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A series of novel nonviral vectors targeting the HER-2/neu gene product human epidermal growth factor receptor-2 (HER2) were constructed and evaluated in breast cancer cell lines to optimize vector formulation for receptor-specific gene transfer. These vectors were DNA/polycation complexes (polyplexes) prepared by mixing, at varying ratios, plasmid DNA carrying a luciferase reporter gene to HerPEI, which is a conjugate of linear polyethylenimine (PEI), a cationic polymer, and trastuzumab (Herceptin®), a HER2-specific monoclonal antibody. Transfection studies were carried out in both HER2 overexpressing Sk-Br-3 and HER2 low-expressing MDA-MB-231 breast cancer cells. The HerPEI polyplexes showed significantly greater transfection activity up to 20-folds than nonderivatized PEI-based polyplexes in the Sk-Br-3 cells. The transfection efficiency of targeted polyplexes was dependent on the trastuzumab:PEI ratio and can be blocked by excess free trastuzumab, suggesting HER2-mediated gene delivery. In contrast, no significant difference in transfection activities was observed between HER2-targeted and nontargeted polyplexes in the HER2 low-expressing MDA-MB-231 cells. The transfection efficiency of HerPEI polyplexes was retained in serum-containing medium. In summary, HerPEI polyplexes have shown promising HER2 receptor-specific gene transfer properties and warrant further evaluation as a tumor-targeted vector for gene therapy. [Copyright &y& Elsevier]

LPDII vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA (polyplexes) complexed with anionic pH-sensitive liposomes. Here, we describe a novel LPDII formulation containing polyethylenimine (PEI) polyplexes complexed with anionic pH-sensitive liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol). The pH-sensitivity of diolein/CHEMS liposomes was evaluated through quantitative fluorescence measurements of calcein release and particle size analysis. The results indicated that diolein/CHEMS liposomes are stable at physiological pH, but undergo rapid aggregation and fluorescence dequenching at pH values ≤5.0. Using a luciferase reporter gene, in vitro transfection of KB oral cancer cells showed that the transfection efficiency of LPDII vectors was superior to other well-characterized polyplexes and lipoplexes. Results further showed that gene delivery using diolein-containing LPDII vectors was dependent on the PEI nitrogen/DNA phosphate (N/P) ratio, the lipid/DNA weight ratio and the cell line being transfected. Replacing PEI with poly-l-lysine as the DNA condensing agent resulted in only a moderate reduction in transfection activity. Moreover, in contrast to LPDII formulations incorporating dioleoylphosphatidylethanolamine (DOPE), the transfection efficiency of diolein-based LPDII vectors was sustained in media containing up to 50% fetal bovine serum. Since diolein-based LPDII vectors mediate efficient gene transfer and retain their transfection activity in the presence of serum, diolein may be a promising alternative to DOPE for the construction of non-viral vectors for in vivo gene delivery. [Copyright &y& Elsevier]

pH-sensitive liposomes are designed to promote efficient release of entrapped agents in response to low pH. In this study, novel pH-sensitive liposomes consisting of cationic/anionic lipid combinations are evaluated for intracellular drug and gene delivery. First, liposomes composed of egg phosphatidylcholine, dimethyldioctadecylammonium bromide (DDAB), cholesteryl hemisuccinate (CHEMS), and Tween-80 (25:25:49:1, mol/mol) were shown to stably entrap calcein at pH 7.4 and undergo rapid content release and irreversible aggregation under acidic pH. Compared to pH-sensitive liposomes incorporating dioleoylphosphatidylethanolamine, these liposomes showed improved retention of pH-sensitivity in the presence of serum. The folate receptor (FR), which is amplified in a wide variety of human tumors, could be targeted by incorporating 0.1 mol% folate-polyethyleneglycol-phosphatidylethanolamine (f-PEG-PE) into liposomes. f-PEG-PE has been shown to facilitate FR-mediated endocytosis of liposomes into KB human oral cancer cells, which express amplified FR. FR-targeted pH-sensitive liposomes produced increased cytosolic release of entrapped calcein, as shown by fluorescence microscopy, and enhanced cytotoxicity of entrapped cytosine-β-d-arabinofuranoside, as shown by an 11-fold reduction in the IC50 in KB cells, compared to FR-targeted non-pH-sensitive liposomes. Furthermore, FR-targeted pH-sensitive liposomes composed of DDAB/CHEMS/f-PEG-PE, combined with polylysine-condensed plasmid DNA, were shown to mediate FR-specific delivery of a luciferase reporter gene into KB cells in the presence of 10% serum. These findings suggest that cationic lipid-containing pH-sensitive liposomes, combined with FR targeting, are effective vehicles for intracellular drug and gene delivery. [Copyright &y& Elsevier]