Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes.
Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA-RNA ribozyme targeted to the HIV-1 5' LTR was investigated, using THP-1, THP-1/HIV-1IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1IIIB cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 microM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1IIIB cells to 5 microM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 microM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 microM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4+ cells immediately after infection with HIV-1IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.
Konopka
K
K
Department of Microbiology, School of Dentistry, University of the Pacific, San Francisco, CA 94115, USA.
Rossi
J J
JJ
Swiderski
P
P
Slepushkin
V A
VA
Düzgüneş
N
N
D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D002412:Cations; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004337:Drug Carriers; D015658:HIV Infections / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000378:metabolism* / Q000494:pharmacology; D046911:Macromolecular Substances; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D016337:RNA, Catalytic / Q000378:metabolism* / Q000494:pharmacology
D002412:Cations; D004337:Drug Carriers; D008081:Liposomes; D046911:Macromolecular Substances; D016337:RNA, Catalytic