| Conventional chemotherapy drugs have poor selectivity,which can easily cause systemic toxic and side effects,and only a small amount of drugs can reach the tumor.Advanced drug delivery systems often employ natural nanomaterials to prepare targeted nanoparticles(NPs)through self-assembly,so as to deliver drugs to the required disease sites and enhance the curative effect.However,it’s difficult for such NPs to maintain stability during blood circulation,chemical coupling macromolecular prodrugs have been developed to make up for this deficiency.When chemically conjugated-NPs reach target cells,drug release still will be affected by the over-stable chemical bonds.In addition,repeated low-dose stimulation may also lead to drug resistance in diseased cells.Therefore,in order to further increase the drug concentration in tumor cells and rapidly exert the therapeutic effect,a serious of stimulus-sensitive prodrug nano-drug delivery systems were designed by using chemical synthesis and self-assembled nanotechnology.The obtained NPs exhibit stability in blood circulation and controlled release-behavior in tumor cells,which will increase the concentration of chemotherapy drugs in tumor sites and reduce systemic toxic and side effects.In this study,doxorubicin(DOX)and galactosylated chitosan(GC)were chemically conjugated to form DOX-conjugated chitosan polymer prodrug(GC-CAD)through acid-sensitive N-cis-aconityl amide linker(CAA),and then NPs were formed by in-situ self-assembly.This paper includes the following sections.Chemical synthesis of GC-CAD,and the preparation and characterization of GC-CAD NPs.DOX was modified by CAA through amide condensation reaction to synthesize CAD intermediate.Then,CAD and GC were chemically conjugated,and the GC-CAD prodrug nano-system was formed by self-assembly.The chemical structures of CAD and GC-CAD were characterized by FT-IR and ~1H-NMR.The nano-size and distribution of GC-CAD NPs were measured by method of dynamic light scattering(DLS),the morphology was observed by transmission electron microscope(TEM),the drug loading content was measured through UV-Vis spectrophotometry,and the drug release performance was measured via DLS.Results confirmed that CAD and GC were successfully conjugated and GC-CAD nano-system was prepared.The average size of the nano-system was 133.7±1.8 nm,and the zeta potential was﹢7.72±0.9 m V.The particle was spherical,which was uniform in size.And these NPs remained stable for up to 7 days.The results showed that drug loading was 14.4%±1.2%.Drug release experiment showed that the release rate and cumulative release of the polymeric prodrug NPs in the slightly acidic microenvironment(p H 5.0)were significantly higher than those in the simulated blood environment(p H 7.4)with p H-responsive property.The biocompatibility of the GC-CAD NPs was evaluated.The bloodbiocompatibility of GC-CAD NPs was studied by hemolysis experiment.At the same time,the MTT assay was conducted to evaluate proliferation inhibition of H9c2,L02and HUVEC cells.Compared with free DOX group,the hemolysis rate of GC-CAD NPs were lower and survival rates for three kinds of normal cells were higher than free DOX after incubation with GC-CAD NPs,which indicated that the synthesized GC-CAD NPs had excellent biological properties.To verify the anti-tumor effect and targeting ability of GC-CAD NPs in vitro,cell proliferation inhibition experiments,cellular uptake and competition binding experiments on hepatocellular carcinoma cell lines Bel-7402,Hep G2,and SMMC-7721 were performed.These results showed that GC-CAD nanosystem could greatly inhibit the proliferation of hepatocellular carcinoma cells,especially the Bel-7402,and the inhibition rate showed a time-and concentration-dependent properties.The result of fluorescence uptake experiments was exhibit a highest uptake ability of Bel-7402 to the nanosystem compared with others.And the competition binding experiments was further confirmed the cell uptake of GC-CAD NPs on Bel-7402 cells was plays a targeting role via mediation of ASGPR.The in vivo bio-distribution and in vivo anti-tumor efficacy of GC-CAD NPs was evaluated in the Bel-7402 xenograft BALB/c nude mice model.The fluorescence signal of the GC-CAD NPs was mainly concentrated in the tumor tissue,which showed splendid in vivo tumor targeting ability.At the same,the results of xenograft BALB/c nude mice of antitumor study showed that GC-CAD NPs performed a better inhibition of tumor growth compared with the free DOX group.It is proved that GC-CAD NPs could reduce the toxic and side effects of DOX,while with an enhanced antitumor effect.Conclusion:We have developed NPs(GC-CAD)by conjugating GC to CAA-modified DOX to synthesis macromolecular prodrug GC-CAD.The preparation process was simple and easy.The prepared GC-CAD NPs possessed the characteristics of acid responsive,showed good biocompatibility and possessed better anti-tumor effects in vitro and in vivo than free DOX.Therefore,GC-CAD NPs has been proven to be have a great potential as an intelligent drug delivery system for the treatment of HCC. |
