Preparation And Evaluation Of PH-sensitive Drug-loaded Nanosystem Based On Carboxymethyl Chitosan

This work regards carboxymethyl chitosan(CMCS)and the daunorubicin(DNR)as the carriers and model drugs,respectively.The drugs were conjugated the carriers through the acid-sensitive imine bonds to form the amphiphilic prodrugs and the nanoparticles can be prepared by self-assembly.The structure of prodrugs was characterized,and the synthetic process conditions were also studied.The physicochemical parameters of nanoparticles were characterized,and the effects of molecular structure parameters on particle size and size distribution were investigated.The stability and pH sensitivity of the nanoparticles were studied and their biological activities were evaluated.The major studies and conclusions are as follows:(1)4-aminobenzaldehyde was transformed into 4-azidobenzaldehyde by azide reaction.The DNR-AZO intermediates were synthesized by Schiff base reaction,using the daunorubicin hydrochloride and 4-azidobenzaldehyde as reactants.The CMCS-PA intermediates were synthesized by amide reaction,using the CMCS and propiolic acid.The polymer prodrugs were synthesized by click reaction with cuprous ions catalyst(Cu~+),using two kinds of intermediates as reactants.The intermediates and prodrugs were successfully characterized by~1H-NMR,FT-IR,MS and the drug loading capacity and grafting ratio of prodrugs were further determined.Moreover,further researches about the synthesis condition of prodrugs indicated that the grafting ratio of prodrugs decreases with the augment of molecular weights of CMCS in the case that the coupling ratio of the two intermediates is constant,and it decreases with the augment of coupling ratio in the case that molecular weights of CMCS is constant.(2)the core-shell nanoparticles with different molecular structure parameters were prepared in aqueous media by self-assembly with assistance of ultrasound.And we determined the critical micelles concentration of nanoparticles by fluorophotometer.The morphology of nanoparticles was determined by transmission electron microscope and the mean size,size distribution,surface potential and variation of size distribution were determined by dynamic light scattering.The circulation stability was evaluated by comparing the results of determination.A high performance liquid chromatography method was used to determine the drug cumulative release of nanoparticles and verify their pH sensitivity.The results of researches exhibited that the nanoparticles have sphere-like shape,low polydispersity index(~0.1),and negatively charged surface(~-25 mV).Under the condition of invariable preparation conditions,the critical micelle concentration decreases with the increase of drug loading capacity,and the particle size also decreases with the increase of the drug loading capacity but increases with the molecular weights of CMCS.The results of analysis of particle size distribution and drug cumulative release curve show that the nanoparticles have good circulation stability and pH stability.(3)The HeLa cells(cancer cells)and L929 cells(normal cells)were acted as the model and control,respectively.The toxicity of nanoparticles and carriers were evaluated by MTT.The cell uptake of nanoparticles was studied by laser confocal microscope and flow cytometry.The results indicate that the non-toxicity of carriers at any concentration of study,and cell toxicity of nanoparticles was less than 10%at each concentration.But the cytotoxicity of nanoparticles and free drugs increases significantly with the time and concentration.And the toxicity of nanoparticles to HeLa cells is higher than L929 cells.The confocal laser scanning microscopy demonstrated that the nanoparticles were uptaken by HeLa cells efficiently and the released drugs in cells were translocated into nuclei gradually to exert anti-cancer effect.Flow cytometry also quantitatively proved the nanoparticles were endocytosed by HeLa cells,which conforms to the confocal laser microscopy results.