Studies On Preparation Of SiRNA Nanoparticles Through Microfluidic Technology

Nano-carriers as efficient delivery system for gene drug have been attracted large researchers attention since it being invented,nevertheless,the development pace were slowing in the past 50 years,which is mainly attributed to the out-dated preparation technology.The disadvantage of conventional nanoparticle synthetic method,such as poor reproducibility,complicated operation and lack of precision and controllability,often results in nano-carriers with a high degree of polydispersity,seriously limiting the development of nanomedicine.Nowadays,applying precisely controllable,reproducible and stabilized microfluidic focusing technology into the synthesis process of nano-carriers is capable of enhancing efficiency?decreasing time and simplifying procedure,which contributes to produce uniform and small nanoparticles.In this study,cationic lipids were selected in combination with PEI to establish P/LNPs for siRNA delivery using two microfluidic chips(MF1,MF2).The stability,security and siRNA delivery efficiency of resulting P/LNPs-MF1,P/LNPs-MF2 were evaluated and investigated.Conventional method prepared P/LNPs(P/LNPsBM)were used as a control.The specific content of my research are summarized as follows:1.Optimization for formulation and preparation procedure of P/LNPs-MFOur research firstly optimized the formulation and preparation procedure to produce homogeneously dispersed P/LNPs-MF.The result showed that DODMA,Egg-PC,Cholesterol,PEI and DSPEmPEG2000(at a molar ratio of 40/19/35/1)were mixed with siRNA at 0.8mg/ml flow rate,followed by sonication to form P/LNPs-MF.These conditions have been identified as optimal parameters for generating P/LNPs-MF with suitbable size?distribution and stabilization.Among the P/LNPs-MF,P/LNPs-MF2 were the best one with particle size of 119.8nm?polydispersity index of 0.096 and surface charge of 13.6mV.P/LNPs-MF2 were proven to be stable even incubated with FBS for 12 h or conserved at 4? for 1 month.In contrast,P/LNPs-BM formed by Conventional mixing method exhibited large and heterogeneous particles.2.Anti-cancer study of P/LNPs-MF in vitroOur research then investigated anti-cancer effects of siRNA encapsulated P/LNPs-MF in vitro,the stability and security?siRNA delivery efficiency? transfection efficiency as well as gene silencing efficiency and anti-cancer cells efficiency of P/LNPs-MF were evaluated and determined.The MTT result showed that the cell viability of all naked P/LNPs remained above 80%.Oppositely,P/LNPs-MF containing siRNA exhibited remarkable repression effect when compared to P/LNPs-BM that rarely killed 20% cancer cells.Over 50% cancer cells were suppressed by P/LNPs-MF2 after 48 h treatment,which is twice as high as that of P/LNPs-BM.In addition,it was observed that P/LNPs-MF2 have the best transfection efficiency in A549?HepG-2 and MCF-7 cells by flow cytometry and laser confocal microscope.The effects of P/LNPs-BM and P/LNPs-MF1 were similar but lower than P/LNPs-MF2.qRT-RCR result showed that P/LNPs-MF2 significantly reduced 60.5% VEGF mRNA expression,which is 9.7% and 17.1% more than P/LNPs-MF1 and P/LNPs-BM,respectively.Western-blot result is consistent with qRT-RCR,indicating that gene silencing effect of P/LNPs-MF2 was more noticeable than that of P/LNPs-MF1 and P/LNPs-BM.3.Anti-cancer study of P/LNPs-MF in vivoOur research finally investigated anti-cancer effects of siRNA encapsulated P/LNPs-MF in vivo.The pharmacokinetic properties? tumor inhibition efficiency as well as gene silencing of P/LNPs-MF were evaluated and determined.Pharmacokinetics result showed that P/LNPs-MF2 successfully avoided reticuloendothelial system phagocytosis and prolonged siRNA circulation time in vivo,all of these contributes to increase siRNA accumulation at tumor site.Tumor inhibition result showed that P/LNPs-MF2 controlled the tumor size to about 300mm3.The growth of tumor would stagnate and began to recession,and the tumor size would shrink to 0.23 g after continuous treatment.However,the tumors were grown to around 800mm3 when treated with P/LNPs-BM.At the meanwhile,tissue section showed that the large amount of tumor cells appeared deformation and death,representing that P/LNPs-MF2 is capable of inducing tumor cells apoptosis compared to P/LNPs-BM that showed slightly inhibition for a part of cancer cells.In vivo gene silencing result further exhibited that P/LNPs-MF2 significantly induced gene silencing,leading to the reduction of VEGF mRNA and the corresponding protein level.Additionally,the physiological characteristics of nude mice as well as other main tissue samples and serum parameters demonstrated that P/LNPs were safety in vivo.P/LNPs-MF is thus confirmed to be safe and efficient delivery vector for siRNA.In conclusion,it was demonstrated that P/LNPs produced by MF method have better physicochemical properties and siRNA delivery efficacy than those made by BM method.Therefore,microfluidic chip can be applied as a novel nano-carriers synthetic method,promoting the development of nano-delivers system,and providing technical support for the next step enlarged production and clinical study.