Preparation And In Vitro And In Vivo Activity Assay Of EGFR-targeted Immunoliposomes For SiRNA Delivery

Gene therapy is recently considered as an effective way in treating both inherited and acquired diseases, especially malignant tumors. RNA interfering (RNAi), an efficient sequence specific gene silencing technology, has sparked an explosion of research in tumor gene therapy field. siRNA (small interfering RNA) is the effective molecule in the RNAi pathways, which is 21~23 bp nucleotide, double-stranded. It can specifically degrade complementary mRNA, thus inhibiting tumor associated gene expression, tumor progression, invasion and metastasis.However, siRNA cannot enter the tumor cells without the help of carriers and carrier is the main problem that hampers the development of gene therapy. So the investigation of the siRNA carrier is the hottest focus of tumor gene therapy. Cationic liposomes have been widely used as the non-viral carriers for siRNA delivery. They are non-toxic, naturally degradable, low-immunogenic and easy-to-produce generously. Recently, they attracted the serious attention of researcher.Previously our group is always going in for the research of preparation and characteristic of cationic liposome for siRNA delivery. We developed Lyophilized PEGylated Immunoliposomes (LPIL), according to PEGlyation, HER2 antibody modification and lyophilization. LPIL containing 2.5% PEG (2.5% PEG LPIL) can specifically delivering siRNA to HER2-overexpressing cancers and slience specific gene expression. As is reported, PEG can significantly enhance the stability of liposomes in plasma and prolong the in vivo half-life period. PEG is arranged in the brush mode with >8 mol% PEGylation for a 100 nm liposomal particle. The brush mode is the ideal configurations that ensures complete coverage of the surfaces of the nanoparticles providing full protection and have an excellent performance in vivo. However, the PEG content of LPIL was low, thus its in vivo application was hindered. Besides, LPIL cannot bear hight content of PEG owing to protect the physical stability of liposomes, and high content of PEG can not only destroy the stability of LPIL and also decrease the siRNA encapsulation efficiency. As a result, we should develop the novel cationic liposomes which possess hight content of PEG, siRNA encapsulation efficiency and good targeting. LPD (liposome-polycation-DNA) is a novel cationic liposomes for siRNA delivery. They composed of nucleic acids, a polycationic peptide and cationic liposome, and were prepared in a self-assembling process. This structure is different from the common liquid phase structure of liposomes, and is a negative core consisting of polycation, siRNA and DNA. More importantly, a post-insertion PEGlyation method was adopted, which provides a high siRNA encapsulation efficiency, liposome stability and was demonstrated to efficiently delivery siRNA to tumor cells. However, nobody investigate the characteristic and in vitro and in vivo activity of antibody-modified LPD. In this study, we finally obtained a final product TLPD-FCC which possesses high potent of PEG and antibody targeting, and investigated the siRNA encapsulation and in vitro and in vivo activity.Firstly, we prepared DOTAP/Chol (1:1 M ratio) cationic liposome, then mixed with protamine, calf thymus DNA, and siRNA in a self-assembling process to form naked LPD. NTLPD or TLPD was finally obtained from naked LPD by PEGlation, antibody modification, respectively. We investigated the effect of antibody type, conjugation strategy and amount on the essential physicochemical properties (such as size, charge, et al.) of liposomes. As a result, TLPD-FC (TLPD conjugated anti-EGFR Fab'with convetional strategy) which had a particle size between 150 and 160 nm, and the average zeta potential was near 10 mV, was used in the subsequent experiments.Secondly, we evaluated the presentation and integrity of Anti-EGFR Fab'on the surface of TLPD-FC by SDS-PAGE. Meanwhile, the siRNA binding af?nity of liposomes, siRNA encapsulation efficiency (EE) and the in vitro gene silencing were examined. Gel retardation assay result showed that naked LPD, NTLPD and TLPD-FC all had powerful binding af?nity to siRNA. The siRNA EE of liposomes was precisely calculated by subtracting unencapsulated siRNA from total siRNA. Consistent with results obtained in the gel retardation assay, the siRNA EE of all liposomes was very high(> 90%), indicating that naked LPD, NTLPD and TLPD-FC have potent encapsulation capacity to siRNA, and PEGylation and antibody conjugation have little adverse impact on siRNA EE for NTLPD and TLPD-FC. Accompanying with increased amount of conjugated antibody, the luciferase gene silencing activity of TLPD-FC in MDA-MB-231 cells gradually increased, TLPD-FCC possessed the best gene silencing activity among TLPD-FC (TLPD-FCA, TLPD-FCB, TLPD-FCC and TLPD-FCD) and was used in the subsequent experiments. Finally, we investigated the characteristic and in vitro and in vivo activity of TLPD-FCC. The size and morphology of liposomes were observed by TEM, which showed that there is no difference between NTLPD and TLPD-FCC in shape and size distribution, suggesting that antibody conjugation have little impact on the structure of liposomes. siRNA serum stability was detected by agarose gel electrophoresis, the result showed that siRNA in TLPD-FC and NTLPD was protected well in aqueous solution of 50% serum. The dynamic light scattering (DLS) showed the strong interaction exsited between naked LPD and bovine serum albumin (BSA), while NTLPD or TLPD-FC had little interaction with BSA and thus had a good stability. In vitro experiment, transfection efficiency and gene silencing were studied. TLPD-FCC possessed signi?cantly increased transfection ef?ciency and gene silencing activity compared with NTLPD. Subsequently, we successfully established EGFR-overexpressing tumor xenograft model, Immunofluorescence staining had been taken to show the high EGFR expression in MDA-MB-231 tumor tissues. In the IVIS imaging system, TLPD-FCC showed a much better accumulation and distribution in the tumor compared with NTLPD. Furthermore, in vivo uptake study showed that TLPD-FCC accumulated profusely throughout the tumors tissues in a pattern consistent with receptor-mediated endocytosis. However, NTLPD showed only minimal binding or uptake. Consistent with the in vitro gene silencing assay, TLPD-FCC showed significantly higher gene silencing activity in vivo than NTLPD.In this study, we obtained TLPD-FCC which could effectively deliver siRNA to EGFR-overexpressing cancer cells. Furthermore, TLPD-FCC showed a significantly enhanced EGFR targeting efficiency and gene silencing activity both in vitro and in vivo, which has the potential possibility to cure breast cancer in clinic.