A Novel Pullulan-based Co-carrier For Drug And Gene

A novel pullulan derivative (PSP) was prepared as a targeted co-delivery system of anti-cancer drug and therapeutic gene in cancer therapy. And the performance of PSP as carrier for hydrophobic drugs and exogenous gene was investigated.1. Synthesis and characterization of PSPStearic acid and polyethylenimine were used to modify pullulan to obtain amphipathic cationic polymer. And’H NMR, FTIR and elemental analysis indicated the successful synthesis of novel bifunctional vector (PSP). The critical micelle concentration of PSP was 58.9 μg/mL. The particle size, zeta potential of PSP nano-micelles were 188.75±3.18 nm and 17.83±0.75 mV, respectively. PSP also showed good buffering capacity, which maybe make PSP nano-micelles escape from endosome easily. The result of MTT and analysis of hemolysis demonstrated that the blank PSP nano-micelles showed excellent biocompatibility.2. Studies on PSP/DOX micellesIn this paper, Doxorubicin (DOX) was loaded in PSP nano-micelles using the dialysis method. With the increase of drug/vector ratio from 1:20 to 1:5, it was found that the encapsulation efficiency (EE) of PSP/DOX micelles decreased and the drug loading content (LC) increased, and the optimal LC (5.10%) and EE (56.07%) were achieved at 1:10 drug/vector ratio. Compared with free DOX, DOX from PSP/DOX micelles showed sustained release. The flow cytometry and confocal microscope images showed that PSP/DOX could be successfully internalized by MCF-7 cells. And the endocytic pathways of PSP/DOX was energy-dependent, associated with clathrin and condensation polymerization of actin mediated endocytosis. MTT assay demonstrated that DOX-loaded nanomicelles displayed slightly higher cytotoxicity in MCF-7 cells than free DOX.3. Studies on PSP/pDNA complexesThe particle sizes of PSP/pDNA complexes at different N/P ratios were between 84.16±6.36 nm and 165.23±0.74 nm, and the complexes were all positively charged, which facilitated gene transfection and effective cellular uptake. Electrophoresis assay indicated that PSP could effectively bind pDNA and protect DNA from degradation by DNase I and serum. In vitro transfection assay, reporter gene was smoothly transported into cells and expressed. PSP exhibited the highest transfection efficiency at the N/P ratio of 10. In addition, MTT assay showed that therapy gene p53 was also successfully delivered into MCF-7 cells by PSP and expressed to induce cell death. To compare Lipo 2000/pDNA and PSP/pDNA complexes, it was found that PSP/pDNA complexes displayed lower cytotoxicity and hemolysis rate.4. Studies of PSP/DOX/p53 and in vivo anti-tumor studies efficacyTEM showed that PSP/DOX/p53 complexes were nearly spherical with average size of around 30 nm. Gel retardation assay demonstrated that the drug-encapsulation displayed no adverse effect on the combination between PSP and pDNA. MTT assay and apoptosis assay revealed that the drug and gene co-delivery system (PSP/DOX/p53) displayed higher cytotoxicity than monotherapy, and induced more cancer cells to apoptosis. In vivo anti-tumor studies displayed that combined therapy demonstrated better anti-tumor efficacy with low levels of side effects. The above results suggested that PSP was a promising co-delivery system for cancer treatments.