Hiroaki Okada

One-step preparation of rifampicin/poly(lactic-co-glycolic acid) nanoparticle-containing mannitol microspheres using a four-fluid nozzle spray drier for inhalation therapy of tuberculosis

We prepared rifampicin (RFP)/poly(lactic-co-glycolic acid) (PLGA) nanoparticle-containing mannitol (MAN) microspheres for inhalation therapy of tuberculosis using a four-fluid nozzle spray drier in one step. The RFP and PLGA acetone/methanol (2/1) solution and MAN aqueous solution were supplied through different liquid passages of the four-fluid nozzle spray drier to obtain MAN microspheres including RFP/PLGA nanoparticles. The mean diameter of RFP/PLGA nanoparticles dispersed in MAN was 213 nm. The (RFP/PLGA)/MAN microspheres showed good in vitro aerosol performance evaluated using a cascade impactor. In an in vitro study of uptake by alveolar macrophage cells evaluated using coumarin 6 as a marker, the PLGA microparticles were more efficiently taken in than the PLGA nanoparticles. In an in vivo study of uptake of RFP by alveolar macrophages in the lungs of rats, the uptake of RFP from the RFP/PLGA microspheres was very small. The uptake of RFP from the (RFP/PLGA)/MAN microspheres, however, was larger, about 4%, at 1 h after administration increasing to 9.3% at 4 h. An in vivo imaging study suggests that the micron-sized PLGA particles were rapidly excreted from the lungs, but that it may be difficult to clear the nano-sized PLGA particles, resulting in their retention. Thus, the RFP/PLGA nanoparticles were recognized by alveolar macrophages and the uptake of RFP increased. This study shows that it is feasible to prepare RFP/PLGA nanoparticle-containing microspheres using a four-fluid nozzle spray drier in one step, and that the particles are retained in the lungs for prolonged periods and targeted to alveolar macrophages.

Cell-penetrating peptide-modified block copolymer micelles promote direct brain delivery via intranasal administration.

ABSTRACTPurposeIn order to develop non-invasive and effective nose-to-brain delivery of drugs, we synthesized Tat analog-modified methoxy poly(ethylene glycol) (MPEG)/poly(ε-caprolactone) (PCL) amphiphilic block copolymers through the ester bond.MethodsWe evaluated the brain distribution of coumarin, acting as a model chemical, after intravenous or intranasal administration of MPEG-PCL. In addition, cellular uptake of coumarin by rat glioma cells transfected with coumarin-loaded MPEG-PCL or MPEG-PCL-Tat was determined. Finally, we determined the brain distribution and biodistribution after intranasal administration of coumarin-loaded MPEG-PCL-Tat.ResultsThe amount of coumarin in the brain after intranasal administration was significantly higher than that after intravenous administration. In addition, cellular uptake of coumarin using MPEG-PCL was the lowest, while cellular uptake of coumarin using Tat-modified MPEG-PCL (MPEG-PCL-Tat) was higher than that of MPEG-PCL. Therefore, the brain distribution of coumarin administered using MPEG-PCL-Tat was significantly greater than that using MPEG-PCL. Then, the coumarin distribution after MPEG-PCL-Tat administration in non-targeted tissues (lung, liver, heart, kidney and spleen) was lower than that after coumarin administration without nanomicelles.ConclusionWe have demonstrated that utilization of nano-sized micelles modified with Tat can facilitate direct intranasal brain delivery.

Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing

The siRNA has been expected to apply for several diseases such as cancer since siRNA specifically silences the disease-associated genes. However, effective gene carriers should be developed to overcome the low siRNA stability in vivo, form stable complexes and facilitate intracellular uptake of siRNA. In this study, to develop a safe and efficient siRNA carrier, stearoyl (STR) peptides with Cys (C), Arg (R), and His (H) residues that can form disulfide cross linkages via Cys (C) were synthesized, and their suitability as siRNA carriers was evaluated. The particle size of STR-CH(2)R(4)H(2)C/siRNA complexes was about 100 nm. The cellular uptake ability after transfection with FAM-siRNA with STR-CH(2)R(4)H(2)C, CH(2)R(4)H(2)C, or STR-GH(2)R(4)H(2)G was significantly higher than that with FAM-siRNA only. STR-CH(2)R(4)H(2)C showed the highest cellular uptake ability when compared with CH(2)R(4)H(2)C and STR-GH(2)R(4)H(2)G. STR-CH(2)R(4)H(2)C did not induce substantial cytotoxicity. The intratumor injection of STR-CH(2)R(4)H(2)C/vascular endothelial growth factor (VEGF) siRNA (siVEGF) complexes achieved a high anti-tumor effect in tumor bearing mice. These results suggest STR-CH(2)R(4)H(2)C has potential of effective siRNA carrier possible to exercise silencing effect in vitro and in vivo.

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery.

To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(epsilon-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, (1)H NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.

Sustained-release of protein from biodegradable sericin film, gel and sponge

A silk protein, sericin, contains 18 kinds of amino acids, mostly polar side chains forming a complex of three principal polypeptides. The major polypeptides exhibit hydrophobic characteristics by forming a β-sheet structure in a hydrate state. As a drug-releasing biomaterial made by an aqueous process without using any cross linker, sericin is expected to form various hydrophobic dosage forms. However, its dosage form, with respect to the molecular weight and concentration of sericin, and its biodegradation behavior has not been studied in detail. In this study, the film, gel and sponge of sericin were prepared and examined to determine the release properties of the charged protein, fluorescein isothiocyanate-albumin (FA). The film and gel, as solid and semisolid forms, respectively, were also evaluated for their biodegradation behavior. For in vitro release, FA was sustained-released from these preparations. The concentration and dosage form markedly affected FA release. For in vivo biodegradation, the sericin preparations implanted subcutaneously in rats gradually decreased in size and weight. Histological examination indicated no marked inflammation at the site. As for in vivo release, FA remained for 3-6 weeks or more in rats. These findings suggest that sericin is suitable for use as a drug-releasing biomaterial.

