brain of AD patients can promote the conformational transformation of?-amyloid protein?A??on the surface of neuron cell membrane and aggregate into toxic oligomer,forming senile plaques.As an important pathological feature of AD,A?protein deposition spots have high concentrations of excessive metal ions,which induce AD by catalytic oxidation.At the same time,a large number of reactive oxygen free radicals produced by redox reaction between A?and metal ions can cause oxidative stress and lead to the occurrence of AD.Thus,the analysis and research of various active substances in the pathological mechanism of the disease is extremely important.However,there are still many challenges in the existing analytical methods:1.The biological environment is complex and there are many components,there are a lot of interfering substances,and the concentration of the active substances is low,so it is very difficult to detect;2.The pathology of AD involves many molecules at the same time,and there are cross reactions between the components;3.The dynamic changes of the living tissue microenvironment are easy to interfere with the detection signals,resulting in inaccurate detection results.4.Ordinary nanomaterials have high toxicity,poor targeting,low uptake efficiency,shallow tissue permeability,and easily affect the biological activity of living cells and living tissues.To solve these problems,it is essential to improve the sensitivity,targeting and selectivity of detection methods,develop the joint detection of multiple active molecules,prepare ratio probes with specific response,improve the stability and biocompatibility of probes,and establish accurate,fast and low detection limit analysis methods.Therefore,a multiple response short peptide molecule has been designed and synthesized.By introducing the fluorescent molecules into the self-assembly of short peptides,a multifunctional fluorescent peptide nanoprobe has been constructed.Using the unique properties of short peptide self-assembled nanomaterials,such as controllable structure,easy modification and biocompatibility,we combine it with fluorescence analysis to realize:1.Targeted imaging analysis and precise joint diagnosis and treatment of tumor related active molecules;2.Accurate and rapid analysis of interaction between active molecules in organisms and sensitive and selective detection of active substances;3.Simultaneous ratio detection and imaging of two kinds of biological ions in living cells and in vivo;4.Simultaneous analysis and research on the pathological toxicity of two kinds of active substances in complex system to AD.Specifically,this dissertation mainly includes the following six chapters:Chapter 1.OverviewThis chapter first introduces the short peptide,self-assembly,construction strategy and application development of short peptide self-assembly.Then,the brief introduction of AD and malignant tumor and the research of related active substances are summarized,and the research progress of diagnosis and treatment of AD and malignant tumor is described.At last,the research purpose,design conception,research content and innovation of this paper are presented.Chapter 2.Bioinspired multivalent peptide nanotubes for sialic acid targeting and imaging-guided treatment of metastatic melanomaIn this chapter,a bioinspired multivalent nanoplatform for the highly effective treatment of the metastatic melanoma is reported.The versatile nanoplatform is designed by integrating indocyanine green and the chemotherapeutic drug?7-ethyl-10-hydroxycamptothecin?into phenylboronic acid?PBA?-functionalized peptide?KLVFFAL?nanotubes?termed as I/S-PPNT?.The binding constant of peptide nanotubes to sialic acid?SA?was 794 M^-11 by using the change of alizarin red fluorescence.The new multivalent nanotubes have a strong affinity for SA,which lays a foundation for I/S-PPNT to accurately target tumor cells for imaging guided combined therapy.I/S-PPNT could precisely target tumor cells through multivalent interaction between PBA and overexpressed SA on the tumor surface.It is demonstrated that I/S-PPNT are efficiently internalized by the B16-F10 melanoma cells in vitro in a PBA grafting density-dependent manner.It is further shown that I/S-PPNT specifically accumulate and deeply penetrate into both the subcutaneous and lung metastatic B16-F10 melanoma tumors.More importantly,I/S-PPNT-mediated combination chemo-and photodynamic therapy efficiently eradicates tumor and suppresses the lung metastasis of B16-F10 melanoma in an immunocompetent C57BL/6 mouse model.The results highlight the promising potential of the multivalent peptide nanotubes for active tumor targeting and imaging-guided cancer therapy.Chapter 3.Glycopeptide nanofiber platform for A?