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Monodispersed PLGA microspheres containing rifampicin (RFP) have been prepared by solvent evaporation method using a Shirasu porous glass (SPG) membrane. The microspheres were spherical and their average diameter was about 2 μm. The loading efficiency of rifampicin was dependent on the molecular weight of PLGA. The higher loading efficiency was obtained by the usage of PLGA with the lower molecular weight, which may be caused by the interaction of the amino groups of rifampicin with the terminal carboxyl groups of PLGA. PLGA with the monomer compositions of 50/50 and 75/25, of lactic acid/glycolic acid, were used in this study. From rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 20,000, rifampicin was released with almost constant rate for 20 days after the lag phase was observed for the initial 7 days at pH 7.4. On the other hand, from rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 5000 or 10,000, almost 90% of rifampicin-loaded in the microspheres was released in the initial 10 days. Highly effective delivery of rifampicin to alveolar macrophages was observed by the usage of rifampicin-loaded PLGA microspheres. Almost 19 times higher concentration of rifampicin was found to be incorporated in alveolar macrophages when rifampicin-loaded PLGA microspheres were added to the cell culture medium than when rifampicin solution was added. [Copyright &y& Elsevier]

Abstract: Although treatment of pulmonary tuberculosis with respirable microspheres (MS) with an incorporated antituberculosis drug is expected to be highly effective, this treatment seems to achieve a much lesser effect than expected in the case of killing Mycobacterium tuberculosis residing in the lungs. To elucidate the reason for this weaker effect, we examined the distribution and accumulation of respirable MS consisting of poly(lactic-co-glycolic) acid (PLGA) in rat lungs. For this, we delivered the PLGA MS containing fluorescent coumarin 6 or an antituberculosis agent, rifampicin (RFP), by insufflation via the trachea and then determined the pulmonary distribution by counting the number of the MS in lung cryosections observed under a microscope. In addition, the uptake of MS by alveolar macrophage (AMϕ) was determined by immunostaining for Mϕ cell marker CD68 and RFP content in the cells. Approximately half of the fluorescent PLGA MS reached the alveoli without entrapment by trachea and primary bronchi and were then ingested by the AMϕ cells up to 24h after insufflation. RFP in a form of PLGA MS was markedly transported into AMϕ at an amount 10 times greater than that for the free RFP powder. However, a large proportion of RFP was eliminated from the lungs by 6h after insufflation. [Copyright &y& Elsevier]

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]