Research On Cerasome-based Carriers For SiRNA Delivery

RNA interference(RNAi)is an very important gene expression regulation mechanism,it has greatly promoted the development of the research of life science since its discovery in 1998.However,the application of RNAi was restricted because its main medium si RNAs are prone to be degraded by nucleases,absorbed by serum protein and eliminated by renal.Therefore,the research of si RNA delivery systems will greatly promote the development of RNAi.The lipid-based delivery system developed rapidly because of its good biocompatibility,low toxicity,high transfection efficiency,etc.However,liposomes were easily be affected by external conditions and environment.And si RNA-induced fusion will produce larger particles which will lead to lower endocytosis susceptibility and poorer vascular mobility.In recent years,researchers had systhesized an organic-inorganic hybrid lipid(cerasome-forming lipid,CFL).The cerasome,which was prepared by this new type lipid exhibted long storage period and well stability,had overcome many defects of the traditional liposomes.And no study had reported the application of CFL for si RNA delivery by now.In addition,whether siRNA can escape from the endosomes to the cytoplasm is the biggest obstacle after si RNAs enter into the cell.A previous study found that a cationic cholesterol derivative containing a hydroxyl head group exhibits well endosome escape efficiency in the delivery of DNA.But whether the hydroxyl head group have the same effect for si RNA delivery is unknown.Therefore,the primary goal of this paper is to validate the role of CFL and cationic lipid with hydroxyl head group in si RNA delivery,and to exploit the advantages of both for develop an effective si RNA delivery system.In this paper,we have synthesized a new type of hydroxylated cationic lipid(HCL)which has similar structure with CFL.Carries prepared with these two lipids will not only verify the HCL function,but also take full advantage of CFL.Thin-film hydration method was used to prepare cationic cerasomes(CCs)with various HCL and CFL mix ratio.In order to overcome the short blood circulation time of single-attribute lipid carrier in vivo,we also have prepared PEGylated cationic cerasomes(PCCs).Detailed characterization of CCs and PCCs demonstrated that the surface atomic layer of polyorganosiloxane of CCs and PCCs could effectively prevent self-aggregation and fusion of the carriers;the in vitro transfection efficiency of CCs was as high as 72.80%;the in vivo fluorescence images exhibited that PCCs have excellent liver accumulation effect;and the liver targeting functional studies showed that PCCs can effectively deliver si RNA to the liver and reduce target protein expression.Former results have shown that 5mol%PEG mix ratio have caused obvious transfection efficiency decrease.But the sphere structure of CCs is not conductive to quantify the PEG amount because the mixed PEG chain will both appear in the inner and outer suface.Researches show that a disc-like bilayer structure nanoparticles used for the study of membrane protein exhibited faster cellular uptake,better endosome escape and longer circulation time.And the structure of this nanoparticles have decided that the PEG chain will all expose on the outer surface of the nanoparticles,hence,it is very conductive to precisely evaluate the influence of PEG mix ratio to the trasfection efficiency.In order to fully take advantages of CFL and HCL,cationic bicellar nanodiscs(NDs)with disc-like structure were prepared with freeze-thaw method by using CFL and HCL.This carrier will also verify whether lipid based carriers with disc like structure could use for si RNA delivery,and test carriers with different PEG mix ratio to the influence of transfection efficiency.The NDs with the Si-O-Si surface structure had not only overcome the shortcoming of the poor stability of nanodiscs prepared by traditional lipids,but also exhibited well endosome escape and transfection efficiency in vitro.Although the NDs did not demonstrated longer circulation time than CCs,considering the structure features of NDs that the PEG chains have completely exposed to the carrier surface,we comprehensively evaluated PEG amount to the influence of transfection efficiency,cellular uptake and in vivo distribution.The results indicated that 1mol% of PEG doping ratio is sufficient to improve the circulation time of carrier and accumulation in target organs in vivo but have slight influence to in vitro transfection efficiency.In summary,The NDs and CCs prepared with the HCL all exhibited well endosome escape efficiency revealed that the hydroxyl head group might be helpful for si RNA endosome escape,and these findings may have a directive significance for the subsequent synthesis of novel cationic lipid.Meanwhile,we successfully utilized the advantages of high stability of cerasomes,and verified the feasibility of cerasome apply in si RNA delivery by preparing cationic cerasomes and cationic bicellar nanodisks.The results provided an experimental basis for exploring a new platform of si RNA delivery with the application of cerasomes.