-sialic acid interaction analysis and highly sensitive detection of A?In this chapter,based on the mechanism of A?and sialic acids?SA?interaction,we report a novel approach for A?-SA interaction analysis and highly sensitive A?detection by mimicing the cell surface presentation of SA clusters through engineering of SA-modified peptide nanofiber?SANF?.The SANF displays well-ordered 1D nanostructure with high density of SA on surface.Using FAM-labeled A?fragments of A?_1-16,A?_16-23,and A?_24-40,the interaction between A?and SA is evaluated by the fluorescence titration experiments.It is found that the order of the SA-binding affinity was A?_1-16>A?_24-40>A?_16-23.Importantly,the presence of full-length A?_1-40-40 monomer triggers a significant fluorescence enhancement due to the multivalent binding of A?_1-40-40 to the nanofiber.This fluorescent turn-on response shows high selectivity and sensitivity for A?_1-40-40 detection and the method is further used for A?aggregation process monitoring and inhibitor screening.Chapter 4.Non-invasive in-situ ratiometric imaging of biometals based on self-assembled peptide nanoribbonIn this chapter,we develop a multifunctional probe based on self-assembling peptide nanoribbon for ratiometric sensing of Zn²⁺,Cu²⁺,or Zn²⁺and Cu²⁺simultaneously.Uniform peptide nanoribbon?AQZ@NR?is rationally designed by co-assembling a Zn²⁺-specific ligand AQZ-modified peptide?AQZKL-7?with peptide KLVFFAL?KL-7?.The nanoribbon further combines with Cu²⁺-sensitive near-infrared quantum dots?QDs?and Alexa 633 as an inner reference molecular,which is endowed with the capability for ratiometric Zn²⁺and Cu²⁺imaging at the same time.The peptide-based probe exhibits good specificity to Zn²⁺and Cu²⁺without interference from other ions.Importantly,the nanoprobe is successfully applied for non-invasive Zn²⁺and Cu²⁺monitoring in both living cell and zebrafish via multicolor fluorescence imaging.This gives insights into the dynamic Zn²⁺and Cu²⁺distribution in an intracellular and in vivo mode,as well as understanding the neurotoxicity of high concentration of Zn²⁺and Cu²⁺.Therefore,the self-assembled nanoprobe shows great promise in multiplexed detection of many other biometals and biomolecules,which will benefit the diagnosis and treatment of AD in clinical applications.Chapter 5.Multifunctional peptide assembled micelles for simultaneous scavenging of A?and reactive oxygen speciesIn this chapter,through a facile approach,we synthesize thioketal modified glutathione?tk-GSH?,which is incorporated into polysialic acid?PSA?to obtain PSA-tk-GSH.Further,functional peptide?LP and TK?are further coupled with PSA-tk-GSH for peptide-polymer.And micelles?MPGLT?are prepared by tip ultrasound assembly method for simultaneous scavenging of?-amyloid protein(A?_1-42)and reactive oxygen species?ROS?.The LD?LPFFD?peptide on the surface of MPGLT,derived from the A?_1-42-42 core sequence,can capture and dissociate A?_1-42aggregates.Moreover tk-GSH on MPGLT surface is reduced with ROS,releasing two GSH,which has stronger reducibility.Therefore,under the upregulation of cell autophagy due to autophagy peptide?TK?,the micelles rapidly enter the cell after capturing A?_1-42,and scavenging ROS with movability to remove them simultaneously.The results show that MPGLT could inhibit the aggregation or degradation of A?_1-42-42 aggregates,have a good response to hydrogen peroxide,and regulate the autophagy.The nanomicelles could quickly enter the cells after combining with A?_1-42,and movably clean ROS,achieving simultaneous scavenging of both.The neurotoxicity of A?_1-42-42 and ROS can be rescued successfully.Chapter 6.Summary and outlookThis chapter summarizes the full contents,and elaborates the innovation and research significance of this paper.In the end,the prospect and assumption related to the research direction are presented.