Redox/pH Dual-responsive PAMAM Drug Delivery System With RGD Modified And Its Mechanism

In this study, we aimed to establish a polymeric drug delivery system that have these functions of long circulation, active targeting, redox and p H responsive drug release. Polyamidoamine dendrimer(PAMAM) was used as the scaffold, poly(ethylene glycol)(PEG) were first conjugated to it by cleavable disulfide bonds and yielded redox and p H dual-responsive PAMAM-PEG(PSSP)conjugates with different PEGylation degree, then Doxorubicin(DOX) was as model antitumor drug to preparation PAMAM-SS-PEG/DOX(PSSP/DOX) complexes. By evaluating antitumor activity of different conjugates in vitro and in vivo, PSSP32 which had the highest PEGylation was found to be the most poteni conjugate. To further improve the active targeting of PSSP32 conjugate and enhance its antitumor activity, c RGDy K peptide was used to modify PSSP32 to yield RGD-PSSP conjugates via bifunctional HS-PEG5000-COOH, then the RGD-PSSP/DOX complexes were prepared. The pharmaceutics properties, cytotoxicity, cell uptake, uptake mechanisms and antitumor activity in vivo were invitagated systematically and deeply.Firstly, with G4.0 PAMAM, SPDP and HS-PEG5000-OCH3 as the starting materials, PSSP conjugates with three different PEGylation degree were prepared, namely PSSP8, PSSP16 and PSSP32, respectively. TLC, 1H NMR and FTIR were used to confirm the construction of conjugates, and the particel size and Zeta potential were measured by dynamic light scattering(DLS). In addition, the redox sensitivity, hemolytic toxicity and in vitro stability in 10% fetal bovine serum(FBS) of PSSP conjugates were investigated. Moreover, in vitro RAW264.7 phagocytic cell uptake study of PSSP conjugates were performed through fluorescence microscope and flow cytometry analysis, and the results showed that PSSP conjugates with PEGylation modification can obviously reduce the uptake by RAW264.7 cell, thus achieving the long circulation in blood and enhance accumulation of PAMAM at tumor sites via the enhanced permeability and retention(EPR) effect. Finally, with the RBITC as a fluorescence probe, the intracellular trafficking of PSSP conjugates was investigated by Confocal Laser Scanning Microscopy(CLSM). The results showed that the conjugates mainly distribution in cytoplasm and lysosome and then could escaped from the lysosome and enter into cytoplasm, which could effectively release the antitumor drug by reductive cleavage of the disulfide bonds in high GSH concentration.Secondly, DOX was selected as a antitumor model durg and PSSP/DOX complexes with three different PEGylation degree were prepared by hydrophobic interaction. The pharmaceutics properities of PSSP complexes were investigated and the results showed these complexes had higher encapsulation efficiency and loading content was about 10%, the particle size and zeta potential of all complexes did not change before and after drug loading. In vitro release studied suggested that PSSP/DOX complexes were obviously redox and p H-sensitive, and the release rate significantly accelerated with the increased PEGylation degrees.Thirdly, B16 and A549 cells were selected as a antitumor cell model and in vitro cytotoxicity, cell uptake and intracellular drug distribution of PSSP/DOX complexes were investigated. The results suggested that with the PEGylation degrees increasing, the cytotoxicity was increased but the cell uptake was decreased, confirming that the intracellular drug release was the key factor which decided the cytotoxicity by combining with the results of in vitro drug release, cytotoxicity and intracellular drug distribution. The cell uptake mechanism of PSSP/DOX complexes was studied by using various endocytosis inhibitors. The results showed that the complexes were entered into cell use two pathways, including clathrin-mediated endocytosis and caveolin-mediated endocytosis. In addition, the cytotoxicity of PSSP/DOX complexes was further studied by acid inhibitors of chloroquine and GSH inhibitors of diethyl maleate(DEM). The results showed that the two inhibitors could restrain the intracellular acid and redox environment, respectively, and result in the cytotoxicity weakened. The results also further manifested that the PSSP/DOX complexes enter into cell by clathrin-mediated endocytosis and caveolin-mediated endocytosis, and then released antitumor drug in two kinds of intracellular environment. Finally, antitumor activity of PSSP/DOX complexes in B16 tumor bearing mice was evaluated by using B16 tumor bearing mice as tumor animal model. The results showed that the complexes could obviously inhibit tumor growth and significantly decreased the DOX side effects.Finally, c RGDy K peptide was used to modify PSSP conjugates to yield RGD-PSSP conjugates via bifunctional HS-PEG5000-COOH, then the RGD-PSSP/DOX complexes were prepared and thier in vitro and in vivo evaluation was carried out. In vitro release results demonstrated that RGD-PSSP/DOX complexes were obviously redox and p H-sensitive, and RGD modification had no influence on the release compared with the PSSP/DOX complexes. Human umbilical vein endothelial cell(HUVEC) and B16 cell were used as the endothelial cell model of tumor neovasculature and tumor cell model, respectively, and the cytotoxicity, cell uptake and uptake mechanism of RGD-PSSP/DOX complexes were investigated. The cytotoxicity results showed that RGD-PSSP/DOX complexes modified with RGD had displayed significant to HUVEC and B16 cells. Cell uptake and uptake mechanism studies revealed that RGD-PSSP/DOX interacted with plasma membrane by specific recognition between integrin avβ3 and RGD peptides, and subsequently be internalized mainly by clathrin-mediated endocytosis and caveolin-mediated endocytosis. Finally, antitumor activity of RGD-PSSP/DOX complexes in B16 tumor bearing mice was evaluated. The results showed that RGD-PSSP/DOX had displayed higher antitumor in vivo compared with PSSP/DOX complexes and could significant reduce the DOX side effects. In addition, the HE staining images of tumor tissues and major organs showed that RGD-PSSP/DOX exhibited almost no toxicity to normal organs, which was a great improvement in safeness compared with DOX solution.