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Abstract: Our previous results on the phagocytic activity of alveolar macrophages (Mϕs) toward poly(lactic-co-glycolic) acid microspheres (PLGA MS) loaded with the anti-tuberculosis agent rifampicin (R-PLGA MS) suggest that the phagocytosis of R-PLGA MS enhances the phagocytic activity of Mϕ cells . To confirm this possibility, we examined the effect of phagocytosis of R-PLGA MS and polystyrene latex (PSL) MS on the phagocytic uptake of fluorescent PSL (F-PSL) MS by cells of the rat alveolar macrophage cell line NR8383 at 37°C. Phagocytic activity was examined in terms of the population of Mϕ cells that had phagocytosed MS (N total) and the total number of MS phagocytosed (n total) by counting the phagocytic Mϕ cells and the MS ingested in optical microscopic fields. Phagocytosis of R-PLGA MS enhanced about 1.5 times the values of N total and n total of the phagocytosis of F-PSL MS under the conditions where the phagocytosis of F-PSL MS did not attain the saturated level. In contrast, the phagocytosis of PSL MS did not enhance the phagocytic activity of Mϕ cells toward F-PSL MS. In conclusion, R-PLGA MS are favorable for drug delivery of anti-tuberculosis agents into alveolar Mϕs due to their ability to up-regulate the phagocytosis of MS. [Copyright &y& Elsevier]

Abstract: Inhalation delivery of poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP–PLGA MS) to alveolar macrophage (Mϕ) cells could be an effective drug delivery system for the treatment of tuberculosis. To examine this possibility, we studied (1) the bactericidal effect of RFP–PLGA MS on Mycobacterium bovis Bacillus Calmette–Guérin (BCG)-infected rat alveolar Mϕ NR8383 cells, and (2) changes in the biochemical events induced in these cells by the uptake of RFP–PLGA MS. The amount of intracellular RFP imported into the Mϕs by RFP–PLGA MS containing 0.25 and 2.50μg RFP/mL was more than twice and ten times, respectively, than that attained with 5.00μg/mL of RFP solution; and the MS exerted more potent bactericidal effect on BCG inside Mϕ cells than 5.00μg RFP/mL solution after incubation for 7days. RFP–PLGA MS little affected the viability of Mϕ cells, whereas the polystyrene latex (PSL) MS used as a reference decreased it significantly. RFP–PLGA MS did not stimulate the production of tumor necrosis factor-α (TNF-α), nitric oxide, interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1) by the Mϕ cells, whereas PSL MS stimulated all of these mediators except IL-10. We conclude that RFP–PLGA MS are bio-safe microspheres due to their “silent” nature when taken into Mϕ cells and that they are promising for the treatment of tuberculosis by pulmonary inhalation. [Copyright &y& Elsevier]

Abstract: A method for exact determination of phagocytic activity of alveolar macrophage (Mϕ) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of Mϕ samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to Mϕ cell membranes during their phagocytosis by Mϕ cells. We found that centrifugation, which was employed to collect Mϕ samples after incubation with MS, affected significantly the efficiency of the various treatments. Of the three treatments, xylene treatment without centrifugation was the most effective to determine the phagocytic activity of Mϕ cells, as xylene dissolved the PSL MS on the cell surface almost completely. This treatment was also effective in the case of poly(lactic-co-glycolic acid) MS (PLGA MS), which have been commonly used as an efficient vehicle for drug delivery system. [Copyright &y& Elsevier]

Abstract: We examined the phagocytic activities of alveolar macrophages (NR8383 cells) toward poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP), the sizes of which were between 1 μm and 10 μm. We found that 1) the phagocytosis was dependent greatly on the particle size and the number of particles added; 2) macrophages phagocytosed considerably the PLGA microspheres loaded with RFP, the diameter of which was between 1 μm and 6 μm, but took up few 10-μm particles; 3) the population of the macrophages that phagocytosed 1-μm or 3-μm particles was larger than that of those phagocytosed 6- or 10-μm particles; 4) a considerable population of macrophages were not able to phagocytose even the 1- and 3-μm particles; 5) the most efficient deliveries of RFP into each macrophage cell and a large population of macrophages were achieved by the phagocytosis of 3-μm particles; and 6) phagocytosis did not affect macrophage viability in 4 h after the start of phagocytosis. [Copyright &y& Elsevier]

Polystyrene microspheres with diameters of 0.2, 0.5, 1.0, 6.0 and 10 μm were added to alveolar macrophages, and their uptake was determined as the amount of superoxide generated from macrophages by the usage of chemiluminescence assay with luminol. The amount of superoxide generated was apparently higher with polystyrene microspheres with a diameter of 1 μm than those with diameters smaller than 1 μm (i.e. 0.2 or 0.5 μm) and with larger than 1 μm (6 or 10 μm). The effects of the functional groups located on the microsphere surfaces upon the uptake by alveolar macrophages were studied with polystyrene microspheres of 1 μm diameter having the primary amine, sulfate, hydroxyl, or carboxyl groups on their surfaces. We found that the macrophages most effectively trapped polystyrene microspheres with primary amine groups, those with carboxyl groups to a slightly lesser extent, and other microspheres much less amounts. The surface properties of these microspheres were determined by measuring their electrophoretic mobility in phosphate buffer solution (pH 7.4) with various ionic strengths. By the analysis of data with Ohshima''s electrokinetic theory for soft particles, the surface charge density and the electrophoretic softness of the microsphere surfaces were determined. All the microsphere surfaces were found to be negatively charged, and those with primary amine groups and carboxyl groups were softer than other microspheres. From these findings, it is suggested that microspheres having soft surfaces are easily accessible to alveolar macrophages, and effectively trapped by macrophages. [Copyright &y& Elsevier]