Yolanda Diebold

Chitosan Nanoparticles as New Ocular Drug Delivery Systems: in Vitro Stability, in Vivo Fate, and Cellular Toxicity

AbstractPurpose. To assess the potential of chitosan (CS) nanoparticles for ocular drug delivery by investigating their interaction with the ocular mucosa in vivo and also their toxicity in conjunctival cell cultures. Methods. Fluorescent (CS-fl) nanoparticles were prepared by ionotropic gelation. The stability of the particles in the presence of lysozyme was investigated by determining the size and their interaction with mucin, by measuring the viscosity of the mucin dispersion. The in vivo interaction of CS-fl nanoparticles with the rabbit cornea and conjunctiva was analyzed by spectrofluorimetry and confocal microscopy. Their potential toxicity was assessed in a human conjunctival cell line by determining cell survival and viability. Results. CS-fl nanoparticles were stable upon incubation with lysozyme and did not affect the viscosity of a mucin dispersion. Invivo studies showed that the amounts of CS-fl in cornea and conjunctiva were significantly higher for CS-fl nanoparticles than for a control CS-fl solution, these amounts being fairly constant for up to 24 h. Confocal studies suggest that nanoparticles penetrate into the corneal and conjunctival epithelia. Cell survival at 24 h after incubation with CS nanoparticles was high and the viability of the recovered cells was near 100%. Conclusions. CS nanoparticles are promising vehicles for ocular drug delivery.

Applications of nanoparticles in ophthalmology

Nanocarriers, such as nanoparticles, have the capacity to deliver ocular drugs to specific target sites and hold promise to revolutionize the therapy of many eye diseases. Results to date strongly suggest that ocular medicine will benefit enormously from the use of this nanometric scale technology. One of the most important handicaps of the eye as a target organ for drugs is the presence of several barriers that impede direct and systemic drug access to the specific site of action. Superficial barriers include the ocular surface epithelium and the tear film, and internal barriers include the blood-aqueous and blood-retina barriers. Topical application is the preferred route for most drugs, even when the target tissues are at the back part of the eye where intraocular injections are currently the most common route of administration. Direct administration using any of these two routes faces many problems related to drug bioavailability, including side effects and repeated uncomfortable treatments to achieve therapeutic drug levels. In this regard, the advantages of using nanoparticles include improved topical passage of large, poorly water-soluble molecules such as glucocorticoid drugs or cyclosporine for immune-related, vision-threatening diseases. Other large and unstable molecules, such as nucleic acids, delivered using nanoparticles offer promising results for gene transfer therapy in severe retinal diseases. Also, nanoparticle-mediated drug delivery increases the contact time of the administered drug with its target tissue, such as in the case of brimonidine, one of the standard treatments for glaucoma, or corticosteroids used to treat autoimmune uveitis, a severe intraocular inflammatory process. In addition, nanocarriers permit the non-steroidal anti-inflammatory drug indomethacin to reach inner eye structures using the transmucosal route. Finally, nanoparticles allow the possibility of targeted delivery to reach specific types of cancer, such as melanoma, leaving normal cells untouched. This review summarizes experimental results from our group and others since the beginnings of nanocarrier technology to deliver drugs to different locations in the eye. Also, it explores the future possibilities of nanoparticles not only as drug delivery systems but also as aides for diagnostic purposes.

Cytokine responses by conjunctival epithelial cells: An in vitro model of ocular inflammation

OBJECTIVES We examined the differential secretion of cytokines by a conjunctival epithelial cell line in response to proinflammatory cytokines to identify the potential contributions during ocular surface inflammation. METHODS A conjunctival epithelial cell line was exposed to IFN-gamma, TNF-alpha, IL-4, or IL-13, and cytokine production was determined in supernatants at different times after exposure. Cell apoptosis was measured by flow cytometry. RESULTS TNF-alpha induced the greatest effect on cytokine secretion, which was time-dependent. TNF-alpha-stimulated secretion of IL-12p40 was significantly increased by 30 min; GM-CSF, MCP-1, IL-6, IL-7, IL-8, and RANTES were significantly increased by 2 h, and IFN-gamma and IL-1alpha by 24 h. After 48 h, TNF-alpha also induced a significant increase in IL-1beta, IL-3, and IP-10 secretion. IFN-gamma significantly enhanced IP-10 and RANTES secretion after 48 h of exposure. Following IL-4 treatment there was a significant increase in eotaxin-1 after 24h, and IL-12p40 and IL-3 after 48 h. IL-13 significantly increased the secretion of eotaxin-1 after 24 h, and IL-8 after 48 h. CONCLUSION Our results suggest that conjunctival epithelial cells are an important source of cytokines and chemokines that are regulated by proinflammatory cytokines and may play an important role in ocular surface inflammation.

