Nobuyuki Morimoto

Hybrid hyaluronan hydrogel encapsulating nanogel as a protein nanocarrier: New system for sustained delivery of protein with a chaperone-like function

Novel hybrid hyaluronan (HA) hydrogel encapsulating nanogels was designed for sustained delivery of protein. HA modified with 2-aminoethyl methacrylate was cross-linked via Michael addition in the presence of cholesteryl group-bearing pullulan (CHP) nanogels. The nanogels were physically entrapped and well dispersed in a three-dimensional network of chemically cross-linked HA (HA gel). Therapeutic peptides and proteins, such as glucagon-like peptide-1, insulin and erythropoietin, were spontaneously trapped in the CHP nanogels in the HA gel just by immersing hybrid hydrogels into the drug solutions. CHP/protein complex nanogels were released from the hybrid hydrogels in a sustained manner both in vitro and in vivo. The release was controlled by the cross-linking density and the degradability of the HA gel, modulated by the initial gelation condition. The synergy between the CHP nanogel as a drug reservoir and the HA gel as a nanogel-releasing matrix of the hybrid hydrogel system simultaneously achieved both simple drug loading and controlled release with no denaturation of the protein drugs. This is a new method of fabricating biodegradable controlled release matrix with molecular chaperone-like activity for therapeutic proteins.

Physical properties and blood compatibility of surface-modified segmented polyurethane by semi-interpenetrating polymer networks with a phospholipid polymer.

Segmented polyurethanes, (SPU)s, are widely used in the biomedical fields because of their excellent mechanical property. However, when blood is in contact with the SPU, non-specific biofouling on the SPU occurs which reduces its mechanical property. To obtain novel blood compatible elastomers, the surface of the SPU was modified with 2-methacryloyloxyethyl phosphorylcholine (MPC) by forming a semi-interpenetrating polymer network (semi-IPN). The SPU film modified by MPC polymer with the semi-IPN (MS-IPN film) was prepared by visible light irradiation of the SPU film in which the monomers were diffused. X-ray photoelectron spectroscopy confirmed that the MPC units were exposed on the MS-IPN film surface. The mechanical properties of the MS-IPN film characterized by tensile testing were similar to those of the SPU film. Platelet adhesion on MS-IPN films was also investigated before and after stress loading to determine the effects of the surface modification on the blood compatibility. Many platelets did adhere on the SPU film before and after stress loading. On the other hand, the MS-IPN film prevented platelet adhesion even after repeated stress loading.

Semi-interpenetrating polymer networks composed of biocompatible phospholipid polymer and segmented polyurethane.

2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers, which have excellent biocompatibility, have been receiving increasing attention in biomedical and bioengineering fields; however, the mechanical strength of the hydrated MPC polymers is not sufficient for use in these fields as a bulk material. Therefore, we hypothesized that a novel material might be realized by reinforcing the MPC polymer network with segmented polyurethane (SPU). Semi-interpenetrating polymer networks (IPNs) composed of crosslinked MPC polymer and SPU were prepared. The mechanical properties of the IPN membrane were significantly improved compared with those of the MPC polymer membrane. Three-dimensional polymer networks of the MPC polymer in the IPNs were observed after solvent extraction of SPU. An X-ray photoelectron spectrum analysis revealed that the MPC units were exposed on the IPN surface. When the IPN was alternately soaked in water and ethanol, the swelling ratio was found to be completely reversible and no disintegration of the network structure was observed. The permeation coefficient of 1, 4-di(2-hydroxyethoxy)benzene through the IPN membrane was 1.11 x 10(-7) cm(-2)s(-1). The amount of adsorbed protein and the number of adherent platelets on the IPN membrane were effectively reduced compared with those on SPU. We concluded that IPNs composed of the MPC polymer and SPU are a new bulk biomaterial, which possesses both blood compatibility and good mechanical properties.

