Search

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca 2+ -uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca 2+ -uptake function. Thus, it is essential for the Ca 2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca 2+ -uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transdermal administration of drugs improves their bioavailability and is capable of systemic and local treatment. To improve the skin permeability of drugs, nano-sized systems have attracted attention as drug carriers for transdermal drug delivery system. We considered that silk fibroin composed of a crystalline region with many hydrophobic amino acids and an amorphous region with many hydrophilic amino acids was useful as a carrier for transdermal administration of a drug because of the balance between hydrophilicity and hydrophobicity. In this study, silk fibroin nanoparticles with mean volume diameters of 42.3 nm were successfully prepared, and storage stability was confirmed by storing the nanoparticle suspension at 4, 32, and 37 °C for a week. At any storage temperature, the mean volume diameter and standard deviation were stable. The polydispersity indexes were 0.19-0.23, and no specific trends were observed. Then, to investigate the transdermal delivery route of the silk fibroin nanoparticles, skin permeability in vivo was evaluated using mice. Six hours after administration, fluorescent substances were observed in the dermis in addition to the stratum corneum, hair follicles and the epidermis around them. This result indicated that fibroin nanoparticles with the mean volume diameter of 40-nm penetrated the stratum corneum and was delivered deep into the skin. Therefore, it was suggested that small nanoparticles prepared using silk fibroin are useful for drug delivery to the dermis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization., (Copyright © 2018 Elsevier Inc. All rights reserved.)

First, an elementary osmotic pump (EOP) with a simple structure was prepared using polyethylene oxide (PEO) and NaCl as an excipient, and the influence of the molecular weight (Mw) of PEO on drug release was investigated. In the dissolution test of EOP, it was observed that the gelated core tablet was pushed out through the orifice. The dissolution profile of EOP was sigmoidal, and despite the short time, a zero-order release region was observed. The gel swelling rate in the zero-order region was independent of the Mw of PEO. It was also found that higher the Mw of PEO, the larger the saturated swelling amount. Next, a push-pull pump (PPP) with almost identical formulation to that of EOP was prepared, and its drug release characteristics were investigated. PPPs were prepared by varying the combination of Mws of PEO in both layers, and their dissolution profiles were compared. It was found that PPP using a low-Mw PEO for the drug layer and PEO with a high-Mw in the push layer showed the longest dissolution profile of the linear region. The obtained findings suggested that the properties of PEO and its hydrogel play a crucial role in the drug release of PPP., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Nondestructive analysis of tablet is of great importance from the aspect of productivity and safety. In terahertz (THz) region, however, the analysis of core of coated tablet has not been progressed. In this study, we have measured a flat-surface push-pull osmotic pump tablet, having no orifice, having bilayer core and a coating film layer. The bilayer core was made from the drug layer and the push layer, and acetaminophen was contained in the drug layer as a model drug. To study its structure and components, we have obtained reflection spectra from the drug layer side and the push layer side measurements using THz time-domain reflection spectroscopy (THz-TDRS). From these results, detection of the peak of acetaminophen in the spectrum from the drug layer side measurements was confirmed. We have made ridges approximated toward the peak using a general method of linear regression analysis in both spectra. Two-sample t-test was applied to their gradients, and significant difference between the drug layer and the push layer was shown. These results suggested that THz-TDRS is applicable to the analysis of structure and component of a coated tablet., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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 © 2013 Elsevier B.V. All rights reserved.)

