Tumor-targeted Drug Delivery By CRGD Modified Palmitic Acid-carboxymethyl Chitosan Based Polymeric Micelles

Object:To realize the effective treatment of cancer, a novel pH-responsive and tumor-targeted amphiphilic polymer palmitic acid-carboxymethyl chitosan modified with cRGD(cRGD-CMCH-PA) was synthesized to prepare micelles loaded with PTX.Methods:(1) cRGD-CMCH-PA was synthesized based on chitosan, and then characterized by the IR spectrum and 1H-NMR spectrum.(2) The critical micelle concentration(CMC) of cRGD-CMCH-PA in different pH PBS(pH 7.4 and pH 5.3) was measured by a fluorescence spectrometer with pyrene as a fluorescence probe. Blank micelles were prepared with thin film dispersion method and then the particle size and Zeta potential in pH 7.4 and 5.3 were measured. The safety of blank micelles was evaluated by hemolysis test and MTT assays.(3) PTX-loaded micelles were prepared and the the encapsulation efficiency and drug loading efficiency were measured. The release characteristics in vitro of Taxol® and cRGD-CMCH-PA/PTX in different pH were then investigated in the dialysis method.(4) Cytotoxicity of micelles was investigated with Luc-A549 cells model by MTT assays. The confocal microscopy, the flow cytometry and the live cell station were used to monitor the cellular uptake of micelles loaded with Coumarin6. The apoptosis of A549 cells treated with PTX in different formulations was detected by flow cytometry.(5) The targeting efficiency of micelles loaded with DiR in subcutaneous tumor-bearing nude mice and orthotopic lung tumor-bearing nude mice were evaluated by IVIS respectively.(6) IVIS and PET/CT were used to evaluate the treatment efficacy of drug-loaded micelles CMCH-PA/PTX and cRGD-CMCH-PA/PTX to subcutaneous tumor –bearing nude mice andl orthotopic lung tumor-bearing nude mice respectively.Results:(1) cRGD-CMCH-PA conjugates were successfully synthesized andthe grafting rate of the palmitic acid and cRGD were respectively 15.0% and 37.5%.(2) The CMC value of cRGD-CMCH-PA blank micelles in pH 7.4 aqueous environment was 2.72×10-3 mg/ml while in pH 5.3 was 1.40×10-2 mg/ml. The blank micelles were obtained with an average diameter of(162.9±1.5) nm and Zeta potential of(-17.0±0.2) mV at pH 7.4,while increased to(5.4±1.2)mV in pH 5.3. What’s more, hemolysis test and the MTT assays showed that the polymer was safe for drug delivery.(3) The optimal prescription of weight ratio of carrier/drug was 10:4 with particle size of(162.9±1.5) nm) and Zeta potential of(-11.4±0.3) mV. Release behavior study in vitro indicated that change of pH value in release media had no significant influence in the release behavior of Taxol®, while the release of cRGD-CMCH-PA/PTX was much faster in pH 5.3 than in pH 7.4.(4) MTT assay showed that cRGD-CMCH-PA/PTX had lower IC50 value against Luc-A549 cell lines compared to Taxol®. Besides, the results of confocal microscopy indicated the uptake amount of cRGD-CMCH-PA/C6 was higher compared to CMCH-PA/C6. The live cell station assay revealed that c RGD micelles presented a significant co-localization with lysosome. It was presented that the capability of PTX inducing apoptosis of A549 cell lines was significantly enhanced when PTX encapsulated with nano-carriers. Furthermore, cRGD modification further facilitated the inducing ability of polymeric micelles.(5) In vivo distribution of Dir loaded micelles test suggested that cRGD-CMCH-PA/DiR showed a better tumor targeting ability than CMCH-PA/DiR.(6) The antitumor assay in vivo revealed that cRGD-CMCH-PA/PTX performed enhanced anti-tumor efficacy in vivo than CMCH-PA/PTX.Conclusion: In this study, a novel amphiphilic and pH-responsive polymer cRGD-CMCH-PA was synthesized for the targeting delivery of the hydrophobic anti-cancer drug PTX. cRGD modification micelles cRGD-CMCH-PA/PTX showed a good anti-tumor efficacy in vitro an d in vivo. It was promising for cRGD-CMCH-PA polymer to be an effective carrier for the delivery of hydrophobic anti-tumor drug.