Synthesis Of Heparin Mimic Glycopolymers And Study For Their Anti-tumor Metastasis Abilities

Heparin is a highly sulfated linear glycosaminoglycan which consists of uronic acid-hexosamine disaccharides,and plays an important role in anti-tumor metastasis.However,the complex structure and anti-coagulation of natural heparin limit its wide application because the long-term use of heparin may cause bleeding,thrombocytopenia,and hypokalemia.Synthetic polymers with pendent oligosaccharides or monosaccharides as their side chains or chain ends,normally were termed as ‘glycopolymers’.The intensive sugar moieties in glycopolymers are able to significantly improve their binding ability with ligands and biological activities by multivalent interactions,known as ‘cluster glycoside effect’.Glycopolymers have been designed to mimic the structure of natural polysaccharides and improve their functions,and have made a lot of achievements for applications of disease treatments such as tumor treatment.So,developing a kind of low-anticoagulant heparin mimic glycoploymer with anti-tumor metastasis activites has become a potential and realistic demand.To date,there are only a few studies on the heparin mimic glycopolymers.Therefore,in this dissertation,various of novel glycopolymers with brush structure were designed and synthesized for anti-tumor fleid.And heparin disaccharides from enzymatic hydrolysis of heparin were used as key raw materials for the generation of glycopolymers: introduced with unsaturated double bonds first,and followed by polymerization.The main topics of our studies are as follows:(1)Polymerizable glucose-containing monomers with unsaturated double bond and a novel polymerizable fluorescent naphthalimide molecule with unsaturated double bond were prepared by chemical synthesis.These two polymerizable monomers were used as hydrophilic end and styrene was used as hydrophobic end were polymerized via RAFT method to prepare amphiphilic glycopolymers.Thereafter,4-acyl chlorobenzene boric was grafted onto the glycopolymer to get novel amphiphilic fluorescent glyco-nanoparticle.The resulting glyco-nanoparticles based on glycopolymer can be self-assembled to uniformed micelle,with good biocompatibility and high selectivity towards tumor cells,holding the potential to be developed as drug carriers.During the in vitro experiments,these glycol-nanoparticles were proven good affinity to tumor cells.(2)Following the polymerization-modification synthetic order,2-aminoethyl methacrylate monomers(AMA)were polymerized into poly-2-aminoethyl methacrylate polymers(PAMA)with varied lengths via reversible addition-fragmentation chain transfer polymerization(RAFT)reaction.By using enzymolysis method by heparin lyase,eight disaccharides have been obtained after separation and purification.Thereafter heparin disaccharides were grafted onto PAMA backbone as pendant sugar side chains and the residual amino groups of PAMA were sulfated to obtain the brush-like sulfated heparin mimic glycoploymers(SGPHD)as targeted products.The experimental results demonstrated that the heparin mimic glycopolymers showed good biological safety,negligible anticoagulant activity and enhanced the antitumor activity.Furthermore,they can inhibit multiple critical stages of tumor development and metastasis of B16 melanoma cells,including migration,adhesion and invasion,which showed greatly potential for the treatments of tumor metastasis.(3)Following the modification-polymerization synthetic order,based on the above studies of glycopolymers,a series of polymerizable saccharide-containing monomers were synthesized.Thereafter,followed the synthesis order from(1),the monosaccharides or oligosaccharide(containing heparin disaccharides)were grafted onto methacryloyl hydrazide,followed by co-polymerization of different monomers,and a list of novel brush-like glycopolymers with different sugar unit combinations were obtained.This synthetic strategy raised the ‘sugar cluster’ density in the glycopolymers,meanwhile,with adjustable composition of the sugar units,providing advantageous for the later screening of biological activities.BGP with the combination of fucose and sialic acid units which can mimic the structure of P-selectin glycoprotein ligand PSGL-1 showed effective binding ability to P-selectin.Two heparin mimic glycopolymers with heparin disaccharides as the side chain showed anti-tumor activities which can even compare to natural heparin,for interrupting several key steps of tumor progression.Moreover,the screened heparin mimic glycopolymer with the coexistence of the three sugar units showed the combined above activities and better safe dose,presenting good anti-metastasis effects in the mouse lung metastasis model.In summary,we have designed and synthesized a series of glycopolymer that can be used in anti-tumor field.The amphiphilic glycopolymer synthesized in part(1)has proved that the glycopolymer can provide good water solubility,biocompatibility and tumor affinity owing to its dense ‘cluster glycoside’,and can be used for tumor cell marker staining.The results have verified that glyopolymers with such structure have advantages for the application of anti-tumor due to their high density sugar structure.And in part(2),the heparin-like glycopolymer SGPHD successfully simulated the similar anti-metastasis activities of heparin without the risk of anticoagulation,which verified the feasibility of the simulation of glycopolymer for improving natural drug’s potency.On the basis of the above two parts,in part(3),with the synthetic order from part(1),the structure of SGPHD was optimized to make the ‘clusters glycoside’ density higher,and a variety of sugar unit were combined.BGP-SFH as a heparin mimic glycopolymer was screened with the best anti-metastasis effect.On the one hand,it was proved that increasing the density of ‘cluster glycoside’ can improve the activity of glycopolymer.On the other hand,BGP-SFH can retain the anti-metastasis activites of heparin while simulating the structure of P-selectin ligand PSGL-1 by adjusting the composition of sugar units,with improved specific anti-adhesion ability.These results demonstrated the great potential of glycopolymers and tailored structural flexibility for physiological activity.They will contribute to the design and development of glycopolymers drugs with better therapeutic efficacy and customized biological activity.