Yoshiaki Kawashima

Improved cellular uptake of chitosan-modified PLGA nanospheres by A549 cells.

The authors have previously developed poly(DL-lactic-co-glycolic acid) (PLGA) nanospheres (NSs) as a nanoparticulate drug carrier for pulmonary administration. The present study demonstrates that chitosan (CS)-modified PLGA NSs (CS-PLGA NSs) are preferentially taken up by human lung adenocarcinoma cells (A549). PLGA NSs prepared using a water-oil-water emulsion solvent evaporation method were surface-modified by adsorption of CS. The physicochemical parameters of PLGA NS, including average size and surface charge, were measured to identify which parameter influenced cellular uptake of PLGA NS. Uptake was confirmed using fluorescence spectrophotometry and was visualized in A549 cells with confocal laser scanning microscopy (CLSM). The cytotoxicities of non- and CS-PLGA NS systems were compared in vitro by MTS assay. Cellular uptake of PLGA NS increased with decreasing diameter to the submicron level and with CS-mediated surface modification. Cellular uptake of PLGA NS was energy dependent, as shown by a reduction in uptake at lower incubation temperatures and in hypertonic growth medium used as an inhibitor of clathrin-coated pit endocytosis. CS-PLGA NSs were taken up by A549 cells in an energy-dependent manner, suggesting a clathrin-mediated endocytic process. CS-PLGA NS demonstrated low cytotoxicity, similar to non-PLGA NS.

Establishing chitosan coated PLGA nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery

The purpose of this paper was to establish the surface modified poly(d,l-lactide-co-glycolide) (PLGA) nanosphere platform with chitosan (CS) for gene delivery by using the emulsion solvent diffusion (ESD) method. The advantages of this method are a simple process under mild conditions without sonication. This method requires essentially dissolving both polymer and drug in the organic solvent. Therefore a hydrophilic drug such as nucleic acid is hardly applied to the ESD method. Nucleic acid can easily form an ion-complex with cationic compound, which can be dissolved in the organic solvent. Thereafter, nucleic acid solubility for organic solution can increase by complexation with cationic compound. We used DOTAP as a cationic compound to increase the loading efficiency of nucleic acid. By coating the PLGA nanospheres with CS, the loading efficiency of nucleic acid in the modified nanospheres increased significantly. The release profile of nucleic acid from PLGA nanospheres exhibited sustained release after initial burst. The PLGA nanospheres coated with chitosan reduced the initial burst of nucleic acid release and prolonged the drugs releasing at later stage. Chitosan coated PLGA nanosphere platform was established to encapsulate satisfactorily wide variety of nucleic acid for an acceptable gene delivery system.

Formulation of nanoparticle-eluting stents by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymeric nanoparticle-eluting stents in porcine coronary arteries.

OBJECTIVES The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. BACKGROUND Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. METHODS We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. RESULTS The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. CONCLUSIONS Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

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.

Chitosan-modified poly(d,l-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects

Abstract Chitosan (CS) surface-modified poly(d,l-lactide-co-glycolide) (PLGA) nanospheres (NS) for a siRNA delivery system were evaluated in vitro. siRNA-loaded PLGA NS were prepared by an emulsion solvent diffusion (ESD) method, and the physicochemical properties of NS were investigated. The level of targeted protein expression and siRNA uptake were examined in A549 cells. CS-modified PLGA NS exhibited much higher encapsulation efficiency than unmodified PLGA NS (plain-PLGA NS). CS-modified PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. siRNA uptake studies by observation with confocal leaser scanning microscopy (CLSM) indicated that siRNA-loaded CS-modified PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The efficiencies of different siRNA preparations were compared at the level of targeted protein expression. The gene-silencing efficiency of CS-modified PLGA NS was higher and more prolonged than those of plain-PLGA NS and naked siRNA. This result correlated with the CLSM studies, which may have been due to higher cellular uptake of CS-modified PLGA NS due to electrostatic interactions. It was concluded that CS-modified PLGA NS containing siRNA could provide an effective siRNA delivery system.

