Study On Microfluidic Based Lipid Nanoparticles Synthesis For SiRNA Delivery

Lipid nanoparticles(LNPs)based small interfering RNA(si RNA)delivery system have been proved effective for various diseases treatment including cancer.Traditional liposome preparation routes rely on macroscopic fluid mixing,which is time consuming and usually results in nanoparticles with poor repeatability.Microfluidic technology can be utilized to facilitate the LNPs synthesize process because of the good liquid controlling ability as well as rapid mixing in microchannel.However,due to the laminar flow at the millimeter level,the mixing efficiency is relatively low and further applications are limited.In this work,we designed a curved microfluidic chip with w-shape herringbone at the bottom of microchannel,which significantly improved the mixing efficiency by inducing chaos between two phases.This device was further employed to synthesis transferrin modified cationic lipid nanoparticles for si RNA mediated gene delivery.The content and results could be described as follows:1.Microfluidic device design and LNPs characterizationTo improve the mixing effect of liquids in microchannel,we manufactured wherringbone in a 200×75 ?m microchannel.The width and depth of w-herringbone were 35 ?m and 50 ?m,respectively.Results of fluorescence mixing experiment demonstrated that our microfluidic device exhibited enhanced mixing ability.To optimize the LNPs synthesize conditions in our microfluidic device,we first explored the influence of total flow rate(TFR),flow rate ratio(FRR),and lipid concentration on LNPs properties.Results indicating that under a condition of lipid concentration =5 mg/ ml,TFR= 735 ?l/ min,FRR= 20,we were able to synthesis microfluidic prepared transferrin lipid nanoparticles(M-Tf-LNPs)sized 110 nm,with a PDI of 0.15 and good stability over a 7-days period.By contrast,lipid nanoparticles synthesized by bulk ethanol injection method(B-Tf-LNPs)show larger size of approximately 200 nm.2.in vitro anticancer study of M-Tf-LNPsSubsequently,we investigated the in vitro cytotoxicity,cellular uptake and protein knocking down efficiency of M-Tf-LNPs.MTT assay was performed to explore the cytotoxicity of unloaded LNPs.The cell viability of blank M-Tf-LNPs treated groups are higher than 90% over a range of 5 ?l/well to 20 ?l/well,demonstrating the low cytotoxicity of M-Tf-LNPs.Results of flow cytometry illustrate that comparing with B-Tf-LNPs,M-Tf-LNPs delivered FAM-si RNA show stronger fluorescence intensity in Hep G2 cells,whereas naked si RNA co-cultured cells exhibit as little uptake as untreated cells,indicating that M-Tf-LNPs can be uptake by tumor cells rapidly.In the confocal microscope experiment we observed that most of the internalized M-Tf-LNPssi RNA located in cytoplasm.Result of western blot shows that M-Tf-LNPs mediated si RNA delivery lead to improved knocking down effect of survivin protein after 48 hours' transfection.3.in vivo anticancer study of M-Tf-LNPsTumor baring mice model was used to evaluate the tumor growth inhibition efficiency of si RNA containing M-Tf-LNPs.Observations obtained by IVIS live imaging system show that after caudal vein injection,M-Tf-LNPs delivered Cy3-si RNA accumulated in the tumor site within 2 hours and maintained rather strong after 24 hours.The changing curve of tumor volume during treatment period shows that M-Tf-LNPs can effectively inhibit the growth of tumor tissue.At the end of treatment,tumors were dissected and weighted.In accordance with previous results,a lower average tumor weight was observed in M-Tf-LNPs treated group,with a tumor suppression efficiency of 73% compared with saline control.We then harvested the main organs of mice for H&E staining.Similar to saline treatment group,no obvious tissue pathological changes can be observed by microscopy in M-Tf-LNPs group.In addition,the body weight variation of M-Tf-LNPs treated mice is limited in 15%.These results indicate the low level in vivo toxicity of M-Tf-LNPsIn summary,we present a novel w-herringbone microfluidic chip which can significantly improve the mixing efficiency of flow stream in microchannel.This device was employed to synthesis monodisperse M-Tf-LNPs,which enables LNPs formation as well as si RNA entrapment in a facilitated and highly controlled manner.Comparing with ethanol injection method producing LNPs,M-Tf-LNPs exhibit enhanced anticancer effect both in vitro and in vivo without visible systematic toxicity.