The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).

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The ability to deliver antigens and immunomodulators specifically to Langerhans cells (LCs) in the skin could impact vaccine development. However, cell-specific targeting of therapeutic molecules remains a challenge in biomedicine. Using phage display technologies, we have developed a protocol that identifies peptides that mediate uptake into target cell types. Employing this approach, we have isolated a 20-mer peptide that mediates specific uptake by immunopotent LCs. The peptide is functional outside the context of the phage and is able to deliver a nanoparticle to LCs in vitro. Although selected on cells in vitro, the peptide is able to direct antigens and genes to LCs in vivo. Liposomes bearing the LC targeting peptide are able to deliver a transcriptionally active gene to LCs in a mouse model. Furthermore, we demonstrate that a low-dose injection into mice of phage bearing the LC-targeting peptide yields faster and higher immune responses against phage-associated antigens than control-phage injections.

Recombinant DNA technology has been employed to produce a polypeptide capable of forming pH responsive hydrophobic microdomains. The design of this peptide is based upon an idealized conceptual model in which electrostatic, hydrophobic, and hydration forces are responsible for the association of amphipathic α‐helical elements. Reduction in solution pH is responsible for reducing electrostatic repulsions between similarly charged residues, promoting the hydrophobic collapse of helical elements. A polymerizable synthetic element (dn31) has been synthesized and inserted into an appropriate expression vector. A clone containing a single copy of the dn31 gene (designated dn31x1) was isolated and the corresponding gene product DN3Lx1 isolated. The physical properties of DN3Lx1 were examined in solution by gel filtration chromatography, CD, and fluorescence probe analysis. It was determined that DN3Lx1 self‐associates in solution with the degree of aggregation dependent on pH and ionic strength. An initial objective of this work was to examine domain organization in higher molecular weight species containing ten or more repetitive sequences. However, attempts to express multiple repeats of DN3Lxn from concatemers were unsuccessful. © 1997 John Wiley & Sons, Inc. Biopoly 41: 521–532, 1997.