| Objective: This study is to construct c RGD peptide modified p H-sensitive drug delivery system collecting intelligent, high efficiency and low cytotoxicity to achieve specific intracellular delivery of doxorubicin(DOX) to tumor cells and neovascular cells and promptly drug release. Its in vitro and in vivo anti-tumor effects were evaluated.Methods: The degradable diblock copolymer poly(ethylene glycol)-poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate)(PEG-PTMBPEC) was synthesized by open-ring polymerization and c RGD peptide was conjugated to the distal end of PEG-PTMBPEC by addition reaction. Their structures were characterized by 1H NMR. Blank micelles and DOX loaded micelles were prepared by dialysis method. Their sizes, zeta potential, PDI, morphous, drug loading contents and encapsulation efficiency were investigated by DLS, TEM and ?uorescence. B16 and human umbilical vein endothelial cells(HUVEC) which over-expressed αvβ3 integrin receptors were chose as the main model cells. The cytotoxicity of blank-micelles, the optimal density of c RGD and IC50 values of various micelles were evaluated by MTT assay. In cellular uptake study, confocal microscopy and flow cytometry were used to qualitative and quantitative analysis. Various inhibitors were used to evaluate the uptake mechanisms of non-target micelles(NT-micelle) and c RGD modified micelles(c RGD-micelle) by flow cytometry. CLSM was further confirmed the p H sensitive of micelles in cells. In vivo antitumor effect of the obtained micelles was performed on B16 tumor-bearing mice. Histological examination for heart specimens were performed using hematoxylin and eosin(H&E) staining.Results: PEG-PTMBPEC and c RGD-PEG-PTMBPEC had successfully synthesized with the purity of 80.3% and c RGD conjugation efficiency was 82.3%. The results suggested that the particle size of the obtained micelles was ranged from 153 to 181 nm and PDI were all below 0.3. The drug loading content was about 10% and encapsulation efficiency was 60%. TEM photos showed that micelles were well-distributed and regularly spherical in shape. The micelles also had good stability in plasma. Ex vitro release experiments indicated that DOX release from polymeric micelles was highly dependent on p H values of the mediums. Blank micelles were proved non-toxic to both tumor cells and normal cells, while DOX-loaded c RGD-micelle exhibited pronounced killing activity especially for B16 and HUVEC. Micelles with 10% c RGD presented 1.82-fold and 1.78-fold lower IC50 value than non-targeted micelles for B16 and HUVEC cells, respectively. Cellular uptake studies revealed that highly efficient uptake of c RGD-micelle was attributed to the receptor-mediated endocytosis. Multiple endocytosis pathways were involved and clathrin-mediated endocytosis was the major cellular entry which would sort micelles to endolysosomes and drug release from the micelles was triggered by its acidic environment. Importantly, micelles could dramatically reduce the systematic toxicity of DOX with the administration drug dose could be reach 15 mg/kg and c RGD-micelle retained excellent tumor killing activity in vivo, as evidenced by the lowest average tumor volumes after 16 th tumor implantation. H&E analysis indicated that drug loaded in micelles could nearly induce the cardiotoxicity at injection dose.Conclusions: In this study, we have successfully constructed c RGD modified p H-sensitive biodegradable micelles based on the synthesis of block copolymers. The prepared micelles have good size distribution, high encapsulated efficiency, well stability in plasma and are sensitive to the p H changes. Both in vitro and in vivo study indicated that the targeted micelles were more potent than the non-targeted micelles. This drug delivery system could achieve the promptly drug release in the tumor cells and could significantly reduce the cardiotoxicity of DOX at high administration dose. Overall, this p H-sensitive micelles decorated with c RGD may be a preferable active drug delivery carrier for cancer therapy. |
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