Development Of Composite Microparticles Co-loaded With SiRNA And Paclitaxelby Supercritical Fluid Technology

In the field of cancer therapy, co-delivery of gene and antitumor drug canachieve a better antitumor effect than single drug, and the development of theco-delivery carrier is very important. In this study, the method of ionic gelation andprocess of suspension-enhanced dispersion by supercritical fluids (SpEDS) were usedto prepare chitosan nanoparticles (CS NPs) and CSNPs/poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) triblock copolymer(PLLA-PEG-PLLA) composite microparticles (MPs), respectively. The operatingparameters for preparation of CS NPs and MPs were optimized, and thecorresponding mean size, morphologies, physicochemical properties and cytotoxicityof the two carriers were characterized; and the study on cytotoxicity of the carriersand antitumor effect of MPs co-loaded with siRNA and PTX were also carried out ata cellular level. The main work of this thesis can be summarized as follows:The operating parameters for preparation of MPs were optimized, and thedynamic light scattering (DLS), scanning electron microscope (SEM), transmissionelectron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR),differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and gelelectrophoresis were conducted to characterize the CS NPs. The result of single factorexperiment revealed that the mean size of CS NPs increased with the increasing inconcentration of CS; CS NPs achieved the smallest mean size when the concentrationof sodium tripolyphosphate (TPP) was1mg/mL, however, the concentration of TPPhad no significant effect on mean size of CS NPs; when the pH of CS increased,mean size of CS NPs decreased first and then increased. At the CS pH value of5.0,the mean size of CS NPs was the smallest; when the ratio of CS/TPP was set asconstant, the mean size of CS NPs increased with the increase in concentration of CSand TPP. After that, the conditions for preparation of CS NPs to fabricate MPs werechose as CS solution of2mg/mL and pH5.0, and TPP solution of1mg/mL. Theresult of FTIR indicated the reaction between phosphates of TPP and amines of CShappened. The result of gel electrophoresis revealed that siRNA could be encapsulated by CS, which could be improved by the addition of TPP, and theencapsulation efficiency was96.97%.The DLS, SEM, TEM, FTIR, DSC, TGA and high performance liquidchromatography (HPLC) were conducted to characterize the MPs. The result offactorial experiments demonstrated that the ratio of solvent/non-solvent had asignificant influence on mean size of MPs. The mean size and Zeta potential of theharvested MPs were consistent with the forecast of contour plot when concentrationof oil phase, ratio of water/oil, rate of emulsion and solvent/non-solvent were set as5mg/mL,0.75:10.00,2mL/min, and0.5:1.0, respectively. The core-shell structure ofMPs could be observed in the TEM image of MPs. The results of FTIR and DSCrevealed that there was almost no changes occurred on functional groups of thematerials, however, some changes on the physical state were observed. The result ofHPLC indicated that the drug load and encapsulation efficiency of MPs increasedwith the increase in different drug dosages, and there were no significant changesoccurred on the mean size and morphology of drug-loaded MPs.In the cytotoxicity evaluation of the carriers, both CS NPs and MPs exhibited agood biocompatibility and had no significant suppression effect on Bcap-37cells.Furthermore, the MPs had no hemolysis effect and acute systemic toxicity. In theexperiments of cellular uptake, cell apoptosis and antitumor effect, it could beobserved that after being co-cultured with Bcap-37cells, the MPs arrived around thenucleus of Bcap-37cells followed by a significant antitumor effect in combinedtherapy achieved by the MPs co-loaded with siRNA and PTX.In conclusion, the optimized MPs with a mean size of about300nm had a goodspherical morphology and a good biocompatibility. Furthermore, the MPs withsiRNA and PTX could arrive around the nucleus of Bcap-37cells and possess a goodantitumor effect, indicating that the MPs have potential in application of co-deliveringgene and antitumor drugs in the therapy of cancer with multidrug resistance.