Ocular delivery of polyphenols: meeting the unmet needs

Producción CientíficaNature has become one of the main sources of exploration for researchers that search for new potential molecules to be used in therapy. Polyphenols are emerging as a class of compounds that have attracted the attention of pharmaceutical and biomedical scientists. Thanks to their structural peculiarities, polyphenolic compounds are characterized as good scavengers of free radical species. This, among other medicinal effects, permits them to interfere with different molecular pathways that are involved in the inflammatory process. Unfortunately, many compounds of this class possess low solubility in aqueous solvents and low stability. Ocular pathologies are spread worldwide. It is estimated that every individual at least once in their lifetime experiences some kind of eye disorder. Oxidative stress or inflammatory processes are the basic etiological mechanisms of many ocular pathologies. A variety of polyphenolic compounds have been proved to be efficient in suppressing some of the indicators of these pathologies in in vitro and in vivo models. Further application of polyphenolic compounds in ocular therapy lacks an adequate formulation approach. Therefore, more emphasis should be put in advanced delivery strategies that will overcome the limits of the delivery site as well as the ones related to the polyphenols in use. This review analyzes different drug delivery strategies that are employed for the formulation of polyphenolic compounds when used to treat ocular pathologies related to oxidative stress and inflammation.Ministerio de Ciencia, Innovación y Universidades - Fondo Europeo de Desarrollo Regional (RTI2018-094071-B-C21)European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Initial Training Network (ITN) “IT-DED3” (H2020-MSCA-ITN-2017) grant agreement No. 76560

Epithelial cells removed in advanced surface ablation (ASA) surgery can be used as a source of corneal samples to perform in vitro studies

Producción CientíficaHuman corneal epithelial cells are needed to study corneal pathophysiology in vitro. Due to the limitations of cell lines, the use of primary cells is highly desirable, but the scarcity of human tissues, along with ethical issues, make it difficult to accomplish all required experiments. In advanced surface ablation (ASA), the central corneal epithelium is removed and discarded. We hypothesized that ASA samples could be used to perform in vitro assays. In this study, 29 samples from patients undergoing ASA were recovered in supplemented DMEM/F12 culture medium, RIPA buffer, or RLT lysis buffer. The first aim was to determine whether cells could be maintained in culture. Although with the explant technique, tissue pieces did not attach to the culture surface, after disaggregation, cells showed high viability (90.0 ± 6.0%), attached to plates, and remained viable for up to 14 days. The second aim was to elucidate if ASA samples could be used to study protein or gene expression. Cytokeratin-3, ZO-1, Ki67, and E-cadherin protein expression were confirmed by immunofluorescence. Total protein (485.8 ± 115.8 μg) was isolated from cells in RIPA buffer, and GAPDH was detected by Western blotting, indicating that samples are adequate for protein studies. RNA (9.0 ± 3.6 μg) was isolated from samples in RLT lysis buffer, and GAPDH gene expression was studied by PCR, confirming that samples were also suitable for gene expression studies. These results suggest that samples obtained from corneal surface ablation procedures may constitute a valuable source of human cells to accomplish in vitro studies.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project FEDER-CICYT MAT2013-47501-C02-R)Ministerio de Ciencia, Innovación y Universidades - Fondo Europeo de Desarrollo Regional (project RTI2018-094071-B-C21

Improved ocular delivery of quercetin and resveratrol : A comparative study between binary and ternary cyclodextrin complexes

Peer reviewe

Topical application of TGF-β-activating peptide, KRFK, prevents inflammatory manifestations in the TSP-1-deficient mouse model of chronic ocular inflammation

Producción CientíficaChronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-β2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-β, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-β, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1−/− mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1−/− mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.Ministerio de Economía, Industria y Competitividad (FEDER-CICYT grant MAT2013-47501-C02-1-R)Massachusetts Lions Eye Research Fund (MLERF, S.M.) and NEI grant EY015472 (S.M.)Junta de Castilla y León - Fondo Social Europeo (project VA098-12

Development of Conjunctival Goblet Cells and Their Neuroreceptor Subtype Expression

