Yusuke Tsukada

Oral nuclear factor-κB decoy oligonucleotides delivery system with chitosan modified poly(D,L-lactide-co-glycolide) nanospheres for inflammatory bowel disease.

Chitosan (CS)-modified poly(D,L-lactide-co-glycolide) (PLGA) nanospheres (NS) were developed and evaluated for use with a nuclear factor kappa B (NF-κB) decoy oligonucleotide (ODN) oral delivery system in an experimental model of ulcerative colitis (UC). Decoy ODN-loaded PLGA NS were prepared by an emulsion solvent diffusion method, and the physicochemical properties of NS were investigated. CS-modified PLGA NS (CS-PLGA NS) showed positive zeta potential, while unmodified PLGA NS (plain-PLGA NS) were negatively charged. Decoy ODN uptake studies with Caco-2 cells using confocal laser scanning microscopy (CLSM) indicated that CS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. Decoy ODN-loaded CS-PLGA NS were able to improve the stability of ODN against DNase I or an acidic medium, such as gastric juice. Daily oral administration of CS-PLGA NS in a rat model significantly improved dextran sulfate sodium-induced diarrhea, bloody feces, shortening of colon length, and myeloperoxidase activity. Furthermore, decoy ODN-loaded CS-PLGA NS were specifically deposited and adsorbed on the inflamed mucosal tissue of the UC model rat. These results suggested that CS-PLGA NS provide an effective means of colon-specific oral decoy ODN delivery in UC.

Histological examination of PLGA nanospheres for intratracheal drug administration.

Polylactide-glycolide (PLGA) nanospheres were reported as useful pulmonary drug delivery carriers for improving the pharmacological effect of drug. This paper describes the pathological and histological examinations of tissues after intratracheal instillation of drug encapsulated PLGA nanospheres. After intratracheally introducing FITC encapsulated PLGA nanospheres (dispersed in the 0.5 ml saline followed by mixing with an equal volume of air) to a rat, FITC was found existing in the rats lungs, liver, kidney, brain, spleen and pancreas as demonstrated by immuno-histo-chemical staining with the dye. In this study, FITC stayed in alveoli at least for 1.5h after the intratracheal administration of the PLGA nanospheres, but the FITC almost disappeared 24h later. In addition, it was found that the PLGA nanospheres were absorbed in the blood immediately (within 0.25 h after the intratracheal administration) through the type 1 alveolar epithelium cell. Furthermore, the PLGA nanospheres were found resistant to uptake by macrophages such as alveolus macrophages and kupffer cells. The results showed that the possibility to induce tissue damage caused by the excessive immune response from the deposition of PLGA nanospheres was very low, because the nanospheres were not treated as foreign substances.

Particle size control of poly(dl-lactide-co-glycolide) nanospheres for sterile applications

Parameters affecting the particle sizes of poly(DL-lactide-co-glycolide) (PLGA) nanospheres produced by the Emulsion Solvent Diffusion (ESD) method were evaluated in this study, so that suitable PLGA nanospheres could be prepared to pass through a membrane filter with 0.2 microm pore size and used as a sterile product. Experimental results demonstrated that the particle sizes of PLGA nanospheres could be reduced by the following efforts. (1) Increase stirring rate of poor solvent. (2) Decrease feed rate of good solvent. (3) Increase poor solvent ratio. (4) Increase the temperature of poor solvent. (5) Decrease polyvinyl alcohol concentration in poor solvent. (6) Increase ethanol concentration in good solvent. (7) Decrease PLGA concentration in good solvent. After optimization, PLGA nanospheres with a mean particle size of 102-163 nm and the 100-98% of filtration fraction could be produced and passed the bacteria challenge tests. This study found PLGA nanospheres can be efficiently prepared as a sterile product.

Prevention of Neointimal Formation After Angioplasty Using Nuclear Factor-κB Decoy Oligodeoxynucleotide-Coated Balloon Catheter in Rabbit Model

Background—Despite the advent of drug-eluting stents, restenosis after endovascular intervention is still a major limitation in the treatment of cardiovascular disease. To regulate the multiple biological mechanisms underlying restenosis, we focused on inhibition of an important transcription factor, nuclear factor-kappaB (NF&kgr;B), using a decoy strategy. Methods and Results—For site-specific application of NF&kgr;B decoy oligodeoxynucleotides into target vessels during angioplasty, we developed a balloon catheter–based delivery system combined with biocompatible nanoparticles as oligodeoxynucleotides carriers. To clarify the therapeutic effect at the site of neointima, balloon angioplasty of the rabbit carotid arteries was performed at 4 weeks after initial endothelial denudation. This delivery system exhibited successful transfer of fluorescence-labeled nanospheres into the neointima in short-term contact with target vessels, and fluorescence could be detected ≥1 week after angioplasty. Consistently, local application of NF&kgr;B decoy oligodeoxynucleotides -loaded nanospheres resulted in significant inhibition of neointimal formation, associated with inhibition of NF&kgr;B binding activity in the injured arteries. The therapeutic effects were caused by inhibition of macrophage recruitment through the suppression of monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and CC chemokine ligand 4 expression and inhibition of vascular smooth muscle cell growth via a decrease in the expression of cyclin A and proliferating cell nuclear antigen. Importantly, application of NF&kgr;B nanospheres accelerated restoration of the endothelial cell monolayer, associated with enhanced expression of phosphorylated Bcl-2 in endothelial cells. Conclusions—A drug-coated balloon catheter using NF&kgr;B decoy oligodeoxynucleotides significantly inhibited the development of neointimal hyperplasia in rabbits. The present study indicates the possibility of a novel therapeutic option to prevent restenosis after angioplasty.

PLGA Nanosphere Technology for Novel Nanomedicine and Functional Cosmetics

Abstract In the recent drug discovery, various drug delivery systems have increasingly played an important role for repositioning existing products and the practical application of biologically active substances such as nucleic acid. Especially, nanosized particles [1–3] such as liposome, micelle, and polymeric nanosphere have been actively researched as drug carriers in recent years. We have been developing poly ( d , l -lactide-co-glycolide) nanosphere (PLGA NS) loaded with bioactive substance. Clinical trials of drugs and devices using our PLGA NS have already been launched in Japan. We also have already succeeded in commercialization of PLGA NS for skincare cosmetics and hair growth tonic. In this chapter, our applications with PLGA NS in the nanomedical system are introduced.