Synthesis And Evaluation Of Functional Legumain-activated Tumor-specific Polymer-DOX Conjugate

Doxorubicin is the most common anticancer agents for treating different kinds of solid tumors. However, its broader therapeutic application is limited by the poor targeting efficiency, short half-life, low drug efficiency and severe toxicity. Compared to the conventional chemotherapeutic agents, nano-scaled polymeric drug delivery systems for cancer therapy have demonstrated many distinct advantages, including versatile physicochemical properties, improved drug solubility, prolonged circulation time through avoiding rapid clearance by the renal and reticuloendothelial systems, decreased side effects, and passive targeting of tumor tissues via the enhanced permeability and retention(EPR) effect,and easy chemical modification or surface functionalization. According to the drug-incorporation mechanisms, polymer nanoparticle carriers can be divided into three categories including polymer-drug conjugates by covalent conjugation、polymer micelles through the hydrophobic interactions, polymer vesicles by physical package.Compared to other categories, polymer-drug conjugates might be cleaved to release pharmacologically active moieties under certain conditions. As a consequence, polymer-drug conjugates typically possess excellent stability against environmental changes and can overcome the shortcomings of other types of nanomedicines. However, such linkages might be too stable even in the tumor cells to release antitumor drugs effectively. The insufficient drug release may impair the overall anticancer efficiency of these polymer-drug conjugates and even hamper their final applications into human bodies. Based on the characteristics of the tumor microenvironment, this dissertation has synthesized a novel functionally legumain-activated tumor-specific polymer-DOX conjugate as drug carriers for tumor-targeted delivery and controlled release of doxorubicin to enhance the anticancer efficacy without systemic toxicity.In this paper, firstly, 4-arm PEG-COOH was prepared through condensation reaction between 4-arm PEG and succinic anhydride, 4-arm PEG-COOH was then activated with NHS, then 4-arm PEG-NHS was conjugated at the N-terminus of the alanine-alanine-asparagine-leucine(AANL). Finally, DOX was conjugated to 4-arm PEG-AANL in presence of PyBOP. The legumain-activated tumor-specific Polymer-drug was termed as 4-arm PEG-AANL-DOX. Another structural similar polymer-DOX conjugate(4-arm PEG-DOX) without enzymatically cleavable linker was also prepared as a control. Their structure were proved by 1H NMR、GPC、MALDI-TOF. The 4-arm PEG-AANL-DOX can self-assemble into micelle with a hydrodynamic radius of 31.6 ± 10.82 nm, which is confirmed by DLS.4-arm PEG-AANL-DOX was significantly accelerated release at the weakly acidic pH in presence of legumain, indicating a desirable enzyme sensitivity of tumor microenvironment; Confocal laser scanningmicroscopy revealed that 4-arm PEG-AANL-DOX could deliver DOX into the cytoplasm and nucleus of cells, more efficiently than that of 4-arm PEG-DOX; the cell proliferation inhibition activity of 4-arm PEG-AANL-DOX was much better than that of 4-arm PEG-DOX to MDA-MB-435 and MCF-7, but lower cytotoxicity to normal cells L929 compared with free DOX; the blood compatibility of the drug carrier demonstrated the excellent blood compatibility of 4-arm PEG-AANL-DOX and the potential application as drug delivery vehicles; Ex vivo doxorubicin fluorescence imaging measurements demonstrate that the 4-arm PEG-AANL-DOX has an improved tumor-targeting delivery as compared with the free DOX and 4-arm PEG-DOX;The result of in vivo antitumor efficiency study demonstrated that 4-arm PEG-AANL-DOX exhibited high antitumor efficacy and excellent safety without systemic toxicity. These characteristics have shown the legumain enzyme-sensitive polymer-DOX system could be a potential multifunctional coordination polymer nanoparticle carrier in the field of cancer therapy.