Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Targeted gene transfer to hepatocellular carcinoma cells in vitro using a novel monoclonal antibody-based gene delivery system.

Gene therapy approaches for the treatment of malignant tumors will require high-level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor-specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor-reactive monoclonal antibody (mAb), designated AF-20, coupled to a DNA-binding cationic amphiphile, cholesteryl-spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high-affinity mAb, AF-20, recognizes a rapidly internalized 180-kd cell-surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF-20 mAb and an isotype-matched control antibody (C7-57) were covalently coupled to cholesteryl-spermine. Binding and internalization of AF-20-cholesteryl-spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody. Following transfection of FITC-labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or beta-galactosidase reporter genes complexed to AF-20-cholesteryl-spermine resulted in high levels of gene expression in AF-20 antigen-positive tumor cells. Very low levels of gene expression were observed using the control compound (C7-57-cholesteryl-spermine), which does not recognize the AF-20 tumor antigen or when AF-20 antigen-negative NIH 3T3 cells were transfected with AF-20-cholesteryl-spermine. Thus, covalent coupling of the AF-20 mAb to cholesteryl-spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF-20 antigen-positive HCC cells.

Efficient gene transfer into mammalian cells with cholesteryl-spermidine.

The naturally occurring polyamine spermidine was covalently conjugated with cholesterol, resulting in a novel cationic compound that mediates efficient gene transfer into mammalian cells. Using reporter plasmids coding for firefly luciferase and beta-galactosidase, a simple procedure was developed allowing highly reproducible and efficient transient and stable transfection of HuH-7 cells. Transfection efficiency could be further increased when a fusogenic peptide derived from the influenza virus hemagglutinin HA2 aminoterminal sequence was included in the cholesteryl-spermidine-DNA complex. Cholesteryl-spermidine (Transfectall) represents a novel cationic compound for efficient transfection of cultured cells in vitro and has the potential to be used for gene transfer in vivo.