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

PENETRATION OF HYDROPHILIC SULFORHODAMINE B ACROSS THE PORCINE CORNEA EX-VIVO

Objective: Sulforhodamine B (SRB) is a hydrophilic tracer whose fluorescence is unaffected by pH unlike that of carboxyfluorescein. Therefore, SRB may serve as a better tracer when there are significant changes in pH. Thus, in this study, the suitability of SRB to assess the barrier properties of the cellular layers of the cornea was examined using a custom-built confocal scanning micro-fluorometer (CSMF). Methods: The dye solution (0.1% SRB) was prepared in PBS-Ca2+and three experiments were performed ex vivo using freshly isolated porcine eyes. First, we investigated the penetration of SRB across the endothelium by injection of the dye into the anterior chamber. Next, we measured the penetration of SRB across the epithelium after exposing the ocular surface to the dye. Finally, we examined the penetration after exposure to the dye with detergent (Tween 20) and exposure to the dye concomitant with microneedle injuries. The dye concentration profiles across the cornea were measured using CSMF.Results: SRB penetrated the corneal endothelium readily into the stroma following injection into the anterior chamber in a time-dependent manner. Despite accumulation in the stroma, SRB did not partition into the epithelium. In agreement with these findings, the dye did not cross the epithelium after topical administration. Co-administration with Tween 20 and injury to the epithelium with microneedles, however, led to penetration of the dye into the stroma.Conclusions: SRB is a hydrophilic dye that can be used as an alternative fluorescent tracer to assess the barrier function of the cellular layers of the cornea

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

Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium: Regulation by Actomyosin Contraction

The authors investigated the assembly and disassembly of adherens and tight junctions in the corneal endothelium using the paradigm of the calcium switch maneuver. They observed that excessive actomyosin contraction is involved in the breakdown of tight junctions upon extracellular Ca2+ removal and further noted that contractility of the actin cytoskeleton close to the apical junctions is necessary for rapid reassembly

Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells

PURPOSE. In corneal endothelial cells, intercellular Ca2+ waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercetlular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells.status: publishe

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