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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: Understanding of the association of phagocytosis of polymers with signaling of innate immunity of macrophages is the major purpose of this study. Polymer conjugates have been utilized for clinical therapy of cancer and infections, such as Mycobacterium tuberculosis, as effective vectors of drug-delivery systems. They are incorporated through phagocytosis into macrophages and activate innate immunity signaling, which plays a crucial role in its therapeutic and side effects. Macrophage phagocytosis of polystyrene latex microspheres was examined and assayed by treatment of macrophages with the cholesterol depletor methyl-β-cyclodextrin (MβCD) or the sphingolipid depletor n-octyl-β-d-glucopyranoside (OGP). Expressions of various mRNAs during phagocytosis were quantified by real-time PCR. Phagocytosis of polystyrene latex microspheres by various macrophages, such as murine monocyte-derived macrophage J774, rat alveolar macrophage NR8383, and murine Kupffer cell KC13-2, was suppressed by treatment with MβCD or OGP in a concentration-dependent manner. The expression of mRNAs of TNFα, IL-1β, IL-6 and CXCL10 genes induced by lipopolysaccharide (LPS) was not suppressed by treatment with MβCD in J774 cells. Moreover, genes that were induced by LPS were up-regulated even in the absence of LPS by the phagocytosis of polymer conjugates, but such up-regulations were not suppressed by the treatment with MβCD. It was shown that lipid rafts play a significant role in incorporation of polymer conjugates through phagocytosis of macrophages, but their association with signal transduction in innate immunity is very limited. [Copyright &y& Elsevier]

Abstract: For the treatment of oral mucositis, carrageenan microspheres containing allopurinol and local anesthetic agents, such as lidocaine hydrochloride, dibucaine hydrochloride and tetracaine hydrochloride were prepared using a spray-drying method. As base materials, κ-carrageenan and ι-carrageenan were evaluated, since carrageenan mitigates bitter taste of lidocaine hydrochloride, dibucaine hydrochloride and tetracaine hydrochloride. The microspheres were spherical and their average diameters were about 10μm. The drug loading efficiency was more than 70%. Allopurinol and local anesthetic agents became amorphous by the spray drying. Allopurinol and the local anesthetic agents were released from the microspheres for at least 400min when ι-carrageenan was used as a base material. On the other hand, the release was prolonged to 600min when κ-carrageenan was used. The microspheres were spread and made membranes at the air/water interfaces immediately after dropped on the water surfaces. The properties of the microspheres such as dispersing efficacy and membrane production on the water surfaces suggest that the microspheres can uniformly cover inner surfaces of oral cavity to prevent and treat oral mucositis. [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: Phagocytosis of alveolar macrophages (Mϕs) toward poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP-PLGA MS) has been shown to be effective for the treatment of tuberculosis. The phagocytosis should be evaluated in terms of that toward reference MS. We chose polystyrene latex (PSL) MS as a reference. In this study, phagocytic activity of cell line NR8383, derived from rat alveolar Mϕ, toward PSL MS with various diameters was examined by incubating the cells for 4h at 37°C with various numbers of PSL MS per Mϕ cell (MS/Mϕ=0.1–10). The results were then compared with those of the phagocytosis toward RFP-PLGA MS. We determined the phagocytic activity by counting the population of Mϕ cells that had phagocytosed MS (N) and the number of particles phagocytosed (n) in microscopic fields. Both N and n for PSL and RFP-PLGA MS increased in general with an increase in MS/Mϕ, but both of these values for PSL MS were smaller than those for RFP-PLGA MS. Phagocytosis of the particles were dependent on the particle size; i.e., of the PSL MS the 6-μm ones were taken up by Mϕ the most, and the RFP-PLGA MS 3μm in diameter seemed to be phagocytosed the most efficiently, although we were not able to determine exactly the phagocytosis of 6- and 10-μm RFP-PLGA MS. From the changes in N and n values with MS/Mϕ, the phagocytosis of RFP-PLGA MS was likely to enhance the phagocytic activity of Mϕ cells, but this effect did not seem to be significant for PSL MS. [Copyright &y& Elsevier]

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]

In order to determine whether the microsphere size affects the phagocytic uptake of microspheres in vitro, polystyrene microspheres with diameters of 0.2, 0.5, 1.0, 6.0 and 10 μm were added to a HL-60 differentiated macrophage-like cell culture system. The extent of the uptake was determined by the amount of superoxide generated from activated macrophages by the usage of a chemiluminescence (CL) assay with luminol. The release amount of superoxide was higher when polystyrene microspheres with diameters between 0.5 and 6 μm (i.e. 0.5, 1, and 6 μm were added to the macrophages than that when those with a diameter 0.2 or 10 μm were added. Especially, polystyrene microspheres with an average diameter of 1 μm were most effectively recognized as foreign materials by the macrophages. The effects of the functional groups on the microsphere surfaces upon the uptake by macrophages were studied using polystyrene microspheres with an average diameter of 1 μm, having primary amine, sulfate, hydroxyl, or carboxyl groups on their surfaces. Polystyrene microspheres having primary amine groups on their surfaces were more effectively engulfed by the macrophages than polystyrene microspheres having other functional groups. Also, polystyrene microspheres coated with human IgG was more easily recognized as foreign materials than intact polystyrene microspheres. [Copyright &y& Elsevier]