Co-Delivery Of Glycyrrhizic Acid And P65 SiRNA With Glycyrrhizic Acid-Modified Lipid Nanoparticles And Its Effect On Ameliorating Hepatic Inflammation

Purpose:To be directed against liver injury caused by hepatitis,which is the pathological basis of varied hepatic diseases,a new strategy of incorporating glycyrrhizic acid(GA)and polyene phosphatidylcholine(PPC)into lipid nanoparticles(GA/PPC-modified LNPs)will be developed as a co-delivery system for co transporting GA and siRNA,which should be capable of promoting cellular uptake,enhancing gene-silencing,reducing cytotoxicity and improving siRNA stability.GA/PPC-modified LNP and p65 siRNA lipoplex designed for targeting NF-κB RelA/p65,a key mediator of inflammation should mitigate acute liver injury with the synergistic anti-inflammatory,anti-oxidant and antiviral activities of GA and p65 siRNA.Method:Incorporating glycyrrhizic acid(GA)and polyene phosphatidylcholine(PPC)into lipid nanoparticles was prepared by thin film dispersion method,and GA/PPC-modified LNP(mLNP)was developed as a nucleic acid delivery system.The size and surface charge were detected by Dynamic Light Scattering.The LNPs were stained with 1%uranyl acetate and imaged using transmission electron microscopy(TEM).The siRNA binding ability,serum stability,cell viability,transfection efficiency and other physico-chemical properties were characterized and the LNP formulation was optimized.Transfected with p65 siRNA via the mLNP on NCTC 1469 cells,western blot(WB),quantitative polymerase chain reaction(q-PCR)and immunofluorescent(IF)staining were performed to assess whether NF-κB p65 gene silencing through mLNP/p65 siRNA lipoplex occurred,and the downstream cytokine mRNA(IL-1β,IL-6,COX-2 and iNOS)of p65 protein could down-regulated.Intraperitoneal administration of LPS to C57BL/6J mice would be employed to induce the animal model of acute liver injury and hepatitis in mice.The mice would be administered intravenously by co-delivery of GA and p65 siRNA via GA/PPC-modified LNP.Western blot assay of mice liver homogenate was carried out to determine whether delivery of p65 siRNA by the mLNPs in mice could effectively suppressed p65 protein expression in liver tissues.Hepatic homogenates would be prepared and then assayed for malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,myeloperoxidase activity(MPO)and glutathione peroxidase(GSH-Px).The IL-6 and IL-1β levels in plasma would be quantified using mouse IL-6 and IL-1β enzyme-linked immunosorbent assay(ELISA).Hepatic homogenates would also be assayed for iNOS and COX-2 levels using Inducible nitric oxide synthase Assay and Mouse COX-2 ELISA.Plasma alanine transaminase(ALT)and aspartate transaminase(AST)activities would be determined using a commercially available kit.The liver tissues would be embedded in paraffin,cut into sections and then stained with hematoxylin and eosin(HE)and the severity of liver damage would be assessed.Moreover,Hepatitis B surface antigen(HBsAg)and Hepatitis B envelope antigen(HBeAg)levels in HepG2.CW cell supernatant would be determined using the National Medical Products Administration(NMPA)approved commercially-available kit post transfected with HBV siRNA and ASO via GA/PPC-modified LNP.Result:Based on transfection of EGFP mRNA or FAM labeled siRNA on HeLa cells,the mLNP formulation was optimized as 0.4GA/PPC LNP,which was considered as the best candidate.The hydrodynamic diameters,the polydispersity index(PDI),GA encapsulation efficiency and the zeta(ζ)potentials of 0.4GA/PPC LNP were in a rational,delivery-beneficial range.0.4GA/PPC LNP possessed great siRNA binding ability,strong siRNA protection from degradation,lower cell toxicity and higher cellular uptake and transfection efficiency.Most of natural products and herbal extracts indeed possess excellent therapeutic efforts while poor pharmacokinetic properties limited their application to our current pharmacopeia,which had a great demand to be delivered by a propriate vector,therefore,natural product transported by mLNPs can bring about important pharmacological improvements and help to enhance the therapeutic effects for RNAi strategy.The results of WB,q-PCR showed that treatment with mLNP/p65 siRNA lipoplex reduced mRNA levels of NF-κB pathway downstream genes(IL-1β,IL-6,COX-2,iNOS)in LPS-stimulated NCTC1469 cells.IF assay showed that 0.4GA/PPC LNP exhibited effective delivery efficiency of p65 siRNA in down-regulating the LPSinduced high level of pro-inflammatory cytokine and mediators.In addition,the HBsAg and HBeAg levels in 0.4GA/PPC LNP-related group were remarkably decreased compared to the control,confirming that 0.4GA/PPC LNP exhibited highly efficient delivery of nucleic acid drugs including siRNA and ASO and additional anti-viral efficacy.Intraperitoneal administration of LPS to C57BL/6J mice was employed to induce the animal model of acute liver injury and hepatitis in mice.The mice were administered intravenously by co-delivery of GA and p65 siRNA via GA/PPC-modified LNP.These in vivo results of MDA,SOD,GSH-PX and MPO are consistent with the in vitro results,suggesting that the 0.4GA/PPC LNP possessed powerful antioxidative activity and improved the redox status.These in vivo results of IL-1β,IL-6,COX-2 and iNOS were consistent with the in vitro results,suggesting that the 0.4GA/PPC LNP possessed powerful anti-inflammatory efforts.The group treated with an equivalent dosage of 0.4GA/PPC/p65 siRNA lipoplex exhibited great efficacy in treating reduced liver dysfunction,further confirm the therapeutic efficacy of 0.4GA/PPC/p65 siRNA lipoplex in acute liver injury.The histological analysis and liver injury score of HEstained liver sections from model mice revealed that LPS-induced liver damage,as evidenced by the hemorrhage and central vein congestion of liver tissue,granulocyte cell infiltration,steatosis,and edema,all of which were ameliorated by 0.4GA/PPC/p65 siRNA lipoplex.Conclusion:The therapeutic strategy of co-delivery of glycyrrhizic acid and p65 siRNA using GA/PPC-modified LNP can effectively play the synergistic anti-inflammatory and antioxidant stress effects of glycyrrhizic acid and p65 siRNA,and further ameliorates the liver injury caused by hepatitis.