DEVELOPMENT AND CHARACTERIZATION OF TOPICAL OPHTHALMIC FORMULATIONS CONTAINING LUTEIN-LOADED MUCOADHESIVE NANOPARTICLES

Objective: To develop and characterize a topical ophthalmic formulation containing chitosan-dextran sulfate nano particles (CDNs) for enhanced ocular bioavailability and stability of lutein.Methods: Lutein-loaded CDNs (LCDNs) were prepared by polyelectrolyte complexation employing oppositely charged polymers, chitosan and dextran sulfate. Effects of the polymer mass ratios, the total amount of polymers, and the amount of EDC and PEG400 on their physicochemical properties as well as the drug release profiles were investigated. The physicochemical stability of LCDNs dispersed in various ophthalmic vehicles and the accompanying microbial contamination were also evaluated.Results: LCDNs possessed a mean size of ~400 nm with a positive surface charge of+30 mV and entrapment efficiency up to 75%. Dissolution profiles followed a Higuchi's square root model, indicating a diffusional release mechanism. LCDNs dispersed in Feldman ophthalmic buffer showed good physical stability with no microbial contamination. The chemical stability of lutein was significantly improved in LCDNs and further improved by the addition of antioxidant in the ophthalmic vehicle.Conclusion: The ophthalmic formulation containing LCDNs, developed in this work, has characteristics suitable for application in ocular surface drug delivery systems.Keywords: Chitosan, dextran sulfate, Nanoparticles, Ophthalmic vehicle, Lutein

A surfactant polymer wound dressing protects human keratinocytes from inducible necroptosis

Chronic wounds show necroptosis from which keratinocytes must be protected to enable appropriate wound re-epithelialization and closure. Poloxamers, a class of synthetic triblock copolymers, are known to be effective against plasma membrane damage (PMD). The purpose of this study is to evaluate the efficacy of a specific poloxamer, surfactant polymer dressing (SPD), which is currently used clinically as wound care dressing, against PMD in keratinocytes. Triton X-100 (TX100) at sub-lytic concentrations caused PMD as demonstrated by the efflux of calcein and by the influx of propidium iodide and FM1-43. TX100, an inducer of necroptosis, led to mitochondrial fragmentation, depletion of nuclear HMGB1, and activation of signaling complex associated with necroptosis (i.e., activation of RIP3 and phosphorylation of MLKL). All responses following exposure of human keratinocytes to TX100 were attenuated by pre- or co-treatment with SPD (100 mg/ml). The activation and translocation of phospho-MLKL to the plasma membrane, taken together with depletion of nuclear HMGB1, characterized the observed cell death as necroptosis. Thus, our findings show that TX100-induced plasma membrane damage and death by necroptosis were both attenuated by SPD, allowing keratinocyte survival. The significance of such protective effects of SPD on keratinocytes in wound re-epithelialization and closure warrant further studies

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells. Exp Eye Res.

PURPOSE. To investigate histamine-induced changes in the phosphorylation of myosin light chain (MLC) and its influence on the barrier integrity of corneal endothelial cells through altered contractility of the actin cytoskeleton. METHODS. Experiments were performed in cultured bovine corneal endothelial cells (BCECs). Phosphorylation of MLC, which increases contractility of the actin cytoskeleton through actomyosin interaction, was assessed by urea-glycerol gel electrophoresis and Western blot analysis. Immunocytochemistry was used to locate phosphorylated MLC in relation to tight junctions. Phosphorylation of the 17-kDa PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17), which inhibits MLC phosphatase, was studied using Western blot analysis. The cortical actin cytoskeleton was visualized by staining with Texas-red phalloidin. Barrier integrity was determined by quantifying horseradish peroxidase (HRP; 44 kDa) flux across cells grown on porous filters. RESULTS. RT-PCR and Western blot analysis confirmed the expression of G␣ q/11 -coupled H1 receptors in BCECs. Exposure to histamine (100 M; 10 minutes) led to phosphorylation of MLC (134% relative to untreated cells) and of CPI-17. Histamine also increased the flux of HRP by sevenfold and disrupted the assembly of the dense cortical actin found in resting cells. PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM; 30 minutes) caused phosphorylation of both MLC and CPI-17. The histamine-induced MLC phosphorylation was reduced by pre-exposure to either ML-7 (50 M), an MLCK (MLC kinase) inhibitor, or chelerythrine (10 M), an inhibitor of PKC. Cotreatment with agents that elevate cAMP in BCECs prevented the histamine-induced MLC phosphorylation and the disruption of the actin cytoskeleton, and increased HRP flux. Phosphorylated MLC in response to histamine or PMA was found in a punctate form in close proximity to ZO-1, a marker of the tight junctional complex. CONCLUSIONS. Histamine induces MLC phosphorylation by activating MLCK and partly inhibiting MLC phosphatase. The latter is facilitated by the phosphorylation of CPI-17. Localization of phosphorylated MLC in proximity to ZO-1 suggests increased contractility of the cortical actin at the tight junctional complex. This contractility oppose the tethering forces and lead to a breakdown of the barrier integrity. Last, elevated cAMP prevents histamine-induced loss of the barrier integrity, not only by blocking inactivation of MLC phosphatase but also by inactivating MLCK. (Invest Ophthalmol Vis Sci. 2006;47: 4011-4018

ATP release through connexin hemichannels in corneal endothelial cells

PURPOSE. Intercellular Ca 2ϩ wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca 2ϩ wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca 2ϩ wave propagation in bovine corneal endothelial cells (BCECs). METHODS. A Ca 2ϩ wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca 2ϩ ] i in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca 2ϩ -sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca 2ϩ wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence. RESULTS. Flufenamic acid (FFA; 50 M) and the connexin mimetic peptide Gap26 (300 M), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 M). These findings suggest that the hemichannel blockers reduce the Ca 2ϩ wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca 2ϩ -free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca 2ϩ removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca 2ϩ . CONCLUSIONS. Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca 2ϩ wave. (Invest Ophthalmol Vis Sci