Nanogel‐based delivery system enhances PGE2 effects on bone formation

Recovery of bone loss is one of the active research issues in bone medicine due to the need for efficient measures for bone gain. We examined here a novel drug delivery system using a nanogel of cholesterol‐bearing pullulan (CHP) in combination with prostaglandin E2 (PGE2). PGE2 or PGE2/CHP, vehicle (saline containing 0.06% ethanol and 0.02% Tween 80) or CHP were injected on to the calvariae of mice once every day for 5 days per week for 4 weeks. Low dosage of PGE2 (0.6 µg) alone or CHP alone did not induce new bone formation in this system. In contrast, PGE2 (0.6 µg)/CHP induced new bone formation. Bone formation activities of PGE2 was enhanced by CHP nanogels only at the site of injection (calvaria) but not in the distant sites of the skeleton, showing that PGE2/CHP could avoid systemic effects. In spite of the fact that previously reported animal models of bone formation by PGE2 were associated with loss of body weight, bone formation based on PGE2/CHP did not associate with loss of body weight. Furthermore, only a single application of PGE2 in combination with nanogel cross‐linking hydrogel sphere (PGE2/CHP‐PEO) induced new bone formation. Thus, nanogel‐based delivery system is an efficient delivery system of bone anabolic agent, PGE2. J. Cell. Biochem. 101:1063–1070, 2007.

Composite nanomaterials by self-assembly and controlled crystallization of poly(2-isopropyl-2-oxazoline)-grafted polysaccharides

We report here new composite nanomaterials by self-assembly and control of crystallization of thermoresponsive poly(2-isopropyl-2-oxazoline) (PIPOZ)-grafted pullulan.

A nonthrombogenic gas-permeable membrane composed of a phospholipid polymer skin film adhered to a polyethylene porous membrane

Polymer membranes are widely used in biomedical applications such as hemodialysis, membrane oxygenator, etc. When the membranes come in contact with blood or body fluids, protein adsorption and cell adhesion occur rapidly. Nonspecific protein adsorption and cell adhesion on the membranes induce not only various bio-rejections but also a decrease in their performance. We hypothesized that a blood compatible gas-permeable membrane could be prepared from polyethylene (PE) porous membranes modified with phospholipid polymers. In this study, poly[(2-methacryloyloxyethyl phosphorylcholine) (MPC)-co-dodecyl methacrylate] (PMD) skin film adhered to a PE porous membrane (PMD/PE porous membrane) was prepared. Elution of PMD was not detected meaning that the PMD film did not detach from the PE porous membrane even after soaking in water for more than 6 months. The permeation coefficient of oxygen gas through the PE membrane with the adhered PMD containing more than 0.20 mole fraction of the MPC unit, was the same as that of the original PE porous membrane. The PMD surface effectively reduced biofouling. We concluded that the PMD/PE porous membrane is useful as a novel membrane oxygenator due to its excellent gas-permeability and blood compatibility.

Polysaccharide nanogel delivery of a TNF-α and RANKL antagonist peptide allows systemic prevention of bone loss

We report here a nanogel-mediated peptide drug delivery system. Low stability is a major drawback towards clinical application of peptide drugs. The W9-peptide, a TNF-alpha and RANKL antagonist, was used as a model for testing the feasibility of cholesterol-bearing pullulan (CHP)-nanogel as the drug delivery system. We found CHP-nanogel could form complex with the W9-peptide and prevents its aggregation in vitro. Murine bone resorption model using low dietary calcium was used to investigate the in vivo effect. Two-time-injection of 24 mg/kg W9-peptide per day with or without CHP-nanogel was given for 7 days. Thereafter, radiological, and histological assessments were performed. The injections of the W9-peptide (24 mg/kg) with CHP-nanogel prevented the reduction in bone mineral density whereas the same dose without CHP-nanogel could not show any inhibitory effect. Histomorphometric analysis of tibiae showed significant decrease of osteoclast number and surface in CHP-W9 complex treated group and the levels of urinary deoxypyridinoline reflected these decrease of bone resorption parameters. Taken together these data shows that CHP-nanogel worked as a suitable carrier for the W9-peptide and it prevented aggregation and increased the stability of the W9-peptide. This study reveals the feasibility of CHP-nanogel-mediated peptide delivery in preventing bone resorption in vivo.

