Ryusuke Nakaoka

Prolongation of the serum half-life period of superoxide dismutase by poly(ethylene glycol) modification

Abstract Superoxide dismutase (SOD) was chemically modified using poly(ethylene glycol) (PEG) with different molecular weights to prepare PEG–SOD conjugates with different extents of modification. The body distribution of the conjugates intravenously injected to mice was investigated to assess the influence of modification on the serum half-life period of SOD. The SOD modification with PEG was effective in lowering the elimination rate of SOD from the blood circulation without any change in the distribution pattern of organs other than the kidney. The molecular weight of PEG used for modification and the modification extent have a minimum effect on the half-life of the SOD. The half-life of the SOD and its PEG conjugates have a similar dependency on the apparent molecular weight as the PEG molecules. This indicates that the half-life of SOD and the PEG conjugates are mainly determined by their molecular size.

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO3)2 and a mixture of (NH4)2HPO4 and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO4 sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.

Size effect on the antibody production induced by biodegradable microspheres containing antigen

Poly(L-lactic acid) (PLLA) microspheres containing a model antigen, ovalbumin (OVA), were prepared by the evaporation method using double emulsion, and fractionated into different sizes by counterflow elutriation. Following the intraperitoneal (i.p.) and subcutaneous (s.c.) injection of the microspheres to mice, the titer of anti-OVA antibody in the serum was measured to assess the size effect on the profile of antibody production. OVA was released from the microspheres for 80 days, irrespective of the microsphere size. In both the s.c. and i.p. immunization, the serum level of anti-OVA IgG antibody in the mice induced by the microspheres containing OVA was higher than that of free OVA when compared at the same dose. The serum level of antibody in the mice i.p. injected with the microspheres tended to increase with the decreasing size. On the other hand, in the s.c. immunization, the microsphere size had little influence on the antibody production. It is possible that the injected microspheres tend to aggregate in the s.c. tissue, disappearing the size effect on the antibody production. Since the amount of microspheres injected increases with the decreasing size when their OVA loading is fixed, the increase in the amount will promote the interaction with immune cells, resulting in an enhanced antibody production. The cell interaction with the microspheres in the peritoneal cavity seems to be influenced by their size to a greater extent than in the s.c. tissue, probably because of their more frequent interaction with immune cells.

Potentiality of gelatin microsphere as immunological adjuvant

This paper describes a new attempt to enhance the production of antibody by delivery of an antigen to phagocytic antigen-presenting cells (e.g. macrophages) using gelatin microspheres. A model protein antigen, human gamma globulin (HGG), was incorporated into microspheres composed of gelatin which have an opsonic ability for macrophage phagocytosis. Subcutaneous injection of the microspheres induced the production of HGG-specific IgG antibody in the mouse serum to a great extent compared with that of HGG in soluble form or in Freunds incomplete adjuvant (FIA) form. There was an optimal concentration of cross-linking agent (glutaraldehyde) for the highest production of antibody. When gelatin microspheres were cross-linked at lower concentrations of glutaraldehyde, they were more extensively swollen in an aqueous solution, leading to an increase in the size of hydrated microspheres because of their lower cross-linking densities. The increased size of microspheres caused a decrease in their macrophage phagocytosis, whereas the release rate of HGG from the microspheres increased as the concentration of cross-linking agent became low. The balance of the two factors, the microsphere susceptibility to macrophage phagocytosis and the rate of HGG release, seemed to affect the efficacy of gelatin microspheres to enhance the antibody production. In addition, incorporation of HGG into gelatin microspheres enhanced the delayed-type hypersensitivity reaction. Moreover, the microspheres developed a strong secondary response in comparison with FIA. The gelatin microspheres induced a minimal inflammatory response around the injection site in contrast to FIA. These findings demonstrate that the gelatin microsphere is promising as an adjuvant to enhance both humoral and cellular immune responses to antigen.

Adjuvant effect of biodegradable poly(DL-lactic acid) granules capable for antigen release following intraperitoneal injection

Ovalbumin (OVA)-containing poly(DL-lactic acid) (PDLLA) granules were prepared with different conditions. Following the intraperitoneal (i.p.) immunization of mice with the granules containing OVA, production of anti-OVA IgG antibody in the mouse serum was investigated. The i.p. injection of the granules induced a strong antibody production compared with that of free OVA, irrespective of the amount of OVA released for initial a few weeks and the period of OVA release. The serum level of IgG antibody induced by the granules was retained at a high level over 16 weeks although the period of OVA release and the amount of OVA released initially were different from each other. The initial OVA release for a few weeks was essential to induce the enhanced antibody production. Comparison of mice immunization by granules with different OVA loadings but at a similar dose revealed that antibody level was higher for the granules with lower loading than for those with the higher loading. However, when the granules were injected after encapsulation into a poly(vinyl alcohol) (PVA) hydrogel tube, the difference in their antibody level became insignificant. Because PVA encapsulation did not affect the OVA release profile, this finding indicates that the injection amount of the granules seems to have influenced the antibody production. We conclude that the release profile of OVA is not always a key factor to enhance the antibody production of OVA-containing granules so far as the initial OVA controlled release is achieved.

