Synthesis,characterization And Biological Evaluation Of Cu(?)-phosphoryldiserine-chitosan

Amino acid zwitterionic polymers not only have the super-hydrophilic and anti-bioadhesive properties of betaine-type zwitterionic polymers such as phosphorylcholine,but also exhibit characteristics such as pH response and metal ion chelation,etc.These facts show that amino acid zwitterionic polymer have important application value in the field of biomedicine.Currently,amino acid zwitterionic polymers are usually synthesized based on amino acid vinyl monomers,and their non-biodegradability limits their applications in vivo.So we synthesized a biodegradable chitosan-based amino acid zwitterionic polymer:phosphoryldiserine-chitosan(PDS-chitosan)by Antherton-Todd reaction,considering the therapeutic potential of Cu ion and the combination ability of serine and Cu ion,we further prepared Cu(?)-phosphoryldisserine-chitosan(Cu(?)-PDS-chitosan),and systematically studied their structures,physical,chemical and biological properties.First,we prepared the triphenylmethyl etherified chitosan(CS-Tr),an oil-soluble chitosan derivative,and coupled the phosphoryl di-tert-butoxycarbonyl serine group(Boc-PDS)to CS-Tr by Antherton-Todd reaction in the oil phase.and then used trifluoroacetic acid(TFA)removes the Tr and Boc protecting groups,and finally obtains the product phosphoryldiserine-chitosan(PDS-chitosan).Considering that the phosphorus chemical shift is different from the conventional P-N bond and appears near0 ppm,we used ³¹P NMR to test the phosphoryldiserine-polyvinylamine(PDS-PVAm),phosphoryl dihydroxy valeric acid chitosan(PDH-chitosan)and phosphoryl di-tert-butoxycarbonyl serine methyl ester-triphenylmethyl ether chitosan(Boc-PDSOM-CS-Tr),and a small molecule model of phosphoryldiserinate-ethylenediamine(Boc-PDS-en),then used the ninhydrin method to test the amino content of PDH-chitosan,the results show that the phosphoryl disylserine(PDS)group is coupled to the amino group of chitosan through a phosphoryl bond,and its phosphorus chemical shift near 0 ppm may be related to the carboxyl group of PDS.Then,we synthesized Cu(?)-PDS-chitosan in the liquid phase using PDS-chitosan and basic copper carbonate,and measured the substitution degree of PDS in PDS-chitosan by inductively coupled plasma spectroscopy(ICP)to be?35%,and Cu(?)-PDS-chitosan has a Cu/P ratio of 0.76.Moreover,Cu(?)-PDS-chitosan is similar to PDS chitosan in that it can self-assemble into nanoparticles in aqueous phase.DLS test results show that the particle size is about 100nm,which is significantly smaller than?200nm of PDS-chitosan,and has better dimensional stability in different p H solutions.In vitro experiments show that Cu(?)-PDS chitosan can generate hydroxyl radicals(·OH)through Fenton reaction in vitro,and degrade methylene blue,which proves its potential value in the field of anti-tumor.In biological evaluation,we used BSA as a model to study the interaction between Cu(?)-PDS chitosan and protein,and the effect of Cu(?)-PDS chitosan on the thermal stability and esterase activity of protein.The results show that Cu(?)-PDS-chitosan is similar to PDS-chitosan,which can maintain the conformation of BSA in aqueous solution almost unchanged and improve its thermal stability and esterase activity.In addition,L929 cytotoxicity test showed that Cu(?)-PDS-chitosan had no obvious cytotoxicity to normal cells.In conclusion,the Cu(?)-PDS chitosan prepared by the method of Atherton-Todd reaction and liquid-phase synthesis shows good biocompatibility,and has a broad application prospect in biomedical fields such as anti-tumor treatment.