Shuntaro Hosaka

Ocular inserts for controlled release of antibiotics

Ocular inserts impregnated with antibiotics (erythromycin and erythromycin estolate) which have sustained release characteristics were prepared, mainly for the purpose of trachoma therapy. In vitro experiments showed that the elution rate of a drug with low solubility in water (erythromycin estolate) is constant when the water content of the hydrogel insert is more than 30%. In the case of a drug with higher solubility (erythromycin), the elution rate depends on the water content. Some in vivo experiments using rabbit eyes were also reported.

Preparation of Microspheres of Poly(Glycidyl Methacrylate) and its Derivatives as Carriers for Immobilized Proteins

Microspheres of poly(glycidyl methacrylate) and its derivatives have been prepared. The diameter can be varied in the range from 0.5 to 5 micron. Proteins were immobilized on the microspheres by various processes. The maximum amount of covalently immobilized gamma-globulin on the microspheres was found to be about 6 mg/m2. It was shown that dyed microspheres with a diameter of 2-4 micron are applicable to immunological agglutination tests on a microplate as carrier particles substituting for red blood cells.

In vivo evaluation of ocular inserts of hydrogel impregnated with antibiotics fop trachoma therapy

Sustained release of antibiotics from hydrogel matrices in the eye was studied for the purpose of developing a new method for trachoma therapy. Copolymers of N-vinylpyrrolidone were moulded into an ocular insert and impregnated with erythromycin or erythromycin estolate. The antibiotic-hydrogel inserts completely suppressed the chlamydia trachomatis infection in the owl monkey eyes. The drug elution rates were a little lower in vivo than in vitro. By comparison of the drug elution rate in the human eye with that in the owl monkey eye, similar therapeutic effect is expected in the treatment of human trachoma.

Polymer Microbeads Bound to C3 Fragments for Detecting and Labeling Cells with C3 Receptors

Monodisperse microbeads (mbs) of a hydrophilic polymer with diameters of 4 microns and 0.2 micron were prepared. To these mbs, IgM or a sugar was bound covalently, and then the mbs were treated with fresh human or mouse serum. This led to the activation of complement and to the binding of C3 fragments to the mbs. C3 fragments fixed on mbs varied with the type of immobilized substance on the mbs and the incubation time of the mbs with fresh serum. Three kinds of mbs binding C3 fragments were prepared: mbs binding C3b, C3bi and C3d (C3hu-mbs); the ones binding C3bi and C3d (C3mu-mbs); and the ones binding only C3b (C3bhu-mbs). They formed rosettes with cells which had the corresponding receptor. When lymphocytes were incubated with C3hu- or C3bhu-mbs with a diameter of 0.2 micron, cap formation of each receptor was observed under a microscope. Once prepared, C3hu-, C3mu- and C3bhu-mbs were stable for more than 4 months.

Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres.

Phagocytosis by polymorphonuclear leukocytes (PMN) was determined by a newly developed technique based on measurement of liberation of a fluorescence substance from PMN phagosomes; 4‐methylumbelliferyl‐beta‐D‐glucuronide (4MUGL), which is a substrate of beta‐glucuronidase in lysosome, was conjugated with a microsphere, and 4‐methylumbelliferone (4MU) liberated from phagocytized 4MUGL‐microspheres was measured. The microspheres were composed of glyceryl‐methacrylate having a diameter of 2.0 μm. Liberating activity of six kinds of 4MUGL‐microspheres containing various amounts of amino and carboxyl groups was compared. Among these six kinds of 4MUGL‐microspheres, four kinds showed activity similar to that of morphological phagocytosis. These four kinds of 4MUGL‐microspheres liberated 4MU into the extracellular fluid from PMN during phagocytosis. Furthermore, they were recognized as a substrate of purified beta‐glucuronidase. 4MUGL‐MS610 showed the highest liberating activity among the four kinds of microspheres. Optimal conditions for phagocytosis by PMN were determined using 4MUGL‐MS610. Total liberation of 4MU from the microspheres increased almost linearly with incubation time with PMN from 0 to 60 min and was linear with 4MUGL‐MS in concentrations up to 4 × 108 microspheres/ml. This liberation was parallel to phagocytosis in a dose‐dependent fashion. During 10‐min incubation 20.4% of 4MU was liberated from 4MUGL‐microspheres with phagocytosis. Seventy‐five percent of the liberated 4MU was distributed in the extracellular fluid. 4MU distributed in the extracellular fluid was not attributable to hydrolysis of unphagocytized microspheres by beta‐glucuronidase extracellularly leaked from PMN by phagocytosis. Also phagocytized 4MUGL‐MS610 by PMN was observed by scanning electron microscopy.

Direct measurement of phagolysosomal esterase activity

A new method of directly measuring esterase activity within phagolysosomes has been developed. Decanoyl fluorescein- binding microspheres were prepared and phagocytosed by human peripheral neutrophils. Within phagolysosomes lysosomal esterase hydrolyzed decanoyl fluorescein on the microspheres, causing the conversion of decanoyl fluorescein- binding microspheres (non-fluorescent) into fluorescein- binding microspheres (fluorescent). The activity of phagolysosomal esterase in intact neutrophils was assayed by the measurement of the fluorescence intensity without rupturing cells. By use of a flow cytometer, esterase activity within phagolysosomes in single cells was measured.

The Application of Luminol-Bound Microspheres for the Quantitative Analysis of Toxic Oxygen within Phagosomes

Hydrophilic microspheres, with a diameter of about 2μm, composed of glyceryl methacrylate and methacrylic acid were prepared. The microspheres could be stably stored in dispersed state and demonstrated little non-specific adherance to cell membranes. Luminol-bound microspheres, prepared by covalently binding luminol to microspheres, were phagocytosed by phagocytic cells without opsonization. Luminol bound to the microspheres was oxidized by toxic oxygen within phagosomes resulting in the generation of chem iluminescence. The chemiluminescence elicited by luminol-bound micro spheres was an effective method of measuring the candidacidal activity of the cells. The difference in phagosomal toxic oxygen versus extracellular toxic oxy gen was examined.

Direct Assay of Phagolysosomal Hydrolase by Fluorogenic Substrate-Binding Microspheres

A new method of directly measuring phagolysosomal hydrolase has been developed. 4-methylumbelliferyl-β-D-glucuronide(4MUGL)-binding microspheres and decanoyl fluorescein(DF)-binding microspheres were prepared. The microspheres were phagocytosed by human peripheral neutrophils, then within phagolysosomes immobilized 4MUGL and immobilized DF were hydro-lyzed by β-glucuronidase and lipase respectively. These reactions were confirmed by the facts that 4MU, which is fluorescent, was released from 4MUGL-binding microspheres and DF binding microspheres turned fluorescent, when the microspheres were phagocytosed. Each enzyme activity within phalysosomes was examined by measuring the increase of fluorescence intensity without cell rupture.