Takeshi Moritera

In vitro phagocytosis of polylactide microspheres by retinal pigment epithelial cells and intracellular drug release

We investigated phagocytosis of biodegradable microspheres containing a drug by retinal pigment epithelial (RPE) cells and drug release within the cells to evaluate the potential usefulness of microspheres for intracellular drug delivery. The biodegradable polymers used were L-lactic acid, and DL-lactic acid with different molecular weights or the copolymers of different monomer compositions. The microspheres containing a non-bioactive fluorescent dye (rhodamine 6GX) as a model drug, were prepared by a solvent evaporation method. The in vitro release of the dye from the microspheres was examined. Phagocytosis of the microspheres by RPE cells was conducted to evaluate the extent of phagocytosis by phase-contrast microscopy and transmission electron microscopy. The RPE cells ingesting the microspheres at different stages were examined by fluorescent microscopy to estimate the intracellular release of the dye. The dye was released with time from every microsphere and the release was controlled by changing the type of polymers constituting microspheres. The microspheres containing the dye were phagocytosed by RPE cells and the dye was released intracellularly with time. The present study indicates that the drug incorporated in the microspheres was delivered into RPE cells by way of phagocytosis and released within the cells. It is concluded that this microsphere system is a promising delivery form capable of drug targeting to RPE cells.

Feasibility of drug targeting to the retinal pigment epithelium with biodegradable microspheres

There are several systems of delivering drugs to cells with phagocytic activity. We studied the possibility of targeting drugs to retinal pigment epithelial (RPE) cells with the use of surface-modified microspheres. A fluorescent dye, 1,4-bis[2-(5-phenyloxazolyl)]-benzene (POPOP), was incorporated into microspheres of poly(lactic acid) for use as a marker to evaluate drug delivery. Phagocytosis of the microspheres, with or without gelatin precoating, was carried out at 37 degrees C and 4 degrees C. The cell-incorporated fluorescence of POPOP was measured, and scanning electron microscopy was used to confirm phagocytosis. At 4 degrees C, no uptake of POPOP was noted; however at 37 degrees C, cell-associated fluorescence was observed to increase for up to 24 hr. In comparison with bare microspheres, gelatin precoating significantly enhanced phagocytosis (P < 0.001) at the same incubation times. These results suggested that drug delivery to RPE cells may be feasible by means of surface-modified polymer microspheres.

Mass Cultivation of Human Retinal Pigment Epithelial Cells with Microcarrier

Microcarrier cell culture permits mass cultivation of anchorage-dependent cells. In this study, mass cultivation of human retinal pigment epithelial (RPE) cells was studied using Cytodex 3 (Pharmacia) as a microcarrier. Human RPE cells were established from aborted fetuses and cultured in Dulbeccos modified Eagles Medium (DMEM). After the 3rd-5th passages, RPE cells were suspended in 50 ml of DMEM in a spinner flask at a density of 2 x 10(5)/ml, and Cytodex 3 was added to the spinner flask at a bead density of 10 mg/ml. Cultures were maintained at 20-50 rpm (final speed) on a magnetic stirrer, and DMEM was added up to 100 ml. Fifty milliliters of DMEM were decanted and replaced with fresh DMEM every 2 days. After 1 week, a cell density of 10(6)/ml DMEM was obtained. Phase contrast microscopy showed bridging formation between microcarriers, which suggests tight cell adhesion. Microcarrier cell culture has a variety of advantages which include greater cell production, use of less medium and less risk of contamination compared to the conventional monolayer culture technique, and it also allows passaging without using proteases. Using this culture system, greater possibilities for wider application of new cell cultures can be expected.

Evaluation of dacryocystorhinostomy using optical coherence tomography and rebamipide ophthalmic suspension.

Purpose To evaluate the surgical outcome of dacryocystorhinostomy (DCR) by measuring the tear meniscus, using optical coherence tomography and rebamipide ophthalmic suspension. Methods Patients with nasolacrimal obstruction and chronic dacryocystitis who were scheduled for an endonasal DCR underwent tear meniscus examinations before and 2 months after surgery. Vertical scans of the inferior menisci were performed before and at 1, 3, 5, 7, and 10 minutes after the instillation of rebamipide ophthalmic suspension. The tear menisci areas were measured with imaging software. Ten young adults without epiphora formed the control group. Results Anatomical success was achieved on 22 sides of 21 patients. The patients’ postoperative tear menisci were significantly smaller than the preoperative menisci at all points during the test, and the response to volume loading in the postoperative patients was corrected to nearly that of the young, healthy adults. Nevertheless, the postoperative meniscus area tended to be larger than that of the young adults at all points. Conclusion The reduced tear meniscus area after DCR reflected the success of the surgical procedure. However, incomplete recovery of the meniscus after the test might suggest a residual disorder of the lacrimal drainage system after DCR.

Breakdown of the blood-retinal barrier after radiofrequency-induced ocular hyperthermia.

To study the possible application of hyperthermia in the treatment of proliferative vitreoretinopathy (PVR), we induced hyperthermia in 15 normal rabbit eyes by a radiofrequency capacitive heating device. The retinal surface of each eye was warmed to a presumed temperature of 41 or 43 degrees C for 30 min. The thermal effect on the blood-retinal barrier (BRB) was evaluated before and 2 h after the hyperthermic regimen by means of vitreous fluorophotometry. Heat treatment to 43 degrees C at the retinal surface for 30 min increased the fluorescein leakage compared with the 41 degrees C treatment. This finding suggests that retinal hyperthermia at 43 degrees C for 30 min may cause breakdown of the BRB.