title: A biomimetic nanovector-mediated targeted cholesterol-conjugated siRNA delivery for tumor gene therapy.
abstract: RNA interference holds tremendous potential as a therapeutic approach of malignant tumors. However, safe and efficient nanovectors are extremely lack for systemic delivery of small interfering RNA (siRNA). The study aimed to develop a biomimetic nanovector, reconstituted high density lipoprotein (rHDL), mediating targeted cholesterol-conjugated siRNA (Chol-siRNA) delivery for Pokemon gene silencing therapy. Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were prepared using thin-film dispersion method and their characteristics were investigated in detail. RHDL/Chol-siRNA complexes at the optimal volume ratio (lipid: Chol-siRNA) exhibited high Chol-siRNA-loading efficiency (~99%), desirable nanoparticle size and excellent stability in serum. In addition, by analyzing Chol-siRNA release profile, rHDL/Chol-siRNA complexes displayed sustained-release characteristic and storage stability. Observations from FACS and confocal microscopic analyses revealed that rHDL-mediated carboxyfluorescein tagged Chol-siRNA (FAM-Chol-siRNA) transfection resulted in highly efficient uptake and specific cytoplasmic delivery of FAM-Chol-siRNA into human hepatocellular carcinoma cell line HepG2 via HDL-receptor mediated mechanism. In vitro cytotoxicity, apoptosis and Western-blot analyses revealed significant cellular growth inhibition and decrease of Pokemon and Bcl-2 protein expression in HepG2 cells treated with Chol-siRNA-Pokemon-loaded rHDL nanoparticles (rHDL/Chol-siRNA-Pokemon complexes), respectively. In in vivo studies, the near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) obviously accumulated in tumor of nude mice after i.v. administration as compared with Cy5-Chol-siRNA-loaded lipoplexes (Lipos/Cy5-Chol-siRNA complexes). Morover, rHDL/Chol-siRNA-Pokemon complexes demonstrated great tumor growth inhibition and significant decrease of Pokemon and Bcl-2 protein expression in vivo. These results suggested that rHDL should be an ideal non-viral tumor-targeting vector for Chol-siRNA transfer, and rHDL-mediated Chol-siRNA-Pokemon delivery might be a promising new strategy for gene therapy in hepatocellular carcinoma.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Ding
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Meiqing
authors.initials: M
authors.lastname: Feng
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Jianping
authors.initials: J
authors.lastname: Zhou
authors.forename: Xuefang
authors.initials: X
authors.lastname: Ding
authors.forename: Xin
authors.initials: X
authors.lastname: Zhou
authors.forename: Congyan
authors.initials: C
authors.lastname: Liu
authors.forename: Ruoning
authors.initials: R
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000628:therapy*; D002784:Cholesterol / Q000737:chemistry*; D008076:Cholesterol, HDL / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D015316:Genetic Therapy; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D053758:Nanoparticles / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D008076:Cholesterol, HDL; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; C485514:ZBTB7A protein, human; D002784:Cholesterol