Bioimaging,Anticancer,and Antibacterial Applications Based On Cell Surface Modification Techniques

The cell surface as a major component of cellular structures participates in a diverse range of biological processes such as metabolic regulation,signal transduction,and cell transport,and plays a critical role in the communication between the cell and the extracellular environment.Systematically dissecting the detailed structures and biological functions of cell surfaces has long been a core mission of modern cell biology.Cell surface engineering,a class of state-of-the-art techniques in this field,aims to deploy various functional units on the cell surface by means of genetic encoding,metabolic labeling,chemical modification,or physical adsorption.In doing so,researchers can visualize/detect biomolecules of interest and manipulate target cells for therapeutic purposes.However,regarding different cell surface modification techniques,their potential applications and unique properties still need to be investigated.Moreover,how to design and construct new modification strategies for cell surface functionalization remains an open challenge.In this thesis,we apply cell surface engineering to fluorescence bioimaging,anticancer therapy,and antibacterial treatment,and develop a series of new cell surface modification strategies.With respect to bioimaging,we aim at two modification techniques based on hydrophobic membrane anchoring and covalent linkage,respectively,and investigate their applications for imaging mammalian cell membranes and bacterial cell walls.To be specific,we construct a red-fluorescent polymeric probe for wash-free plasma membrane imaging.This probe consists of cholesterol?polyethylene glycol(PEG)and a fluorophore cyanine5,wherein the hydrophobic cholesterol serves as an anchor capable of inserting into plasma membranes,and the PEG chain endows the entire probe with good water solubility.This probe is found to rapidly bind to the plasma membranes of various mammalian cells with stable retention for at least 2 h.Using zebrafish embryos as an animal model,we demonstrate that the probe can readily cross the mucus layer and uniformly label the underlying epidermal cell,facilitating the three-dimensional fluorescence imaging of zebrafish embryos.In another study,we develop a chemical modification strategy based on a tyrosinase-mediated oxidative coupling reaction for the functionalization of Gram-positive bacterial cell walls.This technique rests on tyrosinase's ability to convert phenols into quinones,which can then form covalent linkage with teichoic acid in the bacterial cell wall.We prove that the above modification strategy is suitable for selectively imaging,inactivating,separating,and sensing Gram-positive bacteria.As for anticancer applications,we successfully synthesize two plasma membrane-anchorable photodynamic agents.The first one is constructed by using glycol chitosan as a polymeric backbone and modifying its side chains with PEG and protoporphyrin IX(Pp IX,a photosensitizer).The agent is amphiphilic and thus can self-assemble into nanoparticles in aqueous solutions;however,when reaching the tumor cell surface,it undergoes disassembly to expose Pp IX molecules for hydrophobic membrane anchoring.Upon laser irradiation,Pp IX efficiently generates singlet oxygen to disrupt the membrane integrity,leading to rapid cell death.In addition,the compromised plasma membrane concomitantly permits extracellular agents to efficiently enter the cell for enhanced photodynamic therapy.The other photodynamic agent(termed Chol-PEG-Pp IX)is designed to be composed of a cholesterol unit,a PEG chain,and a Pp IX molecule.Similarly,this agent also exhibits an excellent membrane-anchoring ability,and causes damage to plasma membrane integrity under laser irradiation.Besides,we unravel that after long-term incubation,Chol-PEG-Pp IX is internalized by cells through lipid raft-mediated endocytosis,as triggered by the cholesterol unit,and finally accumulates in the endomembrane system.To realize safe intravenous delivery of this agent,we prepare a hybrid liposome whose surface is in situ anchored by Chol-PEG-Pp IX,and demonstrate that this system presents higher tumor accumulation and therapeutic efficacy in mouse models than those of free Chol-PEG-Pp IX.In the last section,we prove that Cho-PEG-Pp IX is also applicable to antibacterial treatments.Unlike most antibacterial materials that bind to bacterial surfaces via electrostatic interaction,Chol-PEG-Pp IX completely relies on hydrophobic interaction for bacterial binding,because the cholesterol and Pp IX units can intercalate into the outer membrane of Gram-negative bacteria in a synergistic manner.Antibacterial experiments reveal that compared with free Pp IX that only shows modest antibacterial efficacy,this agent enables more efficient photodynamic inactivation against both Gram-positive and Gram-negative bacteria.This work not only presents a potent antibacterial agent but also offers solid experimental evidence that bacterial outer membranes can be anchored by hydrophobic units.In summary,this thesis develops a series of physical and chemical cell surface modification strategies for cell surface-localized imaging and therapy applications.These studies greatly enrich our current toolbox for the modification of mammalian and bacterial cell surfaces,and may promote the development of cell surface engineering in the biomedical field.