Synthetic Glycopolymers Modification Of Cell Surface For Regulating Cell Functions

Cell-surface saccharides as the outermost layer of cells are the first molecules to be recognized by the extracellular environments.Therefore,cell-surface saccharides participate in various essential bio-processes,ranging from cell differentiation and immune responses to disease progression and cell apoptosis.However,the complex,uncontrolled and heterogeneous structures of natural glycans have hampered efforts to elucidate the effects of saccharides of specific structure on cell functions.Accordingly it is of particular interest to generate well-defined glycans at the cell surface to achieve the desired cellular phenotypic outcomes or to elucidate the mechanisms of glycan-mediated signaling events at the molecular level.This work focuses on the regulation of cell functions,in particular,neural differentiation of embryonic stem cells(ESCs)and immune responses of macrophages,by modifying cell surfaces with the exogenous synthetic well-defined glycopolymers.First we prepared a new kind of phospholipid-functionalized glycosaminoglycans(GAGs)-mimetic glycopolymers via the combination of reversible addition-fragmentation chain transfer(RAFT)polymerization and post-polymerization(PPM).Then the incorporation of the synthetic well-defined glycopolymers with cell surface and the effect of specific glycopolymer on neural differentiation of mouse ESCs(mESCs)and specific cell signalling pathway were investigated.The obtained results provide guidance for manipulation of other differentiation behaviors of ESCs mediated by cell surface glycan and receptors.The plasma membrane residence half-lives of lipid-functionalized polymers are limited to a few hours,so to achieve more stable attachment of glycans and to facilitate the study of events occurring on longer timescales,a strategy based on membrane-bound HaloTag proteins(HTP)as anchors has been developed in the following study.We synthesized new well-defined glycopolymers by combining protein fusion technique(PFT)with RAFT polymerization and used them for stable cell surface glycosylation aimed at regulating the immune response of tumor-associated macrophages and dendritic cells maturation.This work demonstrates the potential of glycopolymer-engineered tumor cells in cancer immunotherapy.Detailed studies are as follows:(?)Synthesis of lipid-functionalized GAG-mimetic glycopolymers for cell surface engineering.A combination of RAFT polymerization and PPM was used for the first time to prepare glycopolymers containing lipid groups.In the example system presented,a terpolymer(pSMF)of sodium 4-vinylbenzenesulfonate(SS),N-methacryloylglucosamine(MAG)and fluorescein o-methacrylate(FluMA)was first synthesized by RAFT.Maleimide-functionalized 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(MAL-DPPE)was synthesized and attached to pSMF to give the lipid-functionalized glycopolymers,DPPE-pSMF.The 1H NMR spectrum,infrared spectrum and thiols densities assay demonstrated the successful preparation of DPPE-pSMF.Confocal fluorescence images of cell cross-sections demonstrated that DPPE-pSMF with lipid end-group functionality could be inserted into the membrane of HeLa cells while pSMF polymer was endocytosed.The quantity of glycopolymer inserted into the cell membrane depends on the concentrations of the incubating solution.Also the GAG-mimetic glycopolymers incorporated onto cell membranes was shown to be able to promote the growth of L929 cells.(?)Effect of synthetic lipid-functionalized GAG-mimetic glycopolymers on neural differentiation of mESCs.The synthetic DPPE-pSMF with specific structure can retain on the membrane of mESCs rather than being internalized by cells after 1 h of incubation.Besides,DPPE-pSMF showed better activity in promoting neural differentiation.The expression of the neural-specific maker p3-tubulin in DPPE-pSMF-treated cells was?3.8-and?1.9-fold higher compared to natural heparin-and pSGF-treated cells at day 14.The likely mechanism involved in lipo-pSGF-mediated neural differentiation was further investigated by analyzing its effect on fibroblast growth factor 2(FGF2)-mediated extracellular signal-regulated kinases 1 and 2(ERK1/2)signaling pathway which is important for neural differentiation of ESCs.DPPE-pSMF was found to efficiently bind FGF2 and enhance the phosphorylation of ERK1/2,thus promoting neural differentiation.These findings demonstrated that engineering of cell surface glycan using our synthetic lipid-functionalized glycopolymer is a highly efficient approach for neural differentiation of ESCs and this strategy can be applied for the regulation of other cellular activities mediated by cell membrane receptors(?)Combination of PFT technique based on HaloTag protein(HTP)and RAFT polymerization for stably modifying cell surfaces with synthetic well-defined glycopolymers.The plasmid containing HTP gene and transmembrane protein fragment were transfected into HeLa cells.After selecting for 21 days with G418,the HeLa cells stably expressing HTP on cell membranes(HTP-HeLa)were obtained.A key molecule,chloroalkane-grafted chain transfer agent(CPADB-CI),was synthesized and biotin-labeled poly(2-methacrylamide glucopyranose)with chloroalkane end groups(pMB)was prepared by utilizing CPADB-Cl.The H NMR spectrum and SEC data demonstrated the successful preparation of pMB.The successful incorporation of pMB with HTP-HeLa cells was proved by confocal fluorescence images and the glycopolymer can persist for seven days on cell membranes.These results demonstrate the successful specific conjugation of chloroalkane-glycopolymer to HTP and the stable attachment of exogenous glycopolymers on HeLa cell membranes.(?)Effect of synthetic glycopolymers-engineered cancer cells on macrophage polarization and dendritic cells maturation.The "self-homing" of cancer cells to primary or metastatic tumor sites indicates that they could serve as vehicles for self-targeted cancer therapy.Inspired by this,we propose that engineering cancer cells to carry efficient bioactive molecules for in situ activation of immune cells in or near tumor sites to attack tumors is a promising strategy for cancer therapy.First we explored the effect of synthetic glycopolymers-engineered cancers on macrophages polarization and dendritic cells maturation in vitro.By utilizing CPADB-Cl,the RAFT polymerization of the sugar monomers,MAG and N-methacryloylmannosamine(MAM)was carried out to generate well-defined glycopolymers containing a chloroalkane linker(pMAG and pMAM)for conjugation to HTP-HeLa cell surfaces.The H NMR spectra and SEC data demonstrated the successful preparation of pMAG and pMAM.The HTP-HeLa cells were incubated in pMAG and pMAM for 1 h,respectively,to obtain pMAG-HeLa and pMAM-HeLa.It was shown that both of pMAG-HeLa and PMAM-HeLa cells increased expression of the typical marker for M1-type macrophages(CD86)and upregulated secretion of pro-inflammatory cytokines(IL-12p70,TNF-a and iNOS).The maturation of dendritic cells was also promoted.Furthermore,the glycopolymers-engineered B16 cells(pMAG-B16 and pMAM-B16)were obtained to investigate the safety of glycopolymers-engineered cancer cells in vivo.It was shown that after 16 days,no tumor growth were observed in pMAG-B16-and pMAM-B16-administered(s.c.)mice.These results demonstrate the strong potential of glycopolymer-engineered tumor cells in cancer immunotherapy.