Indomethacin (IM) easily forms an amorphous complex with lidocaine (LC) by heat treatment. To know the mechanism involved in the formation of this complex, we studied temperature-dependent phase changes in mixtures of IM and lidocaine hydrochloride (LH), in which the cationic form of LC forms a salt with Cl(-), in various molar ratios by using DSC and NMR. Although heating of the mixture of IM and LC (IM+LC), formed a eutectic mixture, that of IM and LH (IM+LH) did not, and IM in the IM+LH mixture was dissolved into fused LH. Cooling of the fused IM+LH showed the glass transition in all of the samples containing various amounts of IM, suggesting that fused IM+LH took a homogenous amorphous state (IM/LH) below its glass transition temperature, in contrast to the fused IM+LC, which formed the rubber state and/or glass state depending on the molar content of IM. The results of the NMR study showed that IM in IM/LH caused the electronic structure of LH to change in such a way as to become similar to that of LC, but this effect was limited. Hence, mode of interaction of LH with IM is different from that of LC with IM., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We studied temperature-dependent phase changes in the mixture of indomethacin (IM) and lidocaine (LC) in various molar ratios by differential scanning calorimetry (DSC). DSC studies were carried out between -40°C and 180°C heating/cooling/heating cycle at a rate of 10°C/min under a nitrogen gas flow. Although LC in the liquid state was crystallized, fused IM showed a glass transition signal upon cooling. Hence, cooling of fused IM caused it to assume the amorphous state below its glass transition temperature. Heating of the mixture of IM and LC resulted in formation of a eutectic mixture between them, and cooling of the fused mixture caused formation of the amorphous state at any molar ratio examined. It is noteworthy that the amorphous solid of the fused complex of IM and LC was in the rubber state and glass state in the mixtures containing IM between 20% and 40%, whereas there was only the glass state in the samples containing 50% IM and higher. In addition, crystallization of LC took place by cooling the fused sample containing 10% IM., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Crystalline state of pharmaceutical materials is of great importance in the preparation of pharmaceutics because their physicochemical properties affect bioavailability, quality of products, therapeutic level, and manufacturing process. In this study, we have estimated the crystallinity of trehalose dihydrate microspheres by measuring terahertz (THz) spectroscopy. The commercially available trehalose dihydrate takes in general a crystalline state, but trehalose dihydrate microspheres prepared by using spray-drying method are in an amorphous state. We have prepared amorphous anhydrous trehalose by using melt-quenched method from crystalline trehalose dihydrate. We have measured the absorbance of trehalose dihydrate containing amorphous anhydrous trehalose (0%, 25%, 50%, 75%, and 100%) using THz time-domain spectroscopy (THz-TDS) to prepare calibration curves. Using the calibration curves, we have estimated the crystallinity of trehalose dihydrate microspheres prepared by using spray-drying method. Our results suggest that THz-TDS is well suited to distinguish crystallinity differences in pharmaceutical compounds., (Copyright © 2012 Wiley Periodicals, Inc.)

Estradiol is a therapeutic agent for treatment of perimenopausal symptoms and osteoporosis. Conventional oral or intravenous administration of estradiol has many problems, such as, metabolization in gastrointestinal tract and liver, pain by using an injection needle, rapid increase of drug levels in blood and fast clearance with unwanted side effects including thrombosis, endometriosis and uterus carcinoma. The use of nanocarriers for transdermal delivery has been studied because of their ability to deliver therapeutic agents for long time with a controlled ratio, escaping from the first pass effect by liver. In this study, permeability of estradiol-loaded PLGA nanoparticles through rat skin was studied. Higher amount of estradiol was delivered through skin when estradiol was loaded in nanoparticles than estradiol was free molecules. Also, iontophoresis was applied to enhance the permeability of nanoparticles. When iontophoresis was applied, permeability of estradiol-loaded PLGA nanoparticles was much higher than that obtained by simple diffusion of them through skin, since they have negative surface charges. They were found to penetrate through follicles mainly. Also, enhanced permeability effect of estradiol by using nanoparticle system and iontophoresis were observed in vivo. The combination of charged nanoparticle system with iontophoresis is useful for effective transdermal delivery of therapeutic agents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nanoparticles effectively deliver therapeutic agent by penetrating into the rat skin in vivo. Indomethacin (IM) and coumarin-6 were loaded in PLGA nanoparticles with an average diameter of 100 nm. Indomethacin (IM) and coumarin-6 were chosen as a model drug and as a fluorescent marker, respectively. The surfaces of the nanoparticles were negatively charged. Permeability of IM-loaded PLGA nanoparticles through rat skin was studied in vivo. Higher amount of IM was delivered through skin when IM was loaded in nanoparticles than IM was free molecules. Also, iontophoresis was applied to enhance the permeability of nanoparticles. When iontophoresis was applied at 0.05 mA/cm(2), permeability of IM was much higher than that obtained by simple diffusion of nanoparticles through skin. The combination of charged nanoparticle system with iontophoresis is useful for effective transdermal systemic delivery of therapeutic agents., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nanoparticles effectively deliver therapeutic agent by penetrating into the skin. Indomethacin (IM) and coumarin-6 were loaded in PLGA nanoparticles with an average diameter of 100 nm. IM and coumarin-6 were chosen as a model drug and as a fluorescent marker, respectively. The surfaces of the nanoparticles were negatively charged. Permeability of IM-loaded PLGA nanoparticles through rat skin was studied. Higher amount of IM was delivered through skin when IM was loaded in nanoparticles than IM was free molecules. Also, iontophoresis was applied to enhance the permeability of nanoparticles. When iontophoresis with 3 V/cm was applied, permeability of IM was much higher than that obtained by simple diffusion of nanoparticles through skin. The combination of charged nanoparticle system with iontophoresis is useful for effective transdermal delivery of therapeutic agents., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 © 2011 Elsevier B.V. All rights reserved.)

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 © 2011 Elsevier B.V. All rights reserved.)

