Fluorescence investigation of the retinal delivery of hydrophilic compounds via liposomal eyedrops.

We examined the feasibility of using submicron-sized liposomes (ssLips) for retinal delivery of hydrophilic compounds, which would also have a wide range of applications. To evaluate the uptake into conjunctival cell line and the intraocular behavior of hydrophilic compound-containing ssLips after eyedrop application, fluorometric investigation was carried out by using a hydrophilic fluorescence probe, 5(6)-carboxyfluorescein (CF). A relatively high amount of CF (>50%) could be incorporated into an internal phase of ssLips by a calcium acetate gradient method. CF being entrapped within the liposomes markedly enhanced both the uptake of CF into conjunctival cells and CF-oriented emission in the retina in mice after eyedrop application, while the free CF did not clear delivery efficiency in both in vitro and in vivo study. In addition, the cellular uptake and luminescence intensity in the retina were higher when a ssLip formulation composed of L-α-distearoyl phosphatidylcholine was applied than when a ssLip formulation composed of egg phosphatidylcholine was applied. Consequently, ssLips of appropriate composition were considered to have good potential to carry hydrophilic compounds into the retina.

Topical Diclofenac-Loaded Liposomes Ameliorate Laser-Induced Choroidal Neovascularization in Mice and Non-Human Primates.

This study aimed to evaluate the effect of liposomes loaded with diclofenac, a potent cyclooxygenase (COX)-1 and COX-2 inhibitor, on laser-induced choroidal neovascularization (CNV) in mice and non-human primates (common marmosets). CNV was induced by laser irradiation on the unilateral or bilateral eye of each mouse or common marmoset, respectively, under anesthesia. The CNV was visualized using fluorescence labeling with intravenous injection of fluoresceinconjugated dextran (molecular weight = 2,000 kDa), and quantified in the retinal pigment epithelia (RPE)-choroidal flatmounts. Diclofenac-loaded liposome or diclofenac ophthalmic solution was instillated to the eye surface daily for 14 days and 21 days in mice and common marmosets, respectively. In the mouse CNV model, 0.1% diclofenac-loaded liposome eye drops administered four times a day (q.i.d.) significantly reduced CNV formation in the RPE-choroidal flatmounts compared with those in empty liposome eye drops. Diclofenac-loaded liposome (0.1%) eye drops, administered once a day (s.i.d.), twice a day (b.i.d.), and three times a day (t.i.d.), also reduced CNV formation in a frequency-dependent manner. Furthermore, diclofenac-loaded liposome (0.03% and 0.1%) eye drops administered t.i.d. reduced CNV formation in a dose-dependent manner, significantly so at 0.1%. In the common marmoset CNV model, late hyperfluorescence and leakage by fluorescein angiograms was observed within or beyond the lesion borders at 17 days after laser irradiation, and diclofenac-loaded liposome eye drops (0.1% t.i.d.) tended to attenuate the late hyperfluorescence and leakage. Diclofenac-loaded liposomes had significantly reduced CNV formation in the RPE- choroidal flatmounts at 21 days after laser irradiation. In conclusion, diclofenac-loaded liposome eye drops enhance penetration to the RPE-choroid, and reduce the CNV formation. These results suggest that a drug-loaded liposome is a useful tool for drug delivery into the posterior segment of the eye.

Edaravone-loaded liposome eyedrops protect against light-induced retinal damage in mice.

PURPOSE: To investigate the pharmacologic effects of eyedrops containing liposomes loaded with edaravone (3-methyl-1-phenyl-2-pyrazolin-5-1) against light-induced retinal damage in mice. METHODS: Edaravone was incorporated into submicron-sized liposomes (ssLips) by the calcium acetate gradient method. Retinal damage in mice was induced in dark-adapted mice by exposure to white light at 8000 lux for 3 hours. Edaravone-loaded ssLips were dropped into the left eye just before and after light exposure and then three times daily for 5 days after light exposure. Retinal damage was evaluated by recording the scotopic electroretinogram (ERG) and measuring the thickness of the outer nuclear layer (ONL) and by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. The scavenging capacity of reactive oxygen species (ROS) of edaravone-loaded ssLips was determined using a murine cone photoreceptor cell line (661W). The human corneal and conjunctival cell lines were exposed to edaravone-loaded ssLips to determine cytotoxicity. RESULTS: Eyedrop administration of edaravone-loaded ssLips significantly prevented both the decrease in a- and b-wave amplitudes of flash ERG and the shrinkage of the ONL compared with the control group (treated with empty ssLips) after 5 days of light exposure. The edaravone-loaded ssLips prevented the increase in the numbers of TUNEL-positive cells after 48 hours of light exposure. This marked protection was not found in the group treated with free edaravone. Edaravone-loaded ssLips showed a stronger inhibition of in vitro light-induced ROS production and cell death than did free edaravone. The ssLips showed little cytotoxicity toward ocular cell lines. CONCLUSIONS: Edaravone-loaded ssLips protected against light-induced retinal dysfunction by eyedrop administration. Liposomal eyedrops may become one of the therapeutic candidates for drug delivery to posterior eye segments.

Edaravone-loaded liposomes for retinal protection against oxidative stress-induced retinal damage.