Glucose sensor using a phospholipid polymer-based enzyme immobilization method

An electroenzymatic glucose sensor based on a simple enzyme immobilization technique was constructed and tested. The glucose sensor measures glucose concentrations as changes of oxygen concentrations induced by enzymatic reactions. The immobilizing procedure was developed with the purpose of producing wearable biosensors for clinical use. Two types of biocompatible polymers, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (PMD) and MPC copolymerized with 2-ethylhexyl methacrylat, were compared as a sensitive membrane of biosensors. The PMD enzyme membrane had a better response time. Linearity, reproducibility, effect of the concentrations of immobilized enzyme and drifts of sensor characteristics in long-term tests were also investigated. The linear characteristics were confirmed with glucose concentration from 0.01 to 2.00xa0mmol/l, with a coefficient of determination of 0.9999. The average output current for 1xa0mmol/l and the standard deviation were 0.992 and 0.0283xa0μA. Significant changes in the sensors characteristics were not observed for 2xa0weeks when it was kept in a refrigerator at 4 °C. Because of the simple procedure, the enzyme immobilization method is not only useful for wearable devices but also other devices such as micro total analysis systems.

Self-assembled nanogel of hydrophobized dendritic dextrin for protein delivery.

Highly branched cyclic dextrin derivatives (CH-CDex) that are partly substituted with cholesterol groups have been synthesized. The CH-CDex forms monodisperse and stable nanogels with a hydrodynamic radii of approximately 10 nm by the self-assembly of 4-6 CH-CDex macromolecules in water. The CH-CDex nanogels spontaneously trap 10-16 molecules of fluorescein isothiocyanate-labeled insulin (FITC-Ins). The complex shows high colloidal stability: no dissociation of trapped insulin is observed after at least 1 month in phosphate buffer (0.1 M, pH 8.0). In the presence of bovine serum albumin (BSA, 50 mg . mL(-1)), which is a model blood system, the FITC-Ins trapped in the nanogels is continuously released ( approximately 20% at 12 h) without burst release. The high-density nanogel structure derived from the highly branched CDex significantly affects the stability of the nanogel-protein complex.

Effects of cholesterol-bearing pullulan (CHP)-nanogels in combination with prostaglandin E1 on wound healing

The cholesterol-bearing pullulan (CHP)-nanogels are able to trap hydrophobic drugs or proteins inside the nanogels, which is potential in application to drug delivery system and tissue engineering. On the other hand, prostaglandin E1 (PGE1) plays important roles in wound healing and PGE1 ointment has been clinically used to treat chronic skin ulcers and wounds. The purpose of this study is to evaluate effects of CHP nanogels in combination with prostaglandin E1 on wound healing in full thickness skin defect model. A square skin defect (1 x 1 cm(2)) of full thickness was created on the dorsal of Wistar rats. The wound was treated with CHP nanogels without PGE1 (CHP group) or CHP nanogels containing with PGE1 (CHP/PGE1 group) or PGE1 ointment (PGE1 ointment group). In both CHP/PGE1 and PGE1 ointment groups, approximately 6 microg of PGE1 was applied to each wound. In the control group, the wound was untreated. The wound was evaluated in measuring wound area and histologically. In CHP/PGE1 group, the rate of wound size reduction was significantly higher than the ones of other groups. Histologically, CHP/PGE1 promoted neoepithelialization, neovascularization, and wound closure compared to the other treatments. These results suggest that CHP in combination with PGE1 can promote wound healing, which confirms the efficiency of CHP nanogels-based drug delivery system.