| Objective Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in clinic. Chemotherapy plays an important role in the treatment of HCC. Traditional chemotherapy has not been found to be effective in prolonging overall survival of HCC patients, meanwhile it is often associated with significant toxicities and acquired drug resistance. Angiogenesis plays an important role in the progression of HCC, thus antiangiogenesis is believed as a promising strategy for HCC therapy. In this study, a stepwise p H-responsive nanoparticle system, containing charge reversible pullulan-based(CAPL) shell and(?-amino ester)(PBAE)/poly(lactic-co-glycolic acid)(PLAG) core, is designed to be used as carriers of paclitaxel(PTX), a widely used chemotherapeutic drug in clinic, and combretastatin A4(CA4), a known angiogenesis inhibitor, for combining antiangiogenesis and chemotherapy to treat HCC.Contents The contents of this research were mainly divided into three main parts as follows. The first part was the preparation and characterization of CAPL surface-coated PBAE/PLGA(CAPL/PBAE/PLGA) nanoparticles), including the synthesis and chemical structure confirmation of CAPL, and the preparation and characterization of CAPL/PBAE/PLGA nanoparticles. The second part was the in vitro study of CAPL/PBAE/PLGA nanoparticles, including the evaluations of their p H-responsive and charge reversal capabilities, and their cytotoxicity in human hepatoma Hep G2 cells and human umbilical vein endothelial cells(HUVECs). The third part was the in vivo study of CAPL/PBAE/PLGA nanoparticles, including their biodistribution, anti-tumor activity, and anti-angiogenesis effect in Hep G2 tumor-bearing mice.Methods1. The preparation and characterization of CAPL/PBAE/PLGA nanoparticles:CAPL was synthesized from pullulan via two reactions and characterized by the IR, 1H NMR and p H-metric titration methods. PBAE/PLGA nanoparticles were prepared by the solvent emulsion evaporation method, and CAPL was then coated on their surfaces by the charge interactions using incubation method to prepare CAPL/PBAE/PLGA nanoparticles. The morphology of CAPL/PBAE/PLGA nanoaprticles was observed by the transmission electron microscopy(TEM), and their size, size distribution and Zeta potential were determined respectively using the dynamic laser scattering method and Zeta potential analyzer.2. The in vitro studies of CAPL/PBAE/PLGA nanoparticles: The capability of p H-responsive charge reversal of CAPL/PBAE/PLGA nanoaprticles was evaluated by detecting their Zeta potentials in buffer solutions with different p Hs. The cellular uptakes and intracellular localizations of Dil(a membrane fluorescence stain) labeled CAPL/PBAE/PLGA(CAPL/PBAE/PLGA/Dil) nanoparticles in Hep G2 cells were analyzed by the flow cytometry and laser scanning confocal microscope. PTX and CA4 were separately loaded into CAPL/PBAE/PLGA nanoparticles to prepare CAPL/PBAE/PLAG/PTX and CAPL/PBAE/PLGA/CA4 nanoaprticles. The loading contents and encapsulation efficiencies of PTX and CA4 in nanoparticles were detected by ultraviolet spectrophotometry. The in vitro drug releases of PTX and CA4 from CAPL/PBAE/PLGA nanoparticles were studied in buffer solutions with different p Hs using dynamic dialysis method. The effects of CAPL/PBAE/PLGA/PTX and CAPL/PBAE/PLGA/CA4 nanoparticles on the proliferations of Hep G2 cells and HUVECs, and the migration of HUVECs were assessed respectively by MTT assay and wound healing test.3. The in vivo studies of CAPL/PBAE/PLGA nanoparticles: HepG2 cells were injected subcutaneously into the nude mice to construct HCC mouse model. The biodistribution of Cy5.5(an infrared fluorescent dye) labeled CAPL/PBAE/PLGA nanoparticles in Hep G2 tumor-bearing mice was evaluated by the in vivo imaging technique. In addition, the accumulations of CAPL/PBAE/PLGA/Dil nanoparticles in the liver, spleen and tumor tissues were further assessed using the frozen section technique and fluorescence fluorescence microscope. After combination treatment of CAPL/PBAE/PLGA/PTX and CAPL/PBAE/PLGA/CA4(PTX/CA4) nanoparticles via administration of tail vein injection, the tumor growth was detected by measuring the tumor size, the histopathological changes of the organs and tissues of tumor-bearing mice were observed using hematoxylin and eosin stain, and the inhibitory effect against angiogenesis was assessed by the immunohistochemical method.Results1. CAPL was synthesized and its chemical structure was confirmed by the IR and 1HNMR spectroscopy. CAPL/PBAE/PLGA nanoparticles were successfully prepared and had exhibited a typical “core-shell” structure. CAPL/PBAE/PLGA nanoparticles had a small size about 178.1 nm with relatively narrow distribution and Zeta potential of 17.8 m V, and exhibited excellent in vitro stability.2. CAPL/PBAE/PLGA nanoparticles had pH-responsive charge reversal capability. They were generally stable and negatively charged at the physiological p H, but could be hydrolyzed and free the amino group in weakly acidic conditions, thus exposing positively charged PBAE/PLGA nanocores. PTX and CA4 were efficiently loaded into CAPL/PBAE/PLGA nanoparticles and their loading contents were 8.2% and 6.9%, respevtively. PTX and CA4 both displayed significant p H-responsive in vitro releases and their release rates gradually increased with the decrease of the p H value of release medium. Moreover, the accumulation of CAPL/PBAE/PLGA/Dil nanoparticles in Hep G2 cells also exhibited significant p H-selectivity. PTX/CA4 nanoparticles had remarkably synergistic inhibitory effect on the growths of Hep G2 cells and HUVECs. Compared to the mixture of PTX and CA4, PTX/CA4 nanoparticles displayed slightly increased inhibition effect on the migration of HUVECs.3. At 24 h after administration via intravenous injection, CAPL/PBAE/PLGA/Cy5.5 nanoparticles were mostly accumulated in the tumor tissues, and partially in the liver of Hep G2 tumor-bearing nude mice. Compared with other treatment groups, the combination treatment of PTX/CA4 nanoparticles significantly inhibited thegrowth of Hep G2 tumor and depressed the tumor angiogenesis at PTX and CA4 doses of 8 mg/kg and 10 mg/kg. Fortunately, no obvious histopathological changes were observed in the main organs in Hep G2 tumor-bearing mice with treatment of PTX/CA4 nanoparticle.Conclusions CAPL/PBAE/PLGA nanoparticles, which could produce step-by-step responses respectively to weakly acidic tumor microenvironment and endo/lysosome, were prepared and used as carriers of PTX and CA4 for combining antigiogenesis and chemotherapy to treat HCC. CAPL/PBAE/PLGA nanoparticles showed relatively high loading capabilities for PTX and CA4, and efficiently realized their orderly in vitro releases. At cellular levels, CAPL/PBAE/PLGA nanoparticles significantly enhanced synergistic effects of PTX and CA4 on the proliferations of Hep G2 cells and HUVECs, and the migration of HUVECs. In Hep G2 tumor-bearing mice, CAPL/PBAE/PLGA nanoparticles exhibited an excellent hepatoma-targeting capability and remarkably increased synergistic effects of PTX and CA4 on tumor growth and tumor angiogenesis. In summary, CAPL/PBAE/PLGA nanoparticles displayed great potential as a HCC-targeting delivery carrier for antitumor drugs. |
PDF Full Text Request