Current status and approaches to developing press-coated chronodelivery drug systems

The past several decades have seen the development of many controlled-release preparations featuring constant release rates to maintain drug concentrations in the human body, regardless of the patients physiological condition. However, long-term constant drug concentrations in the blood and tissue can cause problems such as resistance, tolerability, and drug side effects. People vary considerably in their physiological and biochemical conditions during any 24 h period, due to the circadian rhythm, and thus, the constant delivery of a drug into the body seems both unnecessary and undesirable. If the drug release profile mimics a living systems pulsatile hormone secretion, then it may improve drug efficacy, and reduce the toxicity of a specific drug administration schedule. Medication and treatments provided according to the bodys circadian rhythms will result in better outcomes. This may be provided by a chronopharmaceutical dosage regimen with pulsatile release that matches the circadian rhythm resulting from a disease state, so optimizing the therapeutic effect while minimizing side effects. The press coating technique is a simple and unique technology used to provide tablets with a programmable lag phase, followed by a fast, or rate-controlled, drug release after administration. The technique offers many advantages, and no special coating solvent or coating equipment is required for manufacturing this type of tablet. The present review article introduces chronopharmaceutical press-coated products from a patient physiological needs perspective. The contents of this article include biological rhythms and pulsatile hormone secretion in humans, the reasons for using pulsatile drug delivery for disease treatment, recent chronopharmaceutical preparations appearing on the market, updated compilation of all research articles and press-coated delivery techniques, factors affecting the performance and drug release characteristics of press-coated delivery systems, and recent challenges for the press coating technique. We also provide a brief overview of press-coating approaches intended for chronotherapy.

A novel method for measuring rigidity of submicron-size liposomes with atomic force microscopy.

There are many useful colloidal drug delivery systems that use liposomes. The rigidity of the carrier particle is one of the most important properties affecting drug delivery effectiveness, assessed by particle stability, release profile of encapsulated drug, and blood circulation time. However, it is difficult to evaluate the rigidity of such fine particles; so far, no useful methods have been reported. We demonstrate a unique method to evaluate the rigidity of liposomes using atomic force microscopy (AFM) and dynamic light scattering (DLS) in this report. We showed that the combination of two types of particle-size measurements, tapping mode AFM in buffer solution with another conventional method such as DLS, is useful for evaluating the rigidity of submicron-size particles such as liposomes.

Hydrophobic ion pairing of an insulin-sodium deoxycholate complex for oral delivery of insulin

Insulin was complexed with sodium deoxycholate to form an insulin-sodium deoxycholate complex (Ins-SD-Comp) using an hydrophobic ion pairing method in aqueous phase to enhance the liposolubility of insulin. In order to obtain the maximal complexation efficiency, the molar ratio of sodium deoxycholate to insulin was found. The zeta potential method was used to confirm the optimal ratio for formation of Ins-SD-Comp. The structural characteristics of Ins-SD-Comp were assessed using the Fourier transform infrared method. The apparent partition coefficient of insulin increased upon the formation of Ins-SD-Comp. Based on the preliminary study, Ins-SD-Comp was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles using an emulsion solvent diffusion method. The maximal encapsulation efficiency of Ins-SD-Comp into PLGA nanoparticles was 93.6% ± 2.81%, drug loading was about 4.8% ± 0.32%, and the mean diameter of the nanoparticles was 278 ± 13 nm. Biological activity and in vivo results revealed that the bioactivity of insulin was not destroyed during the preparation process. Ins-SD-Comp-loaded PLGA nanoparticles have the potential to reduce serum glucose levels and increase the oral bioavailability of insulin.

Engineering of poly(DL-lactic-co-glycolic acid) nanocomposite particles for dry powder inhalation dosage forms of insulin with the spray-fluidized bed granulating system

The objective of the present research is to develop a new particulate preparation process for dry powder inhalation by which peptide-loaded poly(DL-lactic-co-glycolic acid) nanospheres are granulated with sugar alcohol (mannitol, around 4 μm in diameter). A spray-drying fluidized bed granulator was used to form soft matrix composite granules. A nanosphere suspension can be readily obtained by dispersing the composite granules in distilled water. The composite granules showed inhalation performance superior to that of freeze-dried nanosphere powder. More than 50% of composite granules were delivered to the bronchioles and alveoli of a rat. The composite granules of insulin-loaded nanosphere have a strong and prolonged pharmacological effect compared to the inhalation of insulin solution.

Therapeutic Neovascularization by Nanotechnology-Mediated Cell-Selective Delivery of Pitavastatin Into the Vascular Endothelium

Objective—Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. Methods and Results—In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. Conclusions—NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.