It is known that Drug Delivery System (DDS) is useful to remedy against tumors for the reduction of side effects and the effective dosage. However the shape, in particular, the size of drug (medicine) is empirically decided in the present stage, which will be related to a question how much medicine should be dosed. Taking a particular reaction of tumor tissues called the EPR effect into consideration, we try to mathematically describe the behavior (dynamics) of drug in blood vessel by applying several techniques used in mathematics and physics. In this paper, we estimate the configuration of drug which is most effective to remedy for tumors under various conditions. As a result, this model and its simulation will be useful to design the drug in nano-level.

Nanoparticles have widely been studied in drug delivery research for targeting and controlled release. The aim of this article is application of nanoparticles as an inhalable agent for treatment of lung cancer. To deposit effectively deep the particles in the lungs, the PLGA nanoparticles loaded with the anticancer drug 6-{[2-(dimethylamino)ethyl]amino}-3-hydroxyl-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103) were prepared in the form of nanocomposite particles. The nanocomposite particles consist of the complex of drug-loaded nanoparticles and excipients. In this study, the anticancer effects of the nanocomposite particles against the lung cancer cell line A549. Also, the concentration of TAS-103 in blood and lungs were determined after administration of the nanocomposite particles by inhalation to rats. TAS-103-loaded PLGA nanoparticles were prepared with 5% and 10% of loading ratio by spray drying method with trehalose as an excipient. The 5% drug-loaded nanocomposite particles were more suitable for inhalable agent because of the sustained release of TAS-103 and higher FPF value. Cytotoxicity of nanocomposite particles against A549 cells was higher than that of free drug. When the nanocomposite particles were administered in rats by inhalation, drug concentration in lung was much higher than that in plasma. Furthermore, drug concentration in lungs administered by inhalation of nanocomposite particles was much higher than that after intravenous administration of free drug. From these results, the nanocomposite particle systems could be promising for treatment of lung cancer.

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, kappa-carrageenan and iota-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 10 microm. 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 400 min when iota-carrageenan was used as a base material. On the other hand, the release was prolonged to 600 min when kappa-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.

Purpose of the present work was to study in vitro permeation of gold nanoparticles (NPs) through isolated rat skin and intestine. Another objective was to see the effect of particle size on permeation of the gold NP. Gold NP of 15 nm, 102 nm and 198 nm were synthesized and used for study. Franz diffusion cells were used to evaluate permeation of gold NP from rat skin whereas 'intestinal sac' method was used for assessing intestinal permeation. Number density of gold NP was analyzed by UV-vis spectroscopy whereas amount of gold permeated was measured by ICP mass spectrometry. The absorption and localization of gold NP through rat skin was studied by TEM. Qualitative analysis of gold inside of the rat skin was performed by energy dispersive X-ray spectroscopy (EDS). Gold NP showed negative zeta potential. UV-vis absorption spectra of 15 nm, 102 nm and 198 nm gold NP showed lambda(max) at 520 nm, 535 nm and 577 nm, respectively. SEM study revealed spherical morphology of NP. Gold NP showed size dependent permeation through rat skin and intestine. 15 nm gold NP showed higher permeation compared to 102 nm and 198 nm gold NP. Interestingly, 102 nm and 198 nm gold NP showed lag time of 3h and 6h in case of rat skin only. As the size of the gold NP increased, permeability coefficient and diffusion coefficient was found to be decreased. The permeation of gold NP through intestine was higher than that of skin. TEM study of rat skin revealed accumulation of smaller size gold NP in deeper region of skin whereas larger particles were observed mainly in epidermis and dermis. Presence of gold inside of rat skin was confirmed by EDS. Gold NP would be an interesting carrier for transdermal as well as for oral delivery. The study demonstrated initial proof of concept of percutaneous permeation of smaller size gold particles.

A method for exact determination of phagocytic activity of alveolar macrophage (Mvarphi) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of Mvarphi samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to Mvarphi cell membranes during their phagocytosis by Mvarphi cells. We found that centrifugation, which was employed to collect Mvarphi 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 Mvarphi 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.

Phagocytosis of alveolar macrophages (Mphis) 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 Mphi, toward PSL MS with various diameters was examined by incubating the cells for 4h at 37 degrees C with various numbers of PSL MS per Mphi cell (MS/Mphi=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 Mphi 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/Mphi, 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-mum ones were taken up by Mphi the most, and the RFP-PLGA MS 3 microm in diameter seemed to be phagocytosed the most efficiently, although we were not able to determine exactly the phagocytosis of 6- and 10-microm RFP-PLGA MS. From the changes in N and n values with MS/Mphi, the phagocytosis of RFP-PLGA MS was likely to enhance the phagocytic activity of Mphi cells, but this effect did not seem to be significant for PSL MS.

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.

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 microm. 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.