Membrane Fusogenic Liposome-Mediated Nucleic Acid-Quantum Dot Nanoprobes For Spatially-Selective Cell Membrane Engineering

The cell membrane not only acts as a dynamic barrie r to maintain stable internal environment for cells,but also plays a critical role in regulating cell behavior and cell-cell communication.The interactions between biomolecules on membrane,such as lipids,glycans,and proteins,as well as the interactions with cell microenvironment affect a variety of cell functions including signal transduction,metabolic processes,cancer metastasis and autoimmunity.Therefore,cell membrane engineering is of great significance for studying and regulation of cell membrane-based biological processes.In recent years,nucleic acids,protein complexes,polymers,and functional inorganic nanomaterials have been modified onto cell membrane to monitor and regulate important biological processes.To date,cell membrane engineering mainly relies on biochemical modification of the outer side of cell membrane,and only a few reports about modification on the inner side of cell membrane have been reported.The realization of spatial-controlled cell membrane engineering still faces great challenges.To address this issue,our group has developed a liposome fusion-based transport（Li FT）strategy to realize spatial-controlled biorthogonal modification of two sets of functional nucleic acids onto both sides of cell membrane.In order to expand the application of Li FT strategy for cell membrane engineering,this research achieved high spatial-controlled modification of different quantum dots（QDs）on both the inner and outer face of cell membrane using DNA-QD conjugates and fusogenic liposomes.By using this strategy,fluorescence ratiometric probe based on DNA-QD conjugate was modified on the inner face of cell membrane for intracellular pH monitoring.The main contents of this thesis are summarized as follows:1.Surface modification of hydrophobic quantum dots using ligand exchange and DNA conjugationQuantum dots with high fluorescence quantum yield and high photostability have been widely used in biosensing and bioimaging.In this thesis,three core-shell QDs with different emission wavelengths were prepared using high-temperature organic phase method:Cd Se/Zn S QD,Cd Se S/Zn S QD,and Cd Zn Se S/Zn S QD.In order to improve QDs’biocompatibility,we modified QD via surface ligand exchange and obtained hydrophilic 3-mercaptopropionic acid（MPA）and m-PEG₇-thiol（m PEG）co-decorated QD（m PEG/MPA-QD）.Transmission electron microscopy（TEM）images showed that m PEG/MPA-QDs were well-dispersed in water.The morphology and particle size of hydrophilic QD did not change after surface ligand exchange.Then,we prepared three types of DNA-QD conjugates by coupling DNA on m PEG/MPA-QD surface through steric exclusion strategy.2.Fusogenic liposome-mediated DNA-QD conjugates for cell membrane engineeringCell membrane engineering is an important strategy to study cell membrane-related biological processes.To achieve spatial-controlled cell membrane engineering,we used Li FT strategy to realize modification of DNA-QD conjugates on inner face of cell membrane.First,we prepared the fusogenic liposome with DNA-QD conjugates on the inner side of the liposome（Lipo-QD@IF）,and demonstrated that Lipo-QD@IF can transport DNA-QD conjugates onto inner face of cell membrane.We further optimized incubation conditions and revealed that 30 min incubation at25℃achieved efficient modification.The immobilized DNA-QD conjugates can stay on cell membrane for 2 hours.Through DNase I digestion and post-functionalization,we prepared asymmetric dual QDs-modified liposomes and successfully modified Cd Se S/Zn S QD and Cd Zn Se S/Zn S QD on the inner and outer face of cell membrane respectively,achieving asymmetric modification of cell membrane.3.Inner cell membrane-anchored ratiometric sensor based on DNA-QD conjugates for intracellular pH detectionIntracellular pH change is usually associated with physiological and pathological processes of organisms.Therefore,monitoring intracellular pH with high spatial control is of great significance.We immobilized i-motif based pH sensor onto QD to construct an i-motif-QD ratiometric probe.Under acidic conditions,the pH-sensitive DNA strands formed i-motif quadruplex secondary structure,which results in the increase of fluorescence signal of Alexa Fluor 488.The i-motif-Cd Se/Zn S QD probe generatred ratiometric fluorescence change in pH ranging from 5.5 to 7.0,which is suitable for pH detection of biological systems.The i-motif-Cd Se/Zn S QD fluorescence ratiometric probe was further modified onto the inner face of cell membrane using the Li FT strategy.Confocal microscope images demonstrated that the probe can respond to change of intracellular pH.