Liposomal vectors formulated with cationic lipids (cationic liposomes) and fusogenic dioleoylphosphatidylethanolamine (DOPE) have potential for modulating the immune system by delivering gene or antisense oligonucleotide inside immune cells. The toxicity and the immunoadjuvant activity of cationic liposomes containing nucleic acids toward immune effector cells has not been investigated in detail. In this report, we have evaluated the toxicity of liposomes formulated with various cationic lipids towards murine macrophages and T lymphocytes and the human monocyte-like U937 cell line. The effect of these cationic liposomes on the synthesis of two immunomodulators produced by activated macrophages, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), has also been determined. We have found that liposomes formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-: DOTAP, DMTAP, DPTAP, DSTAP) or dimethyldioctadecylammonium bromide (DDAB) are highly toxic in vitro toward phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes. The rank order of toxicity was DOPE/DDAB > DOPE/DOTAP > DOPE/DMTAP > DOPE/DPTAP > DOPE/DSTAP. The ED50's for macrophage toxicity were < 10 nmol/ml for DOPE/DDAB, 12 nmol/ml for DOPE/DOTAP, 50 nmol/ml for DOPE/DMTAP, 400 nmol/ml for DOPE/DPTAP and > 1000 nmol/ml for DOPE/DSTAP. The incorporation of DNA (antisense oligonucleotide or plasmid vector) into the cationic liposomes marginally reduced their toxicity towards macrophages. Although toxicity was observed with cationic lipids alone, it was clearly enhanced by the presence of DOPE. The replacement of DOPE by dipalmitoylphosphatidylcholine (DPPC) significantly reduced liposome toxicity towards macrophages, and the presence of dipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000: 10 mol%) in the liposomes completely abolished this toxicity. Cationic liposomes, irrespective of their DNA content, downregulated NO and TNF-alpha synthesis by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-activated macrophages. The replacement of DOPE by DPPC, or the addition of DPPE-PEG2000, restored NO and TNF-alpha synthesis by activated macrophages. Since macrophages constitute the major site of liposome localization after parenteral administration and play an important role in the control of the immune system, cationic liposomes should be used with caution to deliver gene or antisense oligonucleotide to mammalian cells. Cationic lipids show in vitro toxicity toward phagocytic cells and inhibit in vitro and in situ NO and TNF-alpha production by activated macrophages.

We used a modified adult lung explant technique to directly measure the area of individual airways before and after methacholine (MCh) administration. Lungs were removed from 12-wk-old male Lewis rats under sterile conditions, filled with an agarose-containing solution at 37 degrees C, and cooled to 4 degrees C. Transverse slices (0.5–1.0 mm thick) were cut and cultured overnight. Concentration-response curves to MCh were determined for explant airways from lungs inflated to 25, 50, 75, and 100% total lung capacity (TLC) with a 1.0% agarose solution and to 75% TLC with 0.5 and 2.0% agarose solutions. MCh was added to the medium to achieve final concentrations ranging from 10(-9) to 10(-2) M. Airways were imaged before and 10 min after each increase in MCh concentration with an inverted microscope and video camera, and airway area was determined by computerized image processing. The maximal response (MR) ([1-(minimal area/baseline area)] x 100) and concentration of MCh resulting in 50% MR (EC50) were determined. A total of 217 airways from 3–12 explants per rat constricted in a concentration-dependent manner. Baseline area was larger with both higher lung volumes and agarose concentrations. MR was greatest in the airways from the 25% TLC and 0.5% agarose explants. Although there was considerable heterogeneity toward MCh within rats (EC50 varied up to 5.46 x 10(5)-fold), the median EC50 was similar among all rats (range 1.96 x 10(-6)-5.87 x 10(-4) M). Lung inflation volume and agarose concentration affected baseline area and MR, suggesting that airway-parenchymal interdependence mechanisms are operative in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

No abstract