Effect of chloroquine on phagolysosomal fusion in cultured guinea pig alveolar macrophages: Implications in drug delivery

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Abstract
The aim of this study was to evaluate the effects of chloroquine on phagolysosomal fusion (PLF) in cultured guinea pig alveolar macrophages (AMs). This technique may be of significance for antitubercular drugs, because.the survival of Mycobacterium tuberculosis is linked to evasion of PLF. Guinea pig AMs were obtained from anesthetized animals after exsanguination. The AMs were cultured at a density of 1 x 10(6) cell/mL in 24-well plates after attachment to 13-mm coverslips. Culture conditions were at 37 degrees C, with 95% air/5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% heat-inactivated fetal bovine serum. Rhodamine-dextran (70 kd) was incubated with the cells at 0.25 mg/mL for 24 hours to label the lysosomes. Chloroquine treatment where indicated was performed at 10-20 microg/mL for 1 hour. Fluorescent BioParticles were then added, and PLF was monitored by formation of an orange-yellow fluorescence on fusion of green fluorescent BioParticles with rhodamine-labeled lysosomes. PLF endpoints were measured by scoring for the percentage of orange-yellow cells in the field of view. Image analysis to measure the intensity of the orange-yellow color was performed by obtaining a, b values for 5 x 5 pixel areas using the PhotoAdobe program 4.0.1. The results indicated that the rate of PLF was enhanced by chloroquine. Thus, chloroquine may be used to potentiate the effects of rifampicin. This may be confirmed by studies involving similar dual fluorophore labeling techniques of fluorescein-labeled formulation in macrophages infected with M.tuberculosis. Preliminary studies with the rhodamine-labeled formulation confirmed cellular uptake and persistence for up to 7 days in culture.