Development Of NPs-inlaid Porous PLLA Microspheres Co-loaded With SiRNA And GLP-1 By Compressed CO₂ Antisolvent Process And Their Application In Diabetes Therapy

In the field of diabetes therapy, co-delivery of gene and drug would combine advantages of different drugs. An appropriate design will stimulate the synergistic effect between different drugs which is important to the treatment of diabetes. In this study, a nanoparticles inlaid porous microparticles(NIM) system co-loaded with siRNA and GLP-1 was developed by supercritical fluid technology. The operating parameters for preparing the MPs were optimized, and the corresponding mean size, morphologies, physicochemical properties and cytotoxicity of the co-loaded system were characterized. Meanwhile, a primary evaluation of this co-loaded system was conducted. The main work of this thesis can be summarized as follows:1. The preparation and characterization of the NPs and porous MPs: the NPs was prepared by a ionic gelatin method and the scanning electron microscope(SEM), transmission electron microscopy(TEM), Fourier transform infrared spectroscopy(FTIR) were conducted to characterize the prepared CS NPs. The results showed that when the concentration of the chitosan(CS) was 2 mg/mL, the concentration of the sodium tripolyphosphate(TPP) was 1 mg/mL and the pH of the CS was 5.0, the particle size of the NPs can be controlled within the range of 50~300 nm. The result of FTIR indicated the reaction between phosphates of TPP and amines of CS happened. The operating parameters for preparation of MPs were optimized. Based on the result of optimization experiment, an optimized fabrication condition was obtained with an emulsion flow rate of 4.0 mL/min, a CO2 venting rate of 40 g/min, a ratio of menthol:PLLA of 1:1, and a nozzle size of 0.006 inch. Under this condition, porous microparticles with a rough surface and average particles size of 10.43 μm was prepared. The sustaining atomization time and the atomization efficiency of the prepared MPs was 0.12 s and 96.16±5.21% respectively which indicated an efficient application in the subsequent pulmonary drug delivery.2. Development of the construction of the NIM system and its drug loading property: firstly, the NIM system was prepared under the optimized condition, and SEM, FTIR, XRPD and TEM were conducted to characterize the prepared NIM system. Whenever the drug loading increases from 5.0% to 10.0%, they all have a large geometric diameter(Dg) and a small aerodynamic diameter(Da) which were good for the pulmonary drug delivery. Secondly, the results of confocal laser scanning microscope(LSCM) and the energy disperse spectroscopy(EDS) indicated that the CS NPs can be inlaid in the MPs evenly. Lastly, the drug loading of the siRNA was 94.8% and the drug was released from the CS NPs continuously in 32 h. The actual drug loading of the GLP-1 was increased with the increase of the the theoretical drug loading, and the drug were all released from the MPs in 36 h.3. The study of the NIM system co-loaded with siRNA and GLP-1 on biocompatibility and the hypoglycemic effect: in the cytotoxicity evaluation of the NIM system, CS NPs, MPs and NIM system all exhibited a good biocompatibility and had no significant suppression effect on HepG2 cells. Furthermore, the NIM system had no hemolysis effect or acute systemic toxicity.4. Application of the NIM system in diabetes therapy: a mouse model of type 2 diabetes was built to evaluate the hypoglycemic effect of the NIM system. Results showed that the blood glucose of the NIM system co-loaded with siRNA and GLP-1 was lower than other groups. The hypoglycemic effect of the NIM system at molecular level was further studied: results of the QRT-PCR showed that the DPP-4-mRNA expression of co-loaded system group was significantly inhibited by co-delivery of siRNA and GLP-1. The siRNA of co-delivery group exhibited a significant gene silencing resulting to a better combined therapy effect. These results were consistent with the animal experiment.In conclusion, the optimized NIM system was porous and spherical. The aerodynamic property of the NIM system was good which meant a large Dg and a small Da. CS NPs were inlaid in the MPs evenly. Besides, the drug loading property and the biocompatibility of the NIM system were good. The hypoglycemic effect of the NIM system co-loaded with siRNA and GLP-1 was better than other groups’. All the results above indicated that the NIM system has a potential in the application of co-delivering gene and proteins in the therapy of diabetes, cancers and other diseases.