| Chemotherapy is the major route for tumor therapy. Nowadays, multidrug resistance and side effects of drug limit the effectiveness of chemotherapies in clinic. The strategies and approaches for oncotherapy are to circumvent multidrug resistance (short by MDR) and decrease the toxic and side-effects.The main aims of this research were to design a multifunctional amphiphilic star-shape polymer of lysine-linked di-tocopherol polyethylene glycol succinate. Star-shape copolymers have striking triple functions to prepare a promising nanocarrier in improving the chemotherapy of hydrophobic anticancer drugs by virtue of the following characteristics:long-time circulation, P-gp transporter reversible inhibitor and synergistic anticancer efficacy. Self-synthetic amphiphilic polymers were used as nanocarrier and hydrophobic doxorubicin were used as model drug to self-assemble in aqueous medium to prepare smart nanomicelles. The main contents of this study include the synthesis and identification of the polymer, preparation of the micelles, evaluation of the pharmaceutical pharmacy, cytotoxicity, cellular uptake,endocytosis mechanism, pharmacokinetic study and pharmacodynamics. Meanwhile, we also investigate the oral chemotherapy of nanomicelles in vitro and in vivo.First, lysine-linked di-tocopherol polyethylene glycol succinates were synthesized with vitamin E succinate as hydrophobic group and polyethylene glycol as hydrophilic group with the linker of lysine. Based on the different chain length of polyethylene glycol, the polymer were named as mPEG2000-Lys-di-VES (PLV2K-DOX), mPEG5000-Lys-di-VES (PLV5K-DOX), mPEG2000-NH-Lys-di-VES (PLV2K-NH2-DOX) and TPGS2K was synthesized as reference. Then the chemical structure of the synthesized copolymer was identified by1H NMR. The critical micelle concentration (CMC) of self-synthetic polymer was determined by pyrene as a fluorescent probe. The CMC of the copolymers were around2μg/mL, and with the content of polyethylene glycol, the CMC increased.Self-synthetic amphiphilic polymers as nanomaterials and hydrophobic doxorubicin as model drug to prepare smart nanomicelles, named as PLV2K-DOX, PLV5K-DOX, PLV2K-NH2-DOX, TPGS2K-DOX and TPGS-DOX. The preparation and formulations were optimized and screened based on particle size and encapsulation efficiency. The micelles were prepared by film-thin hydration and the physicochemical properties of nanomicelles were investigated. The encapsulation efficiency of PLV2K-DOX, PLV5K-DOX, PLV2K-NH2-DOX, TPGS2K-DOX and TPGS-DOX micelles were97.11%,96.81%,96.45%,91.68%and91.18%, and the particle size were13.26±2.86,16.4±2.70,34.1±0.90,11.10±0.10and13.5±2.44nm, respectively. DOX-loaded micelles were spherical with uniform size. Zeta potentials were around0mV because of hydrophilic surface of polyethylene glycol. DSC analysis suggested that DOX encapsulated in the core of micelles or DOX loaded in micelles in the state of amorphous state. The in vitro release of DOX was conducted by dialysis bag method. The results implied that the release of DOX from micelles was pH dependent, released rapidly at low pH conditions but slowly at physical condition, which was benefit for smart release of DOX in tumor cells.The cytotoxicity, cellular uptake and endocytosis mechanism of different DOX-loaded micelles were studied in MCF-7cells and MCF-7/Adr cells. In vitro cytotoxicity tests, the self-synthetic copolymers have abilities to inhibit the tumor growth. With the synthetic cytotoxicity of copolymer, DOX-loaded micelles shown significantly cytotoxicity in resistant MCF-7/Adr cells and overcome MDR compared with DOX-Sol. The cellular uptake of DOX-loaded micelles was conducted by flow cytometry and confocal laser scanning microscopy in P-gp over-expressed MCF-7/Adr cells. The DOX-loaded nanomicelles exhibited higher cellular accumulation than DOX-Sol. The P-gp inhibitory trials suggested that self-made nanocarriers have abilities to inhibit the activity of P-gp. Further investigation was conducted to show that self-made polymer was P-gp ATPase inhibitor. With the effect of P-gp inhibition of nanocarrier and bypass the recognition of P-gp by micelles, DOX-loaded micelles significantly improve cellular uptake. The endocytosis of micelles was energy-dependent. The mechanism of endocytosis included caveolae-mediated, clathrin-mediated, caveolae-and clathrn-independent endocytosis and macropinocytosis.UPLC/MS/MS method was developed to determine DOX-Sol and DOX-loaded micelles in rat pharmacokinetics after intravenous injection. In vivo pharmacokinetic results suggested that t1/2of DOX was8.1h, but the t1/2of PLV2K-DOX, PLV5K-DOX, PLV2K-NH2-DOX, TPGS2K-DOX and TPGS-DOX micelles was prolonged to183.8h,94.6h,181.6h,82.4h and42.0h, respectively. The AUC(0-t) of those micelles were10.4,7.5,5.1,7.2and4.9times compared with DOX-Sol, respectively. DOX-loaded micelles could increase the AUC and extend plasma circulation time to arrive the effect of long-time circulation, which could benefit drug to accumulate in target cells through EPR effect to improve the therapy effect. Balb/C mice bearing4T1breast transplanted tumor were used as models to study the antitumor activities of DOX-loaded micelles in vivo. The results implied that DOX-loaded micelles could significantly inhibit the growth of tumor and decrease the toxicity in mice by drug. The inhibitory rate of tumor of PLV2K-DOX, PLV2K-NH2-DOX and TPGS-DOX was62.92%,51.86%and56.95%, which was2.17,1.80and1.97times compared with DOX-Sol.In oral chemotherapy tests, human colon cancer cells Caco-2was selective as model to investigate the uptake and transport of DOX. The results suggested that PLV2K-DOX nanomicelles significantly increase the uptake and transport of DOX in intestinal cells. The transport path of PLV2K-DOX micelles was not opening the tight connection, but through energy dependent, caveolae-mediated and clathrin, caveolin-independent endocytosis. The membrane permeability of PLV2K-DOX was35.75±2.30, which was4.8times of DOX-Sol. So micelles which were used for oral chemotherapy could improve the intestinal permeability and increase the oral bioavailability of DOX. After oral administration of PLV2K-DOX micelles in SD rat, the oral absolute bioavailability of PLV2K-DOX was5times of that in DOX. So micelles could provide effective pathways to improve oral absorption of P-gp substrates. |
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