To optimize the retinal protective effects of submicron-sized liposomes (ssLips) containing edaravone for intravitreal administration, we investigated the effects of liposomal formulation on the pharmacological effects. Loading of edaravone into ssLips of around 50% entrapment efficiency was achieved by a calcium acetate gradient method. The in vitro radical-scavenging capacity of edaravone-loaded ssLip based on egg phosphatidylcholine (EPC-ssLip) and L-α-distearoyl phosphatidylcholine (DSPC-ssLip) was determined in RGC-5, a neuronal precursor cell line that can be differentiated to resemble retinal ganglion cells. Edaravone-loaded EPC-ssLip scavenged intracellular H(2)O(2) radical more strongly than DSPC-ssLip, although there was only a small difference in cellular uptake of edaravone into RGC-5. An in vivo N-methyl-D-aspartate (NMDA)-induced disease model was used to investigate the retinal protective effects in mice. The edaravone-loaded EPC-ssLip significantly reduced NMDA-induced ganglion cell layer (GCL) cell death compared with free edaravone. Such protective effect was small in the case of DSPC-ssLip. These results may be related to the release profile of the edaravone from ssLips across the inner layers of the retina including GCL, indicating effective retinal protection of EPC-ssLip compared to that of DSPC-ssLip. EPC-ssLip is a promising carrier for edaravone in treating oxidative stress-induced retinal diseases.

Physicochemical properties affecting retinal drug/coumarin-6 delivery from nanocarrier systems via eyedrop administration.

PURPOSE: To elucidate the effect of physicochemical properties of nanocarrier systems on drug delivery efficiency to the retina by eyedrop administration in mice, rabbits, and monkeys. METHODS: Submicron-sized liposomes (ssLips) of different particle size, cholesterol content, surface charge, and multilamellar vesicles (MLV) were prepared by the hydration METHOD: Fluorescence probe (coumarin-6)-incorporated liposomes, lipid emulsions, and FITC-labeled polystyrene particles were used to investigate their intraocular behavior after eyedrop administration, using epifluorescence microscopy in mice, rabbits, and monkeys. RESULTS: Delivery efficiency of fluorescent probes to the mouse retina from dropped liposomes was extensively improved by reducing their particle size (<600 nm) and cholesterol content, whereas negligible improvement was observed in the case of MLV. Furthermore, FITC-labeled polystyrene particles and coumarin-6-incorporated lipid emulsions showed an insufficient effect on retinal delivery in mice even if their size was controlled at 110 nm. The highest accumulation of the fluorescent probe in the retina was observed around 30 minutes with any type of ssLip used, followed by the prompt disappearance of their fluorescence within 120 minutes in mice. Changes in the fluorescence intensity of coumarin-6 in rabbits and monkeys were observed in a manner similar to that described in mice. Retinal flat-mount images suggest that coumarin-6 incorporated in ssLip diffused from the iris and ciliary body side to the optic disc side in the retina after eyedrop administration. CONCLUSIONS: The delivery efficiency of coumarin-6 to the retina was altered depending on particle size, constituents, and rigidity. ssLips with appropriate features would be promising drug carriers for retinal delivery through eyedrops.

Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye.

The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eye's posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on l-alpha-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye.

Lig-8, a highly bioactive lignophenol derivative from bamboo lignin, exhibits multifaceted neuroprotective activity.

Lignin is a durable aromatic network polymer that is second only to cellulose in natural abundance. Lig-8, a lignophenol derivative from bamboo lignin, is a highly potent neuroprotectant. It protects human neuroblastoma cells (SH-SY5Y) from hydrogen peroxide (H2O2)-induced apoptosis by preventing caspase-3 activation via either caspase-8 or caspase-9. It exerts this antiapoptotic effect by protecting mitochondrial membrane permeability from damage by H2O2 or the peripheral benzodiazepine receptor ligand PK11195. Lig-8 has been also shown to scavenge the reactive oxygen or nitrogen species in vitro. Furthermore, lig-8 suppresses apoptosis induced by oxygen-glucose deprivation, tunicamycin (endoplasmic reticulum [ER]-stress inducer), or proteasome inhibitor in pheochromocytoma cells. In addition, in vivo, lig-8 reduced intravitreal N-methyl-D-aspartate-induced retinal damage (decreases in retinal ganglion cells and inner plexiform layer thickness) in mice. Lig-8 prevents neuronal damage partly by inhibiting excessive endoplasmic reticulum stress. In this article, we review the protective effects of lig-8 against apoptosis induced by various stimuli. Apoptosis is an active, energy-dependent process through which living cells initiate their own death. It can be induced by a variety of physiological and pharmacological stimuli. Apoptotic cell death is associated with neurodegenerative disorders such as Alzheimer, Parkinson, or Huntington disease as well as glaucoma. We believe that the elucidation of the mechanism of antiapoptotic action of lig-8 may help in finding new approaches to the treatment of neurodegenerative disorders.

Lig-8, a bioactive lignophenol derivative from bamboo lignin, protects against neuronal damage in vitro and in vivo.

Lig-8, a lignophenol derivative from bamboo lignin, potently suppresses oxidative stress-induced apoptosis. Here, we first examined in vitro whether lig-8 protects against neuronal damage induced by oxygen-glucose deprivation (OGD) followed by reoxygenation, tunicamycin [endoplasmic reticulum (ER)-stress inducer], or PSI (proteasome inhibitor). In pheochromocytoma (PC12) cell cultures, lig-8 (1 to 30 microM) concentration-dependently inhibited OGD- and tunicamycin (2 microg/ml)-induced cell deaths (significant at >/=3 microM and >/=1 microM, respectively). In human neuroblastoma (SH-SY5Y) cell culture, the PSI-induced apoptotic cell death and fusion protein accumulation (revealing reduced proteasome activity) was inhibited by lig-8 (30 microM). On the other hand, lig-8 at 30 microM alone did not affect any proteasome activity under resting conditions. In vivo, lig-8 (0.1 nmol/eye) reduced intravitreal N-methyl-D-aspartate (NMDA, 20 nmol)-induced retinal damage (decreases in retinal ganglion cells and inner plexiform layer thickness). Hence, lig-8 protects, partly by inhibiting excessive ER-stress, against neuronal damage in vitro and in vivo.