Preparation And The Research Of Anti-hepatocellular Carcinoma Of EGFR-specific PEGylated Immunoliposomes For Active SiRNA Delivery

Small (short) interfering fragments of RNA (siRNA) are known to inhibit specificprotein synthesis by suppressing target gene expression at the mRNA level by amechanism called RNA interference (RNAi). They are considered prospective anticancerdrugsbecause of their high specific gene silencing efficiency and low toxicity.However,systemic delivery of siRNAs into tumor cells is a challenging task because siRNAsthemselves are unstable in the bloodstream and cannot penetrate cellular. For these reasons,a number of delivery systems have been developed, including lipid-based andpolymer-based systems, peptide conjugates and single-chain fragment variable antibodyfusion protein systems.Among various systems studied, cationic liposomes may be considered as idealvehicles for siRNA delivery, mainly due to their biological inertness, non-toxicity andprotection of siRNAs from nucleases. To further effectively silence the target gene, it is apractical way to develop PEGylated immunoliposomes conjugated with targeting ligands.Huang et al developed high PEG and anisamide targeted LPD (liposome-polycation-DNAcomplex) systems and it was shown to deliver siRNA effciently to tumor cellsoverexpressing sigma receptor. To further extend the application of LPD, based on theresearch of Huang, we first adopted anti-EGFR (epidermal growth factor receptor) Fab’ asthe targeting ligand of LPD and it has been demonstrated to possess potent gene silencingactivity in EGFR overexpressing breast cancers. However, TLPD-FCC did not achievesatisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma(HCC). In this study, some modifcations including increased antibody conjugationeffciency and reduced PEGylation degree were made to TLPD-FCC to increase genesilencing activity in HCC.Firstly, cationic liposomes composed of DOTAP and Chol (1:1molar ratio,10mM)were prepared by the thin flm hydration method, then mixed with protamine, calf thymusDNA, and siRNA in a self-assembling process to form naked LPD. NTLPD or TLPD wasfinally obtained from naked LPD by PEGlation and antibody modification, respectively.Because PEGylation degree and antibody conjugation are the main factors which impacttargeting ability of TLPD, so we prepared immoliposomes of different PEGylation degree (5mol%、7.5mol%、10mol%)and antibody conjugation means(conventional conjugationand post-insertion). We evaluated the characterization of liposomes, such as particle size,zeta potential, Fab’ conjugation and gene knockdown efficiency, then found the liposomeof TLPD-FP75including7.5mol%PEG prepared by post-insertion mean was theoptimization prescription.Secondly, we invested the siRNA encapsulationeffciency(EE), Gel retardation assay,siRNA serum stability, In vitro cellular uptake, Confocal microscopic study, In vitro genesilencing and effect of RhoA silencing on cell migration of TLPD-FP75and NTLPD-FP75.The results of Gel retardationassay indicted the liposomes had powerful bindingaffnity tosiRNA that were unity with siRNA EE (>85%). The results of siRNA serum stabilityproved that encapsulation of siRNA within LPD formulation can protect siRNA fromserum degradation to a great extent (TLPD-FP75144h). Confocal microscopic studyshowed that SMMC-7721cells treated with TLPD-FP75showed higer internalization ofboth the Cy5-siRNA (red fuorescence) and CFPE (green fuorescence) than NTLPD-FP75,which indicated TLPD-FP75efficiently delivering siRNA to the tumor cells and theintracellular delivery was ligand dependent.TLPD-FP75had superior cellular uptake andgene silencing efficiency than NTLPD-FP75indicating TLPD-FP75owed specifictargeting. The effect of RhoA silencing on cell migration demonstrated that TLPD-FP75could specifcally silence RhoA expression and inhibit cell invasion in EGFRoverexpressing SMMC-7721cells, whereas NTLPD-FP75had no such effect.Lastly, we established EGFR-overexpressing tumor euangiotic HCC model, theninvested the tissue distribution, the binding and internalizing and gene silencing in vivo. Invivo distribution assay showed that TLPD-FP75possessed enhanced ability to accumulatein EGFR overexpressing HCC compared with NTLPD-FP75. Furthermore, in vivo uptakestudy showed that TLPD-FP75accumulated profusely throughout the tumors tissues in apattern consistent with receptor-mediated endocytosis. More importantly, TLPD-FP75showed signifcantly higher gene silencingactivity in vivo thanother formulations.In conclusion, we obtained the immunoliposomes TLPD-FP75which couldeffectively deliver siRNA to EGFR-overexpressing tumor cells. Compared withNTLPD-FP75, TLPD-FP75showed a significantly enhanced EGFR targeting efficiency and achieved a superior gene silencing activity both in vitro and in vivo and willpotentially increase the feasibility of HCC gene therapy.