The Effect Of PEGylation On The Interactions Between Polypeptide Based Micelles And Cell Membranes Probed By AFM-SMFS

The interaction of the targeted drug carrier and the cell membrane is essential for the anti-cancer treatment.Most drug carriers are shown to be modified by PEG.PEGylation could prolong the circulation time,reduce the immunogenicity and increase the accumulation in tumor tissue due to EPR effect.Therefore,understanding the influence of PEGylation on the interaction between drug carriers and the cell membrane is important for the design of drug carriers and the efficient drug delivery in the future.However,due to the instantaneous and dynamic process of cell endocytosis,the relevant research is challenging.At present,scientists mainly use imaging technology,flow cytometry and molecular dynamics simulation to study the interaction between PEGylated carriers and cell membrane.However,it is quite difficult to characterize the dynamic process of endocytosis using traditional ensemble methods,such as optical imaging technology and flow cytometry.Moreover,there’s a great difference between parameters used for the theoretical simulation research and the real environment.Atomic force microscopy(AFM)based single molecular force spectroscopy(SMFS)is able to eliminate the averaging effect in the ensemble experiments and monitor the dynamic process.In this thesis,we investigated,at the single particle level,the effect of PEGylation on the polypeptide micelle-cell membrane interactions.In the second chapter,we first constructed the thermo-sensitive elastin-like peptide micelles that can be used for further crosslinking and molecular modification.We successfully obtained the elastin-like peptide(ELP)by gene clone,protein expression and purification.Two kinds of crosslinking agents were used to crosslink micelles into stable nanoparticles that would not change conformation due to concentration and temperature variation.We characterized the crosslinked micelles by particle size measurement and AFM imaging.Secondly,we successfully modified the ligand on the crosslinked micelle surface and measured the modification efficiency of different kinds of molecules.The results showed that the modification efficiency of all molecules reached more than 80%.All the results indicate that we successfully constructed the targeted nanoparticles suitable for the follow-up research by SMFS technology.In the third chapter,the effects of different PEGylation on nanoparticle-cell surface interactions were studied by SMFS.Our results show that as the supporting molecule around the ligand,PEG can effectively improve the binding force between the ligand and the receptor on the cell surface.However,when PEG was used only as the linker between ligand and nanoparticles,PEG could slightly reduce the unbinding force between the ligand and the receptor.We conclude that the modification density of PEG played an important role in ligand-receptor recognition,only when the PEG density is high enough to support and expose the ligands on the nanoparticles,PEG can play its facilitating role in specific binding between ligands and receptors.In the fourth chapter,the effect of PEG on the endocytosis of nanoparticles was studied by the force tracing mode of SMFS.The results showed that the presence of PEG reduced the force and efficiency of targeted nanoparticles’ internalization.The experimental results of control group(PEGylated nanoparticles without ligand modification)showed lower endocytosis force and efficiency,which confirmed the validity of the above conclusion: PEGylation could reduce the cell endocytosis force and efficiency of targeted nanoparticles.Based on these results,we show the effect of PEGylation on the recognition and endocytosis of polypeptide micelles by cells,PEGylation is beneficial to recognition but slightly inhibits endocytosis.These findings will shed lights on the design of nano-drug carriers in the future.