Preparation And Anti-cancer Activity Of Lipid Nanoparticles Encapsulated With PKN3 ShRNA

Recently,RNA interference(RNAi)-based therapies have shown tremendous potential in cancer therapy.Usually,the overexpression Protein kinase N3(PKN3)might induce the enhancement of cell migration and invasive behavior through the phosphatidylinositol 3-kinase(PI3K)signaling pathway,promoting malignant tumor growth and lymph node metastasis.PKN3 is a potential target for the treatment of cancer and lymph node metastasis.Therefore,it is expected that tumor therapy can be achieved by silencing the m RNA of PKN3 and inhibiting the protein expression of PKN3.As a kind of important nano carrier,lipid nanoparticle(LNP)based on ionizable lipids can effectively package and deliver sh RNA,si RNA or m RNA.Lipid nanoparticle(LNP)based on ionizable lipids has the advantages of high transfection efficiency and good biocompatibility,but lack of targeting.The concentration of GSH in tumor tissue and tumor cells is much higher than that in normal cells.To achieve targeted delivery by endowing nano carriers with reduction responsiveness,which is conducive to reducing off target effect and toxic and side effects.Herein,it is expected to develop the ionizable lipid nanoparticles SSLNP encapsulated PKN3 sh RNA(SS-LNP/sh PKN3)with reduction responsiveness and explore the antitumor effect in vitro and in vivo.The specific contents are as follows:Part 1: The reduction responsive lipid nanoparticles SS-LNP encapsulated PKN3 sh RNA(SS-LNP/sh PKN3)was prepared.First,new PKN3 sh RNA sequences(sh PKN3-1 and sh PKN3-2)were designed.Then,ionizable lipid(DDA-SS-DMA)with disulfide bonds and ester bonds were synthesized by three-step reaction.The ionizable lipid was mixed with cholesterol,DSPC,DMG-PEG2000,and PKN3 sh RNA by a microfluidic mixer to prepare a lipid nanoparticle(SS-LNP/sh PKN3).Agarose gel electrophoresis experiment indicated that sh RNA was completely bound with the SS-LNP based on DDA-SS-DMA,when the N/P ratio is 6.Dynamic light scattering(DLS)test results showed that SS-LNP/sh PKN3(Size: 150.0±4.0 nm,PDI: 0.23±0.02,Zeta: +6.0±0.5 m V)possessed excellent physicochemical property and stability.In addition,SS-LNP/sh PKN3 had good stability under physiological conditions,but it was degraded in the presence of glutathione(GSH)and realized the rapid release of sh PKN3.Part 2: The inhibitory effect of SS-LNP encapsulated with PKN3 sh RNA with reduction responsive on breast tumor growth was systematically evaluated.Firstly,the transfection efficiency of nano vector was analyzed by cell transfection experiment.Compared with the commercial transfection reagent Lipo2000,the transfection efficiency of DDA-SS-DMA-based SS-LNP was about 1.7 times that of Lipo2000.CLSM showed that SS-LNP/sh PKN3 successfully escaped from lysosomes and released sh PKN3.It was found that SS-LNP delivery carrier had low cytotoxicity and high biocompatibility and the sh PKN3 interference sequence could not significantly inhibit the proliferation of MDA-MB-231 cells.In Western Blot assay,cell invasion and migration experiments showed that the SS-LNP/sh PKN3-2 treatment group had the highest protein inhibition rate(60.80%)and cell migration inhibition rate(79.92%),indicating that sh PKN3-2 interference sequence had a good ability to silence PKN3 m RNA and inhibited cell migration in vitro.Finally,in vivo anti-tumor assay confirmed that the SSLNP/sh PKN3-2 treatment group had the highest tumor inhibition effect(62.30%)and no obvious toxicity.Part 3: The inhibitory effect of SS-LNP encapsulated with PKN3 sh RNA with reduction responsive on prostate tumor growth was systematically evaluated.Transfection experiments showed that for PC-3 cells,the transfection efficiency of SS-LNP based on DDA-SS-DMA was higher than that of commercial transfection reagent Lipo2000.CLSM showed that SSLNP/sh PKN3 could successfully escape from lysosomes and release sh PKN3.In vitro cytotoxicity and cell proliferation experiments confirmed that SS-LNP delivery vector had good biocompatibility and sh PKN3 interference sequence could not directly affect the proliferation of PC-3 cells.Western Blot assay,cell invasion and migration experiments showed that the SSLNP/sh PKN3-2 treatment group had the highest protein inhibition rate(55.60%)and the highest inhibition rate of cell migration(76.21%),indicating that sh PKN3-2 sequence could significantly inhibit PKN3 m RNA expression and down-regulate PKN3 protein expression.In conclusion,an ionizable lipid nanoparticle SS-LNP based on DDA-SS-DMA with high transfection efficiency and good biocompatibility was successfully prepared.SS-LNP/sh PKN3 can rapidly release PKN3 sh RNA in tumor cells and effectively inhibit the expression of PKN3 protein,which had significant antitumor effect in vivo and the value of further development.