Creation Of Cationic Peptidomimetic Based Lipid Nanoparticles And The Study Of Their Tumor Targeted RNA Interference Effect

Objectives:The objectives of this thesis are:(1)to synthesize and formulate novel lipid functionalized cationic peptidomimetics-based lipid nanoparticles(LNP);(2)to evaluate their efficiency of tumor targeted siRNA delivery in vivo.By measuring the in vitro gene transfection efficiency,we determine the model of amino acid arrangement in the cationic peptidomimetics,and based on these observations,we formulate biocompatible lipid nanoparticles.Furthermore,we compare the siRNA delivery efficiency of the novel lipid molecules,and study the tumor targeted siRNA delivery efficiency,as well as RNAi-mediated tumor suppression effect resulting from STAT3-mediated inhibition of immune checkpoint molecules.Methods:I.Formulation and in vitro study of novel lipid nanoparticles:(1)Formulation: first,we synthesized cholesterol functionalized cationic lipid containing ornithine tripeptide through either linear or branched conjugation(Chorn3).Next,we formulated lipid nanoparticle containing Chorn3,DOPC and PEG2000-C-DMA,and characterized the particles by gel electrophoresis,Zeta potential,dynamic light scattering,and TEM observation.Then,we measured in vitro transfection efficiencyof the lipid nanoparticles including MTT assay,FITC-siRNA transfection,and plasmid(pcDNA-eGFP)transfection.(2)In vitro evaluation of transfection efficiency:we systemically compared 8 Dogo lipids containing oleic acid moieties,and formulated synergistic lipid nanoparticles comprised of Dogo4 and Chorn3(denote DoCh)and characterized;then,we measured in vitro siRNA delivery efficiency such as FITC-siRNA distribution in the cells,and delivery of siSTAT3 in B16F10 melanoma cells.II.Tumor targeted siRNA delivery by DoCh: We established mouse B16F10 melanoma model,and systemically delivered FITC-siRNA/DoCh mixture or naked FITC-siRNA,and measured the fluorescence distribution and fluorescence intensity in tumor tissue and the major organs.To design in vivo the experiment,we first divided the established B16F10-GFP models in untreated group,siControl/DoCh group,siSTAT3/DoCh group and ADR group.After the intravenous injection(i.v.injection),we recorded tumor size and body weight in untreated group and all treated groups,and observed the tumor growth by live imaging.Then we calculated the tumor inhibition efficiency,and measured mRNA and protein level of target gene(STAT3)as well as immune check point gene(PD-L1).Finally,we recorded the survival rate until day 40.Results:I.Chorn3-based LNP: The yield of cholesterol functionalized peptidomimetic Chorn3 lipid was 89.8%.The formulation resulting from Chorn3,DOPC and PEG2000-C-DMA and formed stable,homogenous and biocompatible LNP with diameter of 150 nm.Chorn3 based LNP effectively transferred plasmid DNA to the cells and induced GFP gene expression,with the formulation of 5:1:0.1(molar ratio of Chorn3: DOPC: PEG-2000-C-DMA)showing the highest transfection efficiency.II.Dogo-based LNP: The results of transfection efficiency tests revealed that amongst 8 Dogo-based LNP,Dogo4 had the highest efficiency and lowestcytotoxicity.The synergistic LNP formulated from Dogo4 and Chorn3(denote DoCh)had homogenous particles,with maximum siRNA binding ability and negligible cytotoxicity.The in vitro RNAi effect of DoCh(4:1:0.1)reached 90% knockdown of STAT3 gene expression in B16F10 tumor cells.While the i.v.injection of naked FITC-siRNA showed no signal in tumor tissue,i.v.injection of FITC-siRNA/DoCh LNP mixture showed strong fluorescence signal in the tumor tissue.The FITC-siRNA also accumulated in other organs such as liver and lung.The body weight of untreated group and siSTAT3/DoCh treated group increased by 52% and13%,respectively(p<0.05),while ADR treated group induced 5% decrease of body weight,possibly due to the toxicity of ADR.There is an apparent difference of tumor growth between siSTAT3/DoCh group and untreated group,indicating the inhibitory effect of DoCh mediated siSTAT3 delivery.The tumor inhibition rate of siSTAT3/DoCh was 56.1%,less than that of ADR(80.8%).While all animals in untreated group and siCtrl/DoCh group died,there is 50% survival rate in siSTAT3/DoCh group.The mRNA level of STAT3 in siSTAT3/DoCh treated group decreased 90%,with strong inhibition of PD-L1 expression at both mRNA and protein level.Analysis of liver enzyme content in serum showed that DoCh mediated siRNA delivery do not induce liver toxicity,as the level of ALT/AST in serum did not increase(p<0.001).Conclusion:Chorn3 had excellent biocompatibility and in vitro nucleic acid delivery efficiency.However,Chorn3-based LNP had low efficiency for in vivo siRNA delivery.The synergistic LNP comprised of Chorn3 and Dogo4(DoCh LNP)had remarkable tumor targeting efficiency as well as biocompatibility.DoCh carrier efficiently delivered siRNA to mouse model of B16F10 tumor,induced RNA interference,and inhibit tumor growth.DoCh LNP is a promising platform for tumor targeted delivery of siRNA drugs.