Synthesis Of Redox-responsive Amphiphilic Chitosan Derivatives And Their Characteristics As Nanocarriers

With the development of biomaterials and nanotechnology,the research and application of nanoparticle drug delivery systems(NDDSs)were gradually increased.Polymeric nanoparticles(PNPs)demonstrate some siginificant advantages,such as good stability and drug absorption,targeting to tumor tissue and so on.However,for a number of drugs that need to be released from nanoparticels at specific sites or in a particular organelle,the quick,effective and complete release of the drug after reaching the targeting site is a major problem.The difference in redox potential is regarded as a significant signal to distinguish between the extra-cellular and intra-cellular environment,as well as between normal and tumor tissues.Based on this physiological characteristics,a new drug delivery system 'reduction sensitive polymeric nanocarriers'(RSPNs)was designed,in which reduction sensitive amphiphilic polymers were synthesized to be used as the carrier materials for anti-cancer drugs.In this study,the biodegradable and biocompative carboxymethyl chitosan(CMCTS)as the hydrophilic chain,and fatty alcohol as the hydropholic chain,were connected by DTDP containing disulfide bonds to synthesize CMCTS-ss-R.First,using EDCI as coupling reagent and DMAP as catalyst,DTDP was conjugated with fatty alcohols via ester bonding between one of the carboxyl group of DTDP and the hydroxy group of fatty alcohols to prepare 3,3'-thiodipropionate fatty alcohol monoester(R-ss-COOH).The crude products were purified by column chromatography to obtain R-ss-COOH,which were used as the intermediates for preparation of CMCTS-ss-R.After that,CMCTS-ss-R were synthesized by active acylation reaction.CMCTS was activated by EDCI/NHS,followed by reacting with R-ss-COOH to obtain the final product of reduction sensitive amphiphilic CMCTS-ss-R,which were used as nanocarrier materials.Fatty alcohol with different carbon length(C14-OH,C16-OH,C18-OH);and different molar ratio of sugar residues of CMCTS to R-ss-COOH(nCMCTS:nR-ss-COOH 1:0.1.1:0.2,1:0.3,1:0.4)were used to prepare a series of CMCTS-ss-R with different fatty chain length and degree of distribution(CMCTS-ss-C14?CMCTS-ss-C16?CMCTS-ss-Cl8).Fourier transform infrared spectroscopy(FT-IR),proton nuclear magnetic resonance(1H NMR)and mass spectrometry(MS)confirmed that a series of reduction-responsive intermediates R-ss-COOH and CMCTS-ss-R were successfully synthesized,The degree of substitution(DS)of hydrophobic chain were determined by ninhydrin colorimetric assay.For CMCTS-ss-R synthesized with different molar ratio of sugar residues of CMCTS/R-ss-COOH(nCMCTS:nR-ss-COOH=1:0.1,1:0.2,1:0.3,1:0.4),the DS of hydrophobic chain of CMCTS-ss-C14 were 1.57%,2.10%,1.99%and 3.04%,respectively;CMCTS-ss-C16 were 1.10%,2.04%,3.09%and 3.61%,respectively;CMCTS-ss-C18 were 1.20%,2.30%,3.19%and 4.56%,respectively.The solubility of CMCTS-ss-R were determined in water,PBS(pH 7.4)and other common organic solvents.CMCTS-ss-R were slightly soluble in water and PBS(pH 7.4),and insoluble in DMSO,CHCl3,CH2Cl2 and CH3OH.CMCTS-ss-R could self-assembly into nanoparticles in PBS(pH 7.4)by probe sonication,with diameter of 200?300 nm and narrow size distribution.The zeta potential of the nanoparticles formed by CMCTS-ss-C14,CMCTS-ss-C16 and CMCTS-ss-C18 were in the range of-15.73?-16.95 mV,-19.03?-20.91mV,and-20.53?-23.69 mV,respectively.The critical aggregation concentration(CAC)of CMCTS-ss-R were determined by the steady-state fluorescence probe method using pyrene as probe.For CMCTS-ss-R synthesized with different molar ratio of sugar residues of CMCTS/R-ss-COOH(nCMCTS:nR-ss-COOH=1:0.1,1:0.2,1:0.3,1:0.4),the CAC of CMCTS-ss-C14 were 89.13 ?g/mL,70.71?g/mL,73.98 ?g/mL and 64.58 ?g/mL,respectively;CMCTS-ss-C16 were 61.52 ?g/mL,53.75 ?g/mL,47.27 ?g/mL and 33.74 ?g/mL,respectively;CMCTS-SS-C18 were 61.24?g/mL,47.48 ?g/mL,40.42 ?g/mL and 27.89 ?g/mL,respectively.The CAC decreased with the decrease of molar ratio of CMCTS/R-ss-COOH and the increase of fatty chain length.Different glutathione(GSH)concentration solutions were used to simulate the reduction environment to investigate the redox-sensitive of CMCTS-ss-R nanoparticles.The results showed that CMCTS-ss-R nanoparticles were degraded in higher GSH concentration solutions,which indicated that CMCTS-ss-R would be an excellent reduction-responsive amphiphilic polymers as nanocarriers materials.In order to evaluate the safety of CMCTS-ss-R nanoparticles,their blood compatibility were evaluated by hemolysis test.The results showed that HR%value increased with the decrease of molar ratio of CMCTS/R-ss-COOH and decreased with the increase of the fatty chain length,indicating the blood compatibility become worse with the decrease of DS and length of the fatty chain.Although the blood compatibility of CMCTS-ss-R nanoparticles were slightly worse than that of CMCTS,the HR%values were less than 5%for CMCTS-ss-C14 nanopartilces(the molar ratio of CMCTS-ss-C14/R-ss-COOH were 1:0.1),CMCTS-ss-C16 nanopartilces(the molar ratio of CMCTS-ss-C16/R-ss-COOH were 1:0.1,1:0.2 and 1:0.3),and CMCTS-ss-C18 nanopartilces(the molar ratio of or CMCTS-ss-C18/R-ss-COOH were 1:0.1,1:0.2 and 1:0.3).The hemolysis test demonstrated that the good blood compatability of CMCTS-ss-R.The cytotoxicity of CMCTS-ss-R nanoparticles on L929 cells and A549 cells were investigated by MTT test.The result showed that the nanopartilces have no obvious influence on L929 cell survival at low concentrations.With the increase of nanoparticles concentration or decrease of the molar ratio of CMCTS/R-ss-COOH,the cytotoxicity increased,the IC50 decreased.With the increase of hydrophobic chain length,the cytoxicity decreased,the IC50 increased.The results were similar for A549 cells.Compared with L929 cells,the IC50 for A549 cells were lower.And CMCTS-ss-R nanoparticles have certain inhibitory effect on A549 cells.The results indicated that the nanoparticles were safe.Conclusively,the reduction-sensitive amphiphilic CMCTS-ss-R nanoparticels would be safe carriers for anti-cancer drugs.