High Efficient PH And Redox Dual-responsive Poly(amidoamine) Dendrimer Conjugates For Tumor Intracellular Dually Triggered Drug Release

Objective: The purpose of this study was to establish a smart and effcient polymeric drug delivery system with the characteristics of long circulation, active targeting, p H and redox dual responsive drug release and lysosomal escape, to realize the specific intracellular delivery of chemtherapy drugs to tumor cells and promptly drug release. Meanwhile, its in vitro and in vivo anti-tumor effects and mechanisms were studied and evaluated.Methods: G4 Polyamidoamine dendrimer(PAMAM) was used as the scaffold, and the His-modified PAMAM conjugates(PAMAM-His) with three different modification degrees(1:8, 1:16 and 1:32) were synthesized by controlling the feed ratio of the histidine(His) and PAMAM. Then, anti-tumor drug doxorubicin(DOX) was loaded into the hydrophobic cavity of PAMAM-His via physical encapsulation to obtain PAMAM-His/DOX complexes. By evaluating antitumor activity of different conjugates in vivo, PAMAM-His 1/32 was determined to be the most potential conjugate for further research. The redox-cleavable disulfide bond was introduced to link PAMAM-His and Poly(ethylene glycol)(PEG), yielding the p H and redox dual responsive PSSPH conjugate. To further improve the active targeting of PSSPH conjugate and enhance its antitumor activity, transferrin(Tf) was used to modify PSSPH to yield Tf-PSSPH conjugates via bifunctional HS-PEG2000-COOH. As a control, we also synthesized PP and PPH conjugates, and the PP/DOX, PPH/DOX, PSSPH/DOX and Tf-PSSPH/DOX complexes were prepared. 1H NMR and DLS were used to confirm and describe the conjugates separately. The dialysis bag methods were applied to evaluate drug release behavior in vitro. The the cytotoxicity of blank conjugate and the IC50 values of complexes were evaluated by MTT assay. The intracellular tracking of the four conjugates was investigated by Confocal Laser Scanning Microscopy(CLSM) with the RBITC as a fluorescence probe. Fluorescence microscope and flow cytometry were applied to qualitative and quantitative analyses of DOX cellular uptake by Hep G2 cells. Various inhibitors were used to evaluate the uptake mechanisms and CLSM was used to visualize the subcellular localization of conjugates. We use live-imaging technique and in vivo antitumor activity to characterize the targeting and anti-tumor effects of complexes. Finally, HE staining and Tunel staining were used to evaluate the tissue toxicity and tumor apoptosis.Rusult: PH with three different modification degrees were successfully synthesized and PAMAM-His 1/32 was determined to be the most potential conjugate for further research. PP, PPH, PSSPH and Tf-PSSPH conjugates were synthesized successfully and characteriaed with a size range of 20~30 nm. The complexes had high encapsulation efficiency and their loading contents were reached up to 10%. In vitro release study suggested that Tf-PSSPH had obviously p H and redox dual sensitive release property. All the blank conjugates were non-toxic to both Hep G2 and MCF-7 cells, indicating good biocompatibility. The in vitro cytotoxicity against Hep G2 cells showed that the tumor inhibition rate of Tf-PSSPH/DOX was significantly higher than that of complexes without Tf modification, moreover, the cellular uptake studies also revealed that compared with PSSPH/DOX, Tf-PSSPH/DOX can be much more specifically and efficiently uptaken by cancer cells. Cell uptake mechanism studies revealed that Tf-PSSPH/DOX interacted with plasma membrane by specific recognition between Tf and transferrin receptors, and subsequently was internalized by cells mainly through clathrin-mediated endocytosis. The intracellular tracking of Tf-PSSPH conjugates investigated by CLSM showed that Tf-PSSPH was first distributed in the lysosomes but subsequently escaped from the acidic microenvironment, and then enter into cytoplasm, where the disulfide bonds were cut by reductive cleavage in the high GSH condition to further promote drug release. In vivo study indicated that Tf-PSSPH/DOX could reach the tumor site more efficiently than PSSPH/DOX, and had obvious tumor inhibition effect. H&E analysis indicated that Tf-PSSPH/DOX exhibited almost no toxicity to normal organs, which was a great improvement in safty compared with free DOX. The Tunel staining showed that there were obvious apoptosis in tumors after Tf-PSSPH/DOX treatment.Conclusions: In this study we successfully constructed the Tf-PSSPH/DOX multifunctional targetting drug delivery system. The complexes had high encapsulation efficiency and loading contents, and showed good p H and redox sensitivity in drug release. Tf-PSSPH/DOX could reach the tumor site more efficiently, inhibit tumor growth effectively and significant reduce the cytotoxity of chemotherapy. In short, this multifunction targeted drug delivery system provides a potential application for cancer therapy.