Dry Eye Disease as an Inflammatory Disorder

Purpose: Dry eye disease (DED) is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. It still is a frustrating disease because of the present scarcity of therapies that can reverse, or at least stop, its progression. Methods: A comprehensive literature survey of English-written scientific publications on the role of inflammation in DED. Results: New investigations have demonstrated that a chronic inflammatory response plays a key role in the pathogenesis of human DED. Additionally, correlations between inflammatory molecules and clinical data suggest that inflammation can be responsible for some of the clinical symptoms and signs. Conclusions: Research efforts to clarify its pathophysiology are leading to a better understanding of DED, demonstrating that inflammation, in addition to many other factors, plays a relevant role.

Genetically Engineered Elastin-Like Polymer as a Substratum to Culture Cells from the Ocular Surface

Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices. Materials and Methods: A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox®, and ELP-coated. Cells (104) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed. Results: Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers. Conclusions: The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

Characterization of epithelial primary cultures from human conjunctiva

Primary cultures of human epithelial cells from normal conjunctiva were developed and characterized to determine whether they retained epithelial characteristics. Conjunctival explants were obtained from the upper fornix of healthy donors and cultured in supplemented DMEM/F-12 medium for 5 days. The epithelial outgrowth was maintained for an additional 10 days. Primary cultures were then processed for light microscopy, transmission and scanning electron microscopy (TEM, SEM), and immunocytochemistry. They exhibited typical features of conjunctival epithelium on light microscopy (polygonal morphology, intimate cohesion, production of mucins), TEM (abundant desmosomes, keratin bundles, granules, microvilli), SEM (polygonal shape, microvilli, intimate cohesion), and immunocytochemistry (positivity for the receptor of epidermal growth factor, desmosomal proteins, and cytokeratins). In conclusion, primary cultures developed from normal human conjunctiva maintained the epithelial characteristics in vitro. Because the conjunctiva is a major component of the anterior ocular surface, we propose this in vitro system as suitable for physiopathologic and toxicologic studies.

Expression of MUC5AC in ocular surface epithelial cells using cationized gelatin nanoparticles.

Decreased production of the mucin MUC5AC in the eye is related to several pathological conditions, including dry eye syndrome. A specific strategy for increasing the ocular levels of MUC5AC is not yet available. Using a plasmid specially designed to encode human MUC5AC, we evaluated the ability of hybrid cationized gelatin nanoparticles (NPs) containing polyanions (chondroitin sulfate or dextran sulfate) to transfect ocular epithelial cells. NPs were developed using the ionic gelation technique and characterized by a small size (<200 nm), positive zeta potential (+20/+30 mV), and high plasmid association efficiency (>95%). MUC5AC mRNA and protein were detected in conjunctival cells after in vitro transfection of the NPs. The in vivo administration of the NPs resulted in significantly higher MUC5AC expression in the conjunctiva compared to untreated control and naked plasmid. These results provide a proof-of-concept that these NPs are effective vehicles for gene therapy and candidates for restoring the MUC5AC concentration in the ocular surface.