Safety evaluation of surgical materials by cytotoxicity testing

The cytotoxicity of three kinds of commercially available absorbable hemostats [oxidized cellulose (Surgicel, gauze and cotton types), microfibrillar collagen (Avitene), and cotton-type collagen (Integran)] and one adhesion barrier [sodium hyaluronate and carboxymethyl-cellulose membrane (Seprafilm)] were comparatively assessed by a colony assay using V79 cells and a minimum essential medium (MEM) elution assay in combination with a neutral red assay using L929 cells. Strong cytotoxicity was detected for Surgicel by both the MEM elution assay and the colony assay. For Avitene, both methods revealed weak cytotoxicity. For Seprafilm, no cytotoxicity was detected by the MEM elution assay, while a moderate degree of cytotoxicity was observed in the colony assay. For Integran cytotoxicity was not detected by either the MEM elution or the colony assay. The results of the different methods showed some inconsistency in terms of the degree of cytotoxicity of the materials. It is proposed that the combination of two or more sensitive cytotoxicity testing methods for the evaluation of biomaterials is necessary to avoid false-negative results for biomaterials at the preclinical stage. Furthermore, investigation of the correlation between the cytotoxicity and the extraction period of the surgical materials is helpful for predicting the effect of prolonged in vivo use of biomaterials on surrounding cells, tissues, and organs.

Effects of surface chemistry prepared by self-assembled monolayers on osteoblast behavior

A surface of biomaterials is known to affect the behavior of cells after their adhesion on the surface, indicating that surface characteristics of biomaterials play an important role in cell adhesion, proliferation, and differentiation. To assess the effects of functional groups on biomaterial surface, normal human osteoblasts (NHOsts) were cultured on surfaces coated with self-assembled monolayers (SAMs) containing various functional groups, and the adhesion, proliferation, differentiation, and gap junctional intercellular communication (GJIC) of the NHOsts were investigated. In the case of SAM with terminal methyl groups (hydrophobic surface), NHOst adhesion and proliferation was less prevalent. In contrast, NHOsts were adhered well on SAMs with hydroxyl, carboxyl, amino, phosphate, and sulfate group, which are relatively hydrophilic, their proliferation and differentiation level were dependent on the type of functional groups. Especially, when they were cultured on either SAMs with phosphate or sulfate group, both their alkaline phosphate activity and the calcium deposition by them were enhanced more than those cultured on a collagen-coated dish. More interestingly, GJIC of NHOsts, which has been reported to play a role in cell differentiation as well as homeostasis of cells, were not significantly different among the SAM surfaces tested. These suggest that a specific functional group on a material surface can regulate NHOst adhesion, proliferation, and differentiation via cell-functional group interaction without influencing their homeostasis.

Studies on tumor-promoting activity of polyethylene: Inhibitory activity of metabolic cooperation on polyethylene surfaces is markedly decreased by surface modification with collagen but not with RGDS peptide

Tumor promotion activity of polyethylene (PE) was estimated in terms of the inhibitory potentials on the gap-junctional intercellular communication using the metabolic cooperation assay. The gap-junctional intercellular communication of test cells was inhibited on the PE film, but this inhibitory activity was markedly decreased when the surface of the PE film was immobilized with collagen. These results suggest that the in vivo tumor promotion activity of the untreated PE may be stronger than that of collagen-immobilized PE. On the other hand, surface modification with RGDS peptide, which is well known as the sequence of cell attachment domain in extracellular matrix proteins, did not reduce the promotion activity of PE film. In addition, neither modification with bovine serum albumin nor RGES peptide reduced the activity of PE film. These findings suggest that reduction of the inhibitory activity on gap-junctional intercellular communication by collagen immobilization is not simply due to improved cell adherence via the RGDS sequence but to some cell-cell recognition via collagen molecules essential for the gap-junctional intercellular communication.

Enhanced antibody production through sustained antigen release from biodegradable granules

Abstract A proteinaceous model antigen, ovalbumin (OVA), was incorporated into poly(DL-lactic acid) (PDLLA) granules for sustained release of OVA over about 5 weeks without any large initial burst. When the granules were injected into the mouse peritoneal cavity, the production of anti-OVA IgG antibody in the serum was significantly enhanced and persisted for a long period compared with that induced by injection of OVA in the water-soluble form. Simple mixing of the PDLLA granules with free OVA had no enhancing effect on the antibody response, indicating that OVA incorporation in the granules is essential to enhance IgG production. The secondary response to antibody production was greater for mice given the primary immunization with the OVA-loaded granules than with free OVA. A body distribution study of OVA demonstrated that incorporation of OVA into PDLLA granules reduced its translocation to the blood circulation, leading to prolonged accumulation of OVA in the peritoneal cavity. Macrophages cocultivated with PDLLA granules containing OVA produced a larger amount of interleukin 1 than those cultured with other agents, suggesting that the PDLLA granules effectively activate the cell function for antibody production. It was concluded that the biodegradable granules were useful as a release carrier of antigens to enhance the antibody production.

Antibody production by administration of biodegradable granules incorporating antigen through different injection routes

Abstract Biodegradable poly(DL-lactic acid) granules containing ovalbumin (OVA), a model antigen, were administered by intraperitoneal, subcutaneous, and intramuscular injection to mice to assess the effect of their injection route on the production of OVA-specific antibody in serum. OVA was released in vitro from the OVA-loaded granules over 35 days without any initial burst. A single injection of the granules enhanced the serum level of anti-OVA IgG antibody at an early stage to a great extent compared with that of OVA injection in the water-soluble, free form, although the titer level induced by their intraperitoneal injection was higher than that of other injection routes. The time course of antibody production induced by the granules was independent of the injection route and the enhanced antibody production lasted over a time period of 22 weeks, which was much longer than that of free OVA injection. A body distribution study demonstrated that the injection of OVA incorporated into the granules reduced the serum concentration compared with that of free OVA injection, irrespective of the injection route. It is likely that the granules remained as a depot to gradually release OVA around their injection site, leading to a high adjuvant effect for the enhanced antibody production. In addition, a strong secondary response was observed for every mouse given the primary immunization of the OVA-loaded granules compared with that of free OVA injection, irrespective of their injection route. It was concluded that the granule is a promising immunological adjuvant not only to enhance the antibody production but also to prolong the production.