Modification Of Cell Membranes And Regulation Of Cell Behaviors By Glycopolymers

The interaction and communication of cell-cell and cell-environment are crucial for cell behaviors and physiological processes.These interactons are highly dependent on receptors and ligands expressed on the cell membrane.Therefore,it is expected that cell behaviors can be regulated and specific functions can be achieved through manipulating artificial receptors/ligands on cell membranes via cell surface engineering.The specific interaction of carbohydrate(ligands)and protein(receptors)has great significance in biological processes such as information transmission,cell adhesion,proliferation,differentiation and immune response.However,the complexity of natural carbohydrates limits their research on specific cell behaviors.Therefore,development of cell surface modification methods for presentation of synthesized glycopolymers on cell membranes would allow a better study of the regulation of cell behavior by glycopolymers.This not only expands the methods of constructing glycans on the cell surface,but also has important significance for studying the glycan structure-function relationships and the signaling mechanism mediated by glycans.This paper has carried out a series of work on the topic of "Cell surface modification with glycopolymers:methods construction and regulation of cell behaviors".First,to achieve multiple or dynamic functionalization,a cell surface engineering platform was constructed using synthetic modular polymers.Using this platform,we realized the regulation of various cell behaviors,such as neuronal differentiation and immune responses.In addition,for the specific endothelial cells,we have developed a novel method for the glycosylation of cell membranes by combining metabolic glycan engineering and bio-orthogonal click reaction to study the effects on proliferation and migration of endothelial cellsThe specific research contents are as follows:(1)The construction of cell surface engineering platform by modular polymer.The modular glycopolymers of deoxy-2-(methacrylamido)glucopyranose(MAG,sugare module),oligo(ethylene glycol)-1-adamantanecarbonyl methacrylate(OEGMA-Ada,guest mudule),cholesteryl-methacrylate(MAC,anchoring mudule)and fluorescein Omethacrylate(FluMA,labeling mudule)were synthesized by RAFT and the composition of these polymers were be regulated by changing feed ratio of the monomers.It was demonstrated that these modular polymers are notoxic to L929 cells by CCK-8.What's more,they can be efficiently inserted into the cell membrane via cholesterol units and their residence time on the cell membranes can be regulated by controlling their cholesterol content.For pACGF3 and pACGF4 with high cholesterol content,most of the polymers still stay on HeLa cell membranes after 12 h.The introduction of red fluorescent Rhodamine B-labeled cyclodextrin(CD-RBITC)proved that the modular glycopolmer can undergo host-guest interactions both in solution and on the cell membrane,so that cells can be imaged and further functionalities can be introduced(2)Effect of modular polymers on neural differentiation of mESCs.First,it was demonstrated that modular polymers are notoxic to mESCs and can modify them.The expression of ?3-tubulin(neural-specific marker protein)in the groups treated with pACGFs was very low,while increased significantly by adding seven sulfonate labeled cyclodextrin(CD-S)at day 7.After adding CD-S,GAG analogues were formed and their effects on promotion of neural differentiation appear to increase first and then decrease with increasing cholesterol content.Among them,the expression of ?3-tubulin in pACGF2+CD-S treated cells was?2.2-fold higher than in heparin-treated cells.Furthermore,the binding experiment of basic fibroblast growth factor 2(FGF2)showed that pACGF2 and pACGF3 containing CD-S can enhance the binding capacity of mESCs to FGF2.The results indicated that pACGF2+CD-S,with relatively low cholesterol content,had a greater effect on neural differentiation of mESC.pACGF3+CD-S with high cholesterol content had also good FGF2 binding ability,but showed a poor differentiation promotion effect,which may be the restriction of the polymer on the cell membrane leading to the block of FGF signaling pathway.(3)Effect of modular polymer-modified cancer cells on immune response.The heteroglycopolymers(pACGFs-M)were obtained by the host-guset interaction of pACGFs and appropriate seven mannose labeled cyclodextrin(CD-M).pACGF1-M had a higher specific binding ability than pACGF1 to the CD206 protein(a typical C-type lectin receptor)measured by QCM.U937 cells were then stimulated by pACGFs and pACGFs-M modified HeLa cells.The results showed that the modified HeLa cells could promote the polarization of macrophages to the M1 phenotype,and the promotion effect of pACGFs-M-HeLa was better than that of pACGFs-HeLa,and also increased with the increase of cholesterol content.What's more,the M1 polarization enchanced with the increasing of adamantane content in the polymers.Similarly,the pACGF3-M modified 4T1 cells showed better ability to promote the maturation of mouse dendritic cells than those modified by pACGF3(4)Modification of synthetic GAG-mimicking polymers on EC membranes by combining metabolic gly can engineering and bio-orthogonal reaction and investigation of the effect on enhanced endothelialization.First,the endothelial cells(EC)were successfully labeled with azide groups without affecting proliferation.A GAG-mimicking polymer and a biotin-labeled GAG-mimicking polymer with the DBCO end group(respectively,pGS-DBCO and pGSB-DBCO)were synthesized after several steps of chemical reaction.Avidin-FITC fluorescent labeling proved that PGSB-DBCO can be successfully modified on EC membranes,and the residence time on the cell membrane was more than 48 h.Based on a silicon model,modification of pGS-DBCO on EC membranes could promote the proliferation and migration of ECs,and promote the binding of growth factors(VEGF and FGF2)to ECs.In summary,this paper developed two methods for modifying artificial glycans onto cell membranes,and explored the regulation of specific cell behaviors by specific structure glycans.First,through the combination of hydrophobic insertion modification and supramolecular chemistry,a cell surface engineering platform was successfully constructed,which not only achieved adjustable residence time,but also achieved versatility through host-guest interaction.In addition,through the combination of metabolic glycan engineering and bio-orthogonal reaction,the GAG-mimicking polymers were successfully modified on the endothelial cell membranes,which provided a new idea for promoting endothelialization.