Liposomes and Pulmonary Alveolar Macrophages: Functional and Morphologic Interactions

Abstract

In vitro toxicity of liposomes and their functional and morphologic interactions with rat pulmonary alveolar macrophage (AMs) were investigated using viability (trypan blue exclusion), phagocytic and killing activity (uptake and digestion of live S. cerevisiae), surface adherence, respiratory burst (nitro-blue tetrazolium reduction), and morphometry (computerized image analysis) as indicators. Liposome stability in physiologic solutions and uptake of liposome-encapsulated carboxyfluorescein (CF) by AMs was assessed by fluorescence spectroscopy and microscopy. Liposomes made from saturated phospholipids and cholesterol were stable, whereas liposomes consisting of unsaturated phospholipids without cholesterol lost 30% to 40% of their content over 24 h. However, CF uptake was highest with unsaturated phospholipid preparations, whereas uptake of the three other formulations was comparable. Although liposome exposure did not affect macrophage viability, a reduction in the number of phagocytizing macrophages to 73% of control was noted after 24-h incubation with the highest lipid concentration tested (10 μmol/ml). Phagocytic killing was similar under all circumstances observed. The fraction of intracellularly killed yeast ranged from 32% to 42% for both control and experimental samples. An increase in cell surface area from 166.1 ± 39.9 μm² on day 0 (n = 709) to 196.3 ± 57.6 μm² on day 1 (n = 516) and 211.2 ± 48.0 μm² on day 4 (n = 834) was observed after liposome treatment. The corresponding average cell areas of control samples did not change during the observation period. There was no net cell loss of adherence from monolayers as determined by protein assay. The respiratory burst, indicating generation of intracellular superoxide, was also similar-84% to 92% of experimental and control cells under all conditions showed a strong nitro-blue tetrazolium reduction. In summary, in vitro exposure of AMs to large concentrations of liposomes, although producing an increase in macrophage size, was not associated with aberrant macrophage morphologic features, function, or toxicity for the parameters examined.