Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific antitumor growth activity by radiotherapy

Abstract

Qiushi Liu,1,2 Joohee Jung,3,4 Masaharu Somiya,1,2,5 Masumi Iijima,1,2 Nobuo Yoshimoto,1,2 Tomoaki Niimi,1 Andrés D Maturana,1 Seol Hwa Shin,3,6 Seong-Yun Jeong,3,6 Eun Kyung Choi,3,7,8 Shun’ichi Kuroda1,21Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan; 2The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan; 3Institute for Innovative Cancer Research, ASAN Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 4College of Pharmacy, Duksung Women’s University, Seoul, Republic of Korea; 5Japan Society for the Promotion of Science, Tokyo, Japan; 6ASAN Institute for Life Sciences, ASAN Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 7Department of Radiation Oncology, ASAN Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 8Center for Development and Commercialization of Anti-Cancer Therapeutics, ASAN Medical Center, University of Ulsan College of Medicine, Seoul, Republic of KoreaAbstract: Bionanocapsules (BNCs) are hollow nanoparticles consisting of hepatitis B virus (HBV) envelope L proteins and have been shown to deliver drugs and genes specifically to human hepatic tissues by utilizing HBV-derived infection machinery. The complex of BNCs with liposomes (LPs), the BNC–LP complexes (a LP surrounded by BNCs in a rugged spherical form), could also become active targeting nanocarriers by the BNC function. In this study, under acidic conditions and high temperature, BNCs were found to fully fuse with LPs (smooth-surfaced spherical form), deploying L proteins with a membrane topology similar to that of BNCs (ie, virosomes displaying L proteins). Doxorubicin (DOX) was efficiently encapsulated via the remote loading method at 14.2%±1.0% of total lipid weight (mean ± SD, n=3), with a capsule size of 118.2±4.7 nm and a ζ-potential of -51.1±1.0 mV (mean ± SD, n=5). When mammalian cells were exposed to the virosomes, the virosomes showed strong cytotoxicity in human hepatic cells (target cells of BNCs), but not in human colon cancer cells (nontarget cells of BNCs), whereas LPs containing DOX and DOXOVES (structurally stabilized PEGylated LPs containing DOX) did not show strong cytotoxicity in either cell type. Furthermore, the virosomes preferentially delivered DOX to the nuclei of human hepatic cells. Xenograft mice harboring either target or nontarget cell-derived tumors were injected twice intravenously with the virosomes containing DOX at a low dose (2.3 mg/kg as DOX, 5 days interval). The growth of target cell-derived tumors was retarded effectively and specifically. Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors. These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.Keywords: drug delivery system, liposomes, bionanocapsule, doxorubicin, targeting, chemoradiotherapy