Immunoliposomes Co-delivering Adriamycin And Ribonucleotide Reductase M2siRNAInhibit Hepatocellular Carcinoma Cell Activity

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The chemotherapy combined with gene therapy has received more and more attention, due to their powerful antitumor activity and fewer side effects. However, the successful implementation of combined therapy greatly depends on the suitable choice of drugs, proper formulations carrying both chemotherapeutics and gene drugs, and the special targeting to tumors. We developed targeted liposome-polycaion-DNA complex (TLPD) conjugated with anti-EGFR (epidermal growth factor receptor) Fab’entrapping ADR (adriamycin) and RRM2(ribonucleotide reductase M2) siRNA, ADR-RRM2-TLPD. And the immunoliposome co-delivering ADR and RRM2siRNA could combined inhibit the activity of human hepatocellular carcinoma cells overexpressing EGFR in vitro.The selection of chemotherapeutics and gene drugs is critically important for superior activity of our combined therapy. Both drugs should exert additive or synergistic effects but not antagonistic effects. The choice of ADR and RRM2siRNA in our combined therapy is rational and would achieve additive effect as expects as expected. These two drugs were potent cytotoxic agents. ADR, which is suitable for liposomal drug loading, is the first-line drug for HCC therapy. RRM2is a validated anticancer target and RRM2inhibitors are powerful antitumor reagents, whereas inhibiting the role of RRM2in HCC is rarely investigated.With our experiments, RRM2was found to express in all the HCC cells at different levels, with the highest protein expression in HepG2and SMMC-7721cells, whereas the lowest mRNA and protein expression in Huh7cells. We have interrogated the GEO (Gene Expression Omnibus) for RRM2expression in mouse and human HCC tissue. We found that the RRM2expression of HCC tissue was significantly high than in peritumor and wild type liver tissue in mouse. Moreover, the average RRM2expression in tumor liver tissue was significantly higher than that in non-tumor tissue in the233pairs from the GEO. RRM2was related to the mechanism of tumor resistance in previous studies.We examined the RRM2expression after treatment of SMMC-7721, HepG2and Huh7cells with ADR. That1μg/ml ADR induced the highest expression of RRM2protein at24h was demonstrated in SMMC-7721. And up-regulated RRM2expression respond to ADR was also showed in HepG2and Huh7cells. In addition, immunofluorescence assays demonstrated that RRM2expression increased after ADR treatment, and the assays also clearly showed the location of RRM2protein expression. ADR and RRM2siRNA could have additive effects.Although Heidel et al. characterized the gene silencing activity of the RRM2siRNA in HCC cells, the study did not evaluate its cytotoxicity towards HCC cells. We confirmed that RRM2siRNA was shown to significantly inhibit HCC cells proliferation. And ADR was found to induce significant up-regulated RRM2expression which might contribute to resistance to cell death in HCC cells. So ADR and RRM2siRNA could achieve combined effects in HCC therapy.The RRM2siRNA suppressed RRM2expression in a dose-dependent manner and RRM2expression was inhibited by70-80%with200nM RRM2siRNA. As expected, RRM2siRNA significantly inhibited the proliferation of all the HCC cells in a dose-and time-dependent manner. Furthermore, RRM2siRNA triggered a significant increase in the number of cells in S phase, with a corresponding decrease in the number of cells in G0-G1phase in HepG2cells.The excellent antitumor activity of ADR-RRM2-LPD was better over ADR-NC-LPD in SMMC-7721and Huh7cells. But, the two groups were equally effective in inducing apoptosis in HepG2cells. And RRM2suppression induced senescence in HepG2cells with p53weild-type but not in SMMC-7721cells. The reason may be that senescence induced by RRM2suppression in ADR-RRM2-LPD also inhibited the apoptosis triggered by RRM2suppression, which was consistent with previous studies that senescence could inhibit apotosis in certain conditions.The presence of EGFR antibody was critical to maintaining the special targeting activity of our prepared immunoliposomes to EGFR overexpressing HCC. The data presented here confirmed that ADR-RRM2-TLPD was efficiently bound and delivered to EGFR overexpressing or moderately expressing HCC cells but not to EGFR lowly-expressing HCC cells. Upon cell binding ADR-RRM2-TLPD was readily internalized and released ADR and RRM2siRNA to the cytoplasm, resulting in enhanced growth inhibitory effect and apoptosis compared to ADR-RRM2-NTLPD in EGFR overexpressing or moderately-expressing HCC cells, but not in EGFR lowly-expressing HCC. Although a substantial amount of studies have demonstrated the successful co-delivery of siRNA and chemotherapeutics by using different nanomedicines, to our knowledge, we have shown for the first time that EGFR antibody was demonstrated to successfully promote the intracellular delivery of nanomedicines co-delivering siRNA and chemotherapeutics.ADR-RRM2-TLPD and ADR-RRM2-NTLPD accumulated in tumors due to the enhanced permeability and retention effect. And. ADR-RRM2-TLPD was bound to and internalized in tumor cells via ligand-receptor interactions, while ADR-RRM2-NTLPD remained in the extracellular space and underwent nonspecific endocvtosis and rupture. After intemalization. ADR-RRM2-TLPD readily released ADR and RRM2siRNA to the cytoplasm, resulting in a combined therapeutic effect, including grouth and metastasis inhibition, apoptosis and senescence promotion.In conclusion. ADR-RRM2-TLPD offered the possibility of co-delivering ADR and siRNA specifically and efficiently to EGFR overexpressing HCC. and it represents a potential therapeutic approach toward chemotherapy combined with gene therapy in HCC.