CARBOMER- VERSUS CELLULOSE-BASED ARTIFICIAL-TEAR FORMULATIONS : MORPHOLOGIC AND TOXICOLOGIC EFFECTS ON A CORNEAL CELL LINE

PURPOSE Frequent instillation of artificial tears is the primary disadvantage of the treatment for dry-eye syndrome. Recently gel formulations have been proposed as an alternative to classic cellulose formulations. The higher viscosity of these gels presumably prolongs tear-retention time in the eye and results in fewer daily applications. However, no toxicologic studies with gel formulations have been performed. Our aim was to study the toxic effects of these formulations on corneal cells. METHODS SIRC cells from rabbit cornea were exposed for 30 min, and 1, 3, and 6 h to five commercially available artificial tears, three of them carbomer gel formulations (Lacrivisc, Lacrivisc unit-doses, and Viscotears) and two carboxymethylcellulose (CMC) formulations (Celluvisc and Cellufresh). A cytotoxicity assay and a scanning electron microscopy (SEM) study were used to analyze the putative toxic effects of the formulations. The preservatives of the gel formulations also were tested. RESULTS Carbomer gel formulations, both with and without preservatives, caused more in vitro toxic effects in the corneal cells than did CMC formulations and caused severe damage even after 30 min of exposure. SEM revealed dramatic cell-surface alterations. Preservatives added to Lacrivisc and Viscotears also had toxic effects on cells, whose effects were not significantly different from those of the commercial preparations. CONCLUSION These results demonstrate that in the in vitro study, CMC artificial tears are less toxic than carbomer gel formulations. Questions about the benefits of using high-viscosity gels in the treatment of dry-eye syndrome still remain.

Novel bioadhesive hyaluronan-itaconic acid crosslinked films for ocular therapy.

New hyaluronic acid (HA)-itaconic acid (IT) films have been previously synthesized and used as potential topical drug delivery systems (DDS) for ocular administration. In this study we explored homogeneous and heterogeneous crosslinking reactions of HA using glutaraldehyde (GTA) and polyethylene glycol diglycidyl ether (PEGDE) in the presence of IT, a naturally occurring compound that is non-toxic and readily biodegradable. We have studied the morphology, mechanical properties and in vitro biocompatibility between these new materials and ocular surface cells (human corneal epithelial cell line) and evaluated the biopharmaceutical performance of the designed formulations. Although all the synthesized materials exhibited good mechanical properties, the PEGDE modified films exhibited the best biocompatibility, with in vivo assays showing good adhesive performance and minimal irritation. PEGDE films were also tested for their effects in the treatment of intraocular pressure (IOP) in rabbits using timolol maleate (TM) as the model drug. These results may be useful for further design of novel bioadhesive matrix containing drugs by topical application in ophthalmology.

Structural and Functional Alteration of Corneal Epithelial Barrier Under Inflammatory Conditions

Purpose: The aim of the study was to determine the effect of inflammatory conditions on the expression of tight junction (TJ) and adherens junction (AJ) proteins between human corneal epithelial cells and, consequently, on corneal epithelial barrier integrity. Materials and methods: Zonula occludens proteins ZO-1 and ZO-2, claudin-1 and -2 (CLDN-1 and CLDN-2), occludin (OCLN) as well as E-cadherin (E-cad) expression were analyzed in a human corneal epithelial cell line (HCE) at basal conditions and after stimulation with inflammatory cytokines (TNFα, TGFβ, IL-10, IL-13, IL-17, IL-6), using real time RT-PCR, Western blotting and immunofluorescence. Actin cytoskeleton staining was performed after all stimulations. Transepithelial electrical resistance (TER) and fluorescein transepithelial permeability (TEP) were measured as barrier integrity functional assays. Results: ZO-1, ZO-2, CLDN-1, CLDN-2, OCLN and E-cad were detected in HCE cell membranes at basal conditions. Cytokine stimulation resulted in significant changes in the expression of TJ and AJ proteins, both at mRNA and protein level, a remarkable change in their localization pattern, as well as a reorganization of actin cytoskeleton. Pro-inflammatory cytokines TNFα, TGFβ, IL-13, IL-17 and IL-6 induced a structural and functional disruption of the epithelial barrier, while IL-10 showed a barrier protective effect. Conclusion: Simulated inflammatory conditions lead to an alteration of corneal barrier integrity by modulating TJ, and to a lesser extent also AJ, protein composition, at least In Vitro. The observed barrier protective effects of IL-10 support its well-known anti-inflammatory functions and highlight a potential therapeutic perspective.