The Research Of RNA Interference Of EMs By PLGA-DOTAP As The Nano Delivery Carrier Of SiRNA

OBJECTIVE This study is to investigate the possibility of RNA interference(RNAi) for the treatment of endometriosis(EMs). For the first time, we evaluated the safety of PLGA-DOTAP/siRNA nanoparticles on EMs stromal cells, the feasibility of delivering siRNA to EMs stromal cells. We also investigated whether siRNA exert its biological functions in cells, and then provided the basis for PLGA-DOTAP as the RNAi delivery vehicle used in endometriosis treatment.METHODS 1. Specimens collection: ovarian endometriosis lesions of 6 patients who underwent surgical treatment for ovarian endometriosis in the Tian Jin Central Hospital of Gynecology and Obstetrics were obtained.2. Experimental Procedure:2.1 Primary culture EMs stromal cells, which were identified by immunofluorescence staining. 2.2 PLGA-DOTAP nanoparticles(NPs) were prepared and observed by tansmission electron microscope(TEM). The particle size and zeta potential were measured by laser particle size analyzer. 2.3 Agarose gel electrophoresis was used to investigate siRNA-loading capacity of PLGA-DOTAP NPs with different N/P ratio. 2.4 The cytotoxic effect of PLGA-DOTAP/siRNA with different N/P ratio was tested by CCK-8 assay. 2.5 Confocal laser scanning microscope(CLSM) was used to detect transfection ability of PLGA-DOTAP/siRNA to EMs stromal cells. 2.6 PLGA-DOTAP/mi R-142-3p mimic and PLGA-DOTAP/STAT3 siRNA were transfected to EMs stromal cells, respectively. m RNA expression were measured by realtime PCR after 48 hours of transfection.RESULTS 1. The TEM image clearly showed a well-defined core-shell structure of the PLGA-DOTAP hybrid nanoparticles. PLGA-DOTAP NPs had a mean diameter of 231.2±10.5 nm and a zeta-potential of 28.3±1.5 mv.2. At N/P=3 the binding efficiency is low. When the N/P ratio was increased to 6:1 or larger, PLGA-DOTAP encapsulated siRNA with high efficiency. PLGA-DOTAP had cytotoxicity on EMs stromal cells, and the toxicity increased with the increase of PLGA-DOTAP dosage. When concentration of transfected RNA was 100nmol/L,the cytotoxicities of PLGA-DOTAP in N/P=10:1 or 15:1 were less than Lipo(P < 0.05). CLSM had shown that PLGA-DOTAP/siRNA was able to deliver ds RNA into cytoplasm, and the morphology of the cells had no obvious change after transfection.3. After 6 hours of mi R-142-3p mimic transfection, contents of intracellular mi R-142-3p mimic in PLGA-DOTAP/mimic group and Lipo group were increased(P<0.01). When put into the same amount of RNA, content of intracellular mi R-142-3p mimic in PLGA-DOTAP group was more than that in Lipo group. 48 hours after transfection, the expression levels of gp130 m RNA were decreased in both PLGA-DOTAP/mimic group and Lipo group(P<0.05). When transfected RNA was 100nmol/L, expression of gp130 m RNA in Lipo group was lower than PLGA-DOTAP/mimic group(P<0.05).4. After 48 hours of STAT3 siRNA transfection, the expression of STAT3 mRNA were decreased in both PLGA-DOTAP/STAT3 siRNA group and Lipo group(P<0.05). When RNA was 100nmol/L, expression of STAT3 m RNA in Lipo group was lower than PLGA-DOTAP group(P<0.05).CONCLUSION 1. PLGA and DOTAP can be self-assembled to form nanoparticle which remains stable. When the N/P?6:1, PLGA-DOTAP can encapsulate siRNA with high efficiency to form PLGA-DOTAP/siRNA nanoparticles2. PLGA-DOTAP/siRNA NPs can successfully deliver siRNA to EMs stromal cells efficiently, and show little cytotoxicity to the cells.3. The efficiency of delivery ds RNA by PLGA-DOTAP/ds RNA NPs was higher than Lipofectamine 2000.4. PLGA-DOTAP/siRNA NPs had sustained release property. siRNAs delivered by nanoparticles can play the biological functions by targeting the specific m RNA.5. It shows bright future and broad development space for the treatment of EMS by RNAi using the delivery carrier of PLGA-DOTAP.