Liposome targeting to human immunodeficiency virus type 1-infected cells via recombinant soluble CD4 and CD4 immunoadhesin (CD4-IgG).

HIV-infected cells producing virions express the viral envelope glycoprotein gp120/gp41 on their surface. We examined whether liposomes coupled to recombinant soluble CD4 (sCD4, the ectodomain of CD4 which binds gp120 with high affinity) could specifically bind to HIV-infected cells. sCD4 was chemically coupled by 2 different methods to liposomes containing rhodamine-phosphatidylethanolamine in their membrane as a fluorescent marker. In one method, sCD4 was thiolated with N-succinimidyl acetylthioacetate (SATA) and coupled to liposomes via a maleimide-derivatised phospholipid. In the other method, the oligosaccharides on sCD4 were coupled to a sulfhydryl-derivatised phospholipid, utilizing the bifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH). The association of the liposomes with HIV-1-infected or uninfected cells was examined by flow cytometry. CD4-coupled liposomes associated specifically to chronically infected H9/HTLV-IIIB cells, but not to uninfected H9 cells. CD4-coupled liposomes also associated specifically with monocytic THP-1 cells chronically infected with HIV-1 (THP-1/HIV-1IIIB). Control liposomes without coupled CD4 did not associate significantly with any of the cells, while free sCD4 could competitively inhibit the association of the CD4-coupled liposomes with the infected cells. The chimeric molecule CD4-immunoadhesin (CD4-IgG) could also be used as a ligand to target liposomes with covalently coupled Protein A (which binds the Fc region of the CD4-IgG) to H9/HTLV-IIIB cells. The CD4-liposomes inhibited the infectivity of HIV-1 in A3.01 cells, and also bound rgp120. Our results suggest that liposomes containing antiviral or cytotoxic agents may be targeted specifically to HIV-infected cells.

Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides.

The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non-dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH-sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected lack of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo.

In vivo gene delivery. Efficient transfection of T lymphocytes in adult mice.

We have demonstrated that a single intraperitoneal injection of cationic liposomes complexed to a chloramphenicol acetyltransferase (CAT) gene expression plasmid can transfect the majority of splenic Thy 1.2+ T lymphocytes, as well as significant numbers of bone marrow-derived hematopoietic cells, in adult mice. CAT activity was detected in the spleen for at least 2 weeks, and there was no evidence of treatment-related toxicity. Some degree of tissue-specific transgene expression was achieved by varying the cationic lipid used.

Targeting of liposomes to HIV-1-infected cells by peptides derived from the CD4 receptor.

Liposomes can be targeted to HIV-infected cells by either reconstituting transmembrane CD4 in the membrane or covalently coupling soluble CD4 to modified lipids. We investigated whether synthetic peptides could be used as ligands for targeting liposomes. A synthetic peptide from the complementarity determining region 2 (CDR-2)-like domain of CD4 could bind specifically to HIV-infected cells and mediate the binding of peptide-coupled liposomes to these cells. A peptide from the CDR-3-like domain of CD4 inhibited HIV-induced syncytia formation, but failed to target liposomes to infected cells. This apparent discrepancy may be due to the requirement for a conformational change in the CD4 receptor for the CDR-3 region to interact with the HIV envelope protein. Our results demonstrate the feasibility of using synthetic peptides to target liposomes containing antiviral drugs to HIV-infected cells.

Sterically stabilized pH-sensitive liposomes. Intracellular delivery of aqueous contents and prolonged circulation in vivo.

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.