The Binding Of Glycopolymers With Different Chain Lengths To Immune Cell Surface Receptors And Their Effects On Immune Cell Behavior

The interaction between polysaccharide and immune cell surface receptor plays an important role in regulating immune cell behavior and activating immune response to resist pathogens and tumors.However,the high heterogeneity of natural polysaccharide structure prevents people from further studying the molecular mechanism of polysaccharide-receptor interaction and its influence on cell behavior.With the rapid development of controlled free radical polymerization technology,synthetic glycopolymers have definite and controllable molecular structures.Therefore,through these chemical synthesis methods,glycopolymers with different structures,such as glycopolymers with different chain lengths,can be obtained.We can study the influence of glycopolymer structures on immune cell behavior driven by the interaction between glycopolymers and immune cell surface receptors,and on the interaction between cancer cells and immune cells,which provides a theoretical basis for developing immunotherapy drugs and immune strategies based on glycopolymers.This work focuses on the theme of "The binding of glycopolymers with different chain lengths to immune cell surface receptors and their effects on immune cell behavior".In view of the importance of glycopolymers in regulating immune cell behavior and activating immune response,and the lack of basic research on the influence of glycopolymers chain length on immune cell behavior,this thesis studies the binding of glycopolymers with different chain lengths to immune cell surface receptors,the effects of glycopolymers with different chain lengths on immune cell behavior,and the effects of glycopolymers with different chain lengths on the interaction between cancer cells and immune cells.First,the glycopolymers(pMAG)with different chain lengths were synthesized by reversible addition-fragmentation chain transfer polymerization(RAFT).The effects of glycopolymer chain length on the interaction between glycopolymer and immune cell surface receptors were studied through experiments and computer simulation.Then,we further studied the influence of glycopolymer chain length on dendritic cell maturation,macrophage polarization and aggregation,which provided a theoretical basis for the development of immunotherapeutic drugs based on glycopolymer.In addition,we modified glycopolymers with different chain lengths on the surface of mouse melanoma cell through cell surface engineering,and studied the influence of the chain length of glycopolymers on the interaction between cancer cells and immune cells.Moreover,we studied the promotion of immune cell activation by glycopolymer modified cancer cells and the inhibition of tumor growth in vivo,which provided theoretical basis and new ideas for tumor immunotherapy strategies.The detailed research contents are as follows:(Ⅰ)Synthesis of glycopolymers with different chain lengths and their binding to receptors on immune cell surface.Glycopolymers(pMAG,named as P1～P4)with different chain lengths were prepared by RAFT method with Nmethacryloylglucosamine(MAG)as monomer.Determinations of 1H NMR,FT-IR and size exclusion chromatography(SEC)prove the successful synthesis of glycopolymers with different chain lengths.The results of quartz crystal microbalance(QCM)show that short-chain glycopolymers(DP<20)bind more DC-SIGN protein and TLR4-MD2 protein than long-chain glycopolymers(DP>20)when the mass of glycopolymers(or the number of sugar rings)is equal.The results of molecular docking simulation show that the affinity of short-chain glycopolymers to DC-SIGN and TLR4-MD-2 is stronger than that of long-chain glycopolymers,and the utilization rate of ligand sites on short-chain glycopolymers is higher.(Ⅱ)Effect of glycopolymer chain length on immune cell behavior.The morphological changes of dendritic cells(DC2.4)prove that glycopolymers can promote the maturation of dendritic cells,and the effect of glycopolymers on the maturation of dendritic cells is concentration-dependent.The expression of CD80 and CD86 on the surface of dendritic cells was further detected by flow cytometry.The results show that short-chain glycopolymers could promote the maturation of dendritic cells more effectively than long-chain glycopolymers under the same amount of sugar rings,because the utilization rate of ligand site on short-chain glycopolymers is higher.And when the number of effective ligand sites of long-chain glycopolymers is the same as that of short-chain glycopolymers,both short-chain glycopolymers and long-chain glycopolymers have the same effect on promoting the maturation of dendritic cells.The expression of CD86 and CD206 on the surface of macrophages(U937)was detected by flow cytometry.The results show that pMAG could promote the polarization of macrophages to M2 type,which is also concentration-dependent.When the amount of sugar rings is the same,the effect of short-chain glycopolymers on promoting macrophage polarization is better than that of long-chain glycopolymers,which is also due to the higher utilization rate of ligand sites on short-chain glycopolymers than longchain glycopolymers.When the expression of TLR4 on macrophage surface was inhibited by TAK-242,pMAG promoted U937 cell polarization to M1 type,which indicates that pMAG has a bidirectional effect on macrophage polarization.When pMAG binds to TLR4,it promotes the polarization of macrophages to M2 type,and when TLR4 is inhibited,pMAG promotes the polarization of macrophages to M1 type.These results provide a theoretical basis for developing immunotherapeutic drugs based on glycopolymers.(Ⅲ)The interaction between cancer cells modified by glycopolymers with different chain lengths and immune cells,and enhancement of immune activation by glycopolymers modified cancer cells.At First,we constructed B16 cell lines(named as HTP-B16)which stably expressing HTP(Halo Tag Protein)on its surface through gene transfection and G418 screening.HA immunofluorescence staining results show that the construction is successful.Then we incubated cells with p(MAG-co-biotin)and FITC-avidin.The results of fluorescence imaging show that glycopolymer with chloroalkane can be successfully modified on the surface of HTP-B16 cells.We further synthesized glycopolymers with different chain lengths using chain transfer agents containing chlorinated alkanes.Determinations of 1H NMR,FT-IR and SEC prove that the glycopolymers are successfully synthesized.The interactions between B16 cells modified by glycopolymers with different chain lengths and macrophages(RAW264.7)were monitored by living cell dynamic imaging and analysis system.The results show that the chain length of glycopolymers modified on the surface of cancer cells needs to reach an appropriate length to effectively promote the interaction between cancer cells and immune cells.When glycopolymers with appropriate length are modified on the surface of cancer cells(cancer cells were incubated with the same mass of glycopolymes),short-chain glycopolymers promote the contact between cancer cells and immune cells more effctively than long-chain glycopolymers.The expression of CD86 and CD206 on the surface of macrophages proves that the glycopolymers modified on the surface of cancer cells could promote the activation of immune cells.In the in vivo experiment study of mouse,the cancer cells modified by glycopolymers and irradiated by 60Co were injected into mice.The results of tumor volume show that cancer cells modified by glycopolymers can enhance the immune response in vivo and inhibit tumor growth to a certain extent,which shows the application potential of glycopolymers in tumor immunotherapy,and provides a new idea for tumor immunotherapy.