Pulmonary Surfactant Inhibits Cationic Liposome-Mediated Gene Delivery to Respiratory Epithelial CellsIn Vitro

Abstract

Cationic lipid-mediated transfection of the alveolar epithelium in vivo will require exposure of plasmid DNA and cationic lipids to endogenous surfactant lipids and proteins in the alveolar space. Effects of pulmonary surfactant and of surfactant constituents on transfection in vitro of two respiratory epithelial cell lines (MLE-15 and H441) with a plasmid encoding the luciferase reporter gene were studied using two cationic lipid formulations: 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/cholesterol (DMRIE/C) and 1,2-dioleoyl-3-trimethylammonium propane/dioleoyl phosphatidylethanolamine (DOTAP/DOPE). Gene expression, as assessed by luciferase activity, decreased as increasing concentrations of natural surfactant were added to cationic lipid-DNA complexes. Incorporation of phospholipids DOPC/DOPG or surfactant proteins SP-B or SP-C in the cationic lipid formulation inhibited transfection. A fluorescent lipid mixing assay was used to determine the effects of surfactant proteins SP-B and SP-C on mixing between cationic lipid-DNA complexes and surfactant lipid vesicles. Mixing between DOPC/DOPG vesicles and cationic lipid-DNA complexes in the absence of added proteins amounted to 10-20%. Addition of SP-B or SP-C increased the mixing of DOPC/DOPG vesicles with DOTAP/DOPE-DNA complexes, but not DMRIEC-DNA complexes. These results demonstrate that pulmonary surfactant lipids and proteins inhibit transfection with cationic lipid-DNA complexes in vitro, and may therefore represent a barrier to gene transfer in the lung.

Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus

“…8,22,23 Others have reported that Alveofact, a natural pulmonary surfactant, forms a barrier to cationic lipid-mediated gene transfer. [11][12][13] We examined whether this also held true for our lipoplexes.…”

“…Instead, at concentration ratios between 0.16 and 1.8 a significant increase was observed. Also, Duncan et al 11 have reported an increase in gene expression in the presence of Alveofact. They attributed this to binding of surfactant protein B and C, present in small amounts in Alveofact, to the surface of the cationic lipoplexes, which enhances their binding to epithelial cells.…”

“…Only a few papers have shown that pulmonary surfactants, which line the alveoli, decrease the gene transfer activity of cationic liposomes. [11][12][13] However, these authors did not elucidate in detail the underlying mechanism for this inhibition in activity.…”

Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome–DNA complexes

“…Viral vectors, the current favorites for intracellular DNA delivery, suffer from immunogenicity, nonspecificity, and inherent risks of complications such as the interference with the activity of tissue-specific promoters (8)(9)(10). Nonviral vectors such as cationic lipids͞liposomes (11) are nonspecific, demonstrate low efficiency compared with viruses, and can be inactivated by pulmonary surfactants (12) and serum proteins (13). Cytotoxic reactions such as pulmonary inflammation (14), antiproliferative activity in proteoglycan-deficient cells (15), production of reactive oxygen intermediates (16), and inhibition of NO and tumor necrosis factor-␣ production in activated macrophages (17) were reported after cationic lipid͞liposome treatments.…”

Effects of altering dosing on cationic liposome-mediated gene transfer to the respiratory epithelium

“…To this end novel vectors, both viral and nonviral, are being extensively studied. 5,6 Further, it is clear that a number of potential biological extra-and intracellular barriers to gene transfer exist, [7][8][9][10][11] and ways to overcome these are being intensively investigated. While these improvements in gene transfer technology are awaited, we asked whether simple changes to existing clinical protocols might be able to increase expression levels.…”