The Anti-tumor Effect Of MBD1-siRNA Plasmid Loading PLGA/poloxamer Nanoparticles To Pancreatic Cancer

Objective To investigate the anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles to pancreatic cancer in vitro and in vivo.Methods (1)The siRNA sequence of MBD1 gene was designed and synthesized and inserted it into plasmid. Nanoparticles were prepared by a modified solvent diffusion technique. Nanoparticle characterization was investigated at different synthesis condition through dynamic light scattering and transmission electron microscopes. Plasmid DNA encapsulating and loading efficiencies were determined by spectrofluorometry. Stability of release of MBD1 siRNA plasmid DNA, DNaseⅠdigestion and cytotoxicity were also examined.(2) The transfection efficiency, gene expression level and anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer in vitro were tested. Flow cytometry was used to test the transfection efficiency of MBD1-siRNA plasmid loading PLGA/poloxamer. Distribution of nanoparticles in the pancreatic cancer cells was examined by fluorescence microscopy. MTT tests were used to test the anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles at different concentrations and times. RT-PCR was used to examine the change of MBD1 mRNA level after transfection. Western blot was carried to test the change of MBD1 protein level after transfection. Flow cytometry, TUNEL, and Hoechst staining were carried to test the apoptosis of tumor cells. Flow cytometry was carried to examine the cell cycle changes of tumor cells.(3) The transfection efficiency, gene expression level and antitumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer in vivo were tested. Tumor animal models were established by subcutaneous injection at subaxillary. MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles were delivered by intraperitoneal injection. Tumor volume was calculated through the whole process after injection of nanoparticles. Distribution of nanoparticles in the pancreatic cancer cells was examined by transmission electron micrography and fluorescence microscopy. RT-PCR was used to examine the change of MBD1 mRNA level after transfection. Western blot was carried to test the change of MBD1 protein level after transfection. Flow cytometry and TUNEL were carried to test the apoptosis of tumor cells. Flow cytometry was carried to examine the cell cycle changes of tumor cells. The change of tumor microstructure was tested through pathological examination.Results (1) No significant differences in particle size were observed between MBD1-siRNA plasmid loading PLGA/poloxamer (210.1±24.3 nm) and water loading PLGA/poloxamer (187.1±21.9 nm), suggesting that pDNA encapsulation did not affect particle size. Transmission electron micrography showed that the nanoparticles were spherical and were of almost equal size. No difference in the averageδpotential was found between MBD1-siRNA plasmid loading PLGA/poloxamer and water loading PLGA/poloxamer. MBD1-siRNA plasmid loading PLGA/poloxamer exhibited a similar biphasic plasmid DNA release pattern, characterized by a first initial rapid release (>30% of pDNA within the first day) followed by a slower, continuous release (90% released in 11 days). No DNA fragments were detected from the plasmid DNA released from MBD1-siRNA plasmid loading PLGA/poloxamer. MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles showed the protective effect against DNaseⅠ. PLGA/poloxamer nanoparticles did not exert toxic effects on the cells.(2)Though the intensity of transfection was low, the number of cells transfected was high and was maintained in a very similar manner throughout the duration of the experiment, which may be explained by the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles. Relative cell viability levels decreased as the concentration of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles increased from 1 mg/ml to 5 mg/ml. From days 3 to 7, the relative viability of BxPC-3 cells was among 20% and 35% compared to that of controls at a MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles concentration of 2mg/ml, which also points to the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer. In MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated BxPC-3 cells, MBD1 gene expression decreased gradually from day 2 and MBD1 protein expression also decreased from day 2 and completely disappeared at day 5. Apoptotic cells were observed by immunostaining for the presence of DNA fragments and the apoptosis rate was 24.19% in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells, which was higher than that of the blank control cells (4.79%).The percentage of S-phase in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells (33.68%) was higher than that of the blank control cells (8.03%).(3) The distribution of PLGA/poloxamer nanoparticles in tumors was confirmed by transmission electron micrography and fluorescence microscopy. In MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group, MBD1 gene expression decreased gradually from day 2 and MBD1 protein expression also decreased from day 2 and completely disappeared at day 5, which also points to the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer. Apoptotic cells were observed by immunostaining for the presence of DNA fragments and the apoptosis rate was 21.53% in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group, which was higher than that of the blank control cells. The percentage of S-phase in the MBDl-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells (15.74%) was higher than that of the blank control cells (8.01%). Death cells were found in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group through pathological examination. The volume of tumors of the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group was smaller than naked plasmid treated group and water loading PLGA/poloxamer nanoparticles treated group.Conclusions MBD1 is an ideal target for treatment of pancreatic cancer. PLGA/poloxamer nanoparticles have controlled release quality. MBD1 siRNA plasmid can be successfully transfected into tumor cells and the MBD1 nanoparticle compound can inhibit cell growth and induce apoptosis. The MBD1 nanoparticle is a promising candidate for gene therapy of pancreatic cancer.