Suppression of tumor growth by systemic delivery of anti-VEGF siRNA with cell-penetrating peptide-modified MPEG-PCL nanomicelles.

Small interfering RNAs (siRNAs) have potential applications for many diseases, such as cancer, since siRNAs can specifically silence disease-associated genes. However, effective siRNA carriers need to be developed to overcome the low siRNA stability in vivo, to form stable complexes and to facilitate intracellular uptake. In this study, to develop a carrier for systemic siRNA delivery, we prepared methoxy poly(ethylene glycol) (MPEG)/polycaprolactone (PCL) diblock copolymers conjugated with a cell-penetrating peptide, Tat, via a disulfide linkage, and evaluated their ability as an siRNA carrier. The particle size of MPEG-PCL-SS-Tat/siRNA complexes was approximately 100-200 nm. The cellular uptake ability after transfection with FAM-siRNA with MPEG-PCL-SS-Tat was significantly higher than that with FAM-siRNA only. MPEG-PCL-SS-Tat did not induce substantial cytotoxicity. Intravenous injection of MPEG-PCL-SS-Tat/anti-vascular endothelial growth factor (VEGF) siRNA (siVEGF) complexes achieved a high anti-tumor effect in tumor-bearing mice. These results suggest that MPEG-PCL-SS-Tat is a potentially effective siRNA carrier for silencing genes in vitro and in vivo.

Therapeutic Effects on Atopic Dermatitis by Anti-RelA Short Interfering RNA Combined with Functional Peptides Tat and AT1002

Atopic dermatitis (AD) has high morbidity and poor prognosis because safe and effective treatments are scarce. Recently, short interfering RNA (siRNA) has shown promise as an effective treatment for targeting specific aberrantly expressed genes. However, naked siRNAs are too inefficient because of various enzymatic, membrane, and cellular barriers. We previously reported that a Tat analog acting as a cell-penetrating peptide, combined with AT1002, which reversibly increases paracellular transport of molecules across the epidermal barrier in epidermis-disrupted mice and enhances the skin permeation of water-soluble siRNA. In the present study, to develop a novel treatment for AD, we determined the intradermal permeation of siRNAs and the antiallergic effects of a siRNA that silences RelA, a member of the nuclear factor-κB family, using Tat and AT1002 peptides in an AD mouse model. We first showed that the Tat analog and AT1002 delivered siRNA into the skin of ICR mice and, upon topical application to the AD-induced ears of NC/Nga mice, changed zonula occludens protein 1 expression. In addition, the silencing effects on the mRNA of RelA in JAWS II cells transfected with siRNA oligonucleotides for mouse RelA, complexed with Tat, were as effective as a commercial vector. Furthermore, the ear thickness, clinical skin severity, topical cytokine levels, and serum IgE production in AD model mice treated with anti-RelA siRNA with Tat and AT1002 were improved.

Application of a Four-fluid Nozzle Spray Drier to Prepare Inhalable Rifampicin-containing Mannitol Microparticles

The purpose of this study was to use a four-fluid nozzle spray drier as a new one-step method for preparing rifampicin (RFP)-containing mannitol microparticles. A RFP-acetone/methanol (2:1) solution and aqueous solutions of mannitol (MAN) were simultaneously supplied through different liquid passages of a four-fluid nozzle spray drier and then dried to obtain MAN microparticles containing RFP. Using a cascade impactor, the in vitro aerosol performance of RFP powder and RFP-MAN microparticles with 1:5, 1:10, and 1:20 ratios was compared. The in vivo retention of RFP in the lungs of rats after intratracheal administration of 1:20 RFP-MAN microparticles was also compared. The RFP-MAN microparticles had better aerosol performance than RFP powder and delivery to the lung stages improved as the fraction of MAN was increased. For the 1:20 RFP-MAN microparticles, deposition in stages 2–7 was approximately 43%, which is sufficient for treatment. Approximately 8% of the RFP-MAN microparticles were deposited in stages 6–7, which corresponds to alveoli containing alveolar macrophages. The initial retention of RFP in the lung following pulmonary delivery of 1:20 RFP-MAN microparticles was higher than following oral or intravenous administration of RFP, but the elimination was rapid, resulting in the disappearance of RFP from the lung within 4xa0h. The plasma concentration–time profile of RFP after intratracheal administration of 1:20 RFP-MAN microparticles was consistent with the profile for RFP retention in the lung. Addition of cholesterol or phosphatidylcholine to RFP had little effect on its retention in the lung. The RFP-MAN microparticles were effective for delivery of RFP to the lung, but the RFP rapidly removed from the lung into the blood circulation. This study demonstrated that RFP-containing MAN microparticles prepared in one step using the four-fluid nozzle spray drier efficiently deliver RFP to the lung, although methods must be developed to prolong its retention and improve targeting to alveolar macrophages.

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine.

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes.

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80 nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80 nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100 nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.