PURPOSE. To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS. Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiff's reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and ␤-adrenergic receptors. RESULTS. At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M 2 -and M 3 -muscarinic and ␤ 2 -adrenergic receptors were found in stratified squamous cells, but M 1 -muscarinic and ␤ 1 -adrenergic receptors were not detected. At days 17 and 60, M 2 -and M 3 -muscarinic receptors were found in goblet cells, whereas M 1 -muscarinic receptors were in stratified squamous cells. ␤ 1 -and ␤ 2 -Adrenergic receptors were found on both cell types. ␤ 3 -Adrenergic receptors were not detected. CONCLUSIONS. In conjunctiva, nerves, M 2 -and M 3 -muscarinic, and ␤ 1 -and ␤ 2 -adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open. (Invest Ophthalmol Vis Sci. 2000;41:2127-2137 T he tear film mucus layer consists of high molecular weight glycoconjugates including mucins, which are secreted mainly by conjunctival goblet cells. This layer plays an important role in protecting the ocular surface from exogenous agents (bacterial or chemical) and provides lubrication during all types of eye movements. 1 Goblet cells can release their secretory granules in a reflex response mediated by the activation of either parasympathetic or sympathetic nerves that surround them. 2,3 Previous reports from this laboratory showed the localization of nerve fibers adjacent to goblet cells in rat conjunctiva. 5 Use of immunofluorescence techniques demonstrated that M 2 -and M 3 -, but not M 1 -muscarinic acetylcholine receptors (MAchRs), are present on goblet cells and are located on membranes subjacent to secretory granules. VIP type 2 receptors (VIPR2s) are located in the basolateral membranes of goblet cells. 3 Although the role of the sympathetic agonists in stimulating goblet cell secretion is unknown, ␤ 1 -and ␤ 2 -adrenergic receptor (␤AR) subtypes appear to be present in goblet cells as well as in stratified squamous cells. Morphologic studies in developing conjunctiva suggest that based on changes in the acidity of glycoproteins in the secretory granules, goblet cells may differentiate from basal epithelial cells in the forniceal zone. 7 Watanabe et al

Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium

PURPOSE. To study the presence of muscarinic and ␣-and ␤-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line. METHODS. Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M 1 -, M 2 -, and M 3 -muscarinic and to ␣ 1A -, ␣ 1B -, ␣ 1D -, ␣ 2A -, ␣ 2B -, ␣ 2C -, ␤ 1 -, ␤ 2 -, and ␤ 3 -adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-␣ and/or interferon (IFN)-␥. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS. By immunofluorescence microscopy, all receptor subtypes, except the ␣ 2C -adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M 2 -and M 3 -muscarinic receptors and ␣ 1A -, ␣ 1B -, ␣ 1D -, ␣ 2A -, ␣ 2B -, ␣ 2C -, ␤ 1 -, and ␤ 3 -adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M 1 -muscarinic and ␤ 2 -adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M 2 and M 3 -muscarinic and ␣ 1A -, ␣ 2A -, ␣ 2B -, ␤ 1 -and ␤ 2 -adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M 2 -muscarinic and ␣ 1B -and ␣ 2B -adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN␥ and/or TNF␣. CONCLUSIONS. The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation. (Invest Ophthalmol Vis Sci. 2005;46:504 -513

0 Novel anti-inflammatory liposomal formulation for the pre-ocular tear film: In vitro and ex vivo functionality studies in corneal epithelial cells

In ocular surface inflammatory diseases, such as dry eye disease, long-term symptom relief requires targeting the inflammation itself rather than treating only the surface-associated dryness with artificial tears. Therefore, we included an anti-inflammatory agent in an unpreserved liposome-based (LP) formulation used as artificial tears. Our aim was to characterize and study its in vitro and ex vivo cell uptake and functionality. Human corneal epithelial (HCE) cells were used to study MPA-LP37 induced effects after 60 min of exposure, using blank LP and non-LP MPA formulations as controls. A fluorescent labeled LP formulation was used to determine uptake by HCE cells and localization in ex vivo porcine corneas. The LP formulation complied with the required physicochemical properties and had no cytotoxicity on HCE cells after 60 min of exposure. HCE cells showed LP-associated fluorescence at 24, 48, and 72 h after 60 min of exposure, and the LP-associated fluorescence was uniformly distributed throughout the porcine corneal epithelium immediately after 5 min of exposure. MPA44 LP increased protein expression and nuclear translocation of progesterone receptor in comparison with controls as determined by Western blotting and immunofluorescence. Moreover, MPA-LP significantly reduced the cell proliferation rate and IL-6 and IL-8 production 48 h after the exposure period, as determined by the alamarBlue assay and ELISA, respectively. None of these effects were evident in blank LP-exposed cells and non-LP MPA formulation reduced only IL-6 production. Our results suggest that the LP50 based formulation, used to replenish the lipids of the tear film, can be loaded with anti-inflammatory agents that can be delivered into the cells and activate specific drug receptors. These agents can reduce inflammatory cytokine production and may be effective in the treatment of inflammatory processes associated with ocular surface diseases

Three-Dimensional Human Cell Culture Models to Study the Pathophysiology of the Anterior Eye

In recent decades, the establishment of complex three-dimensional (3D) models of tissues has allowed researchers to perform high-quality studies and to not only advance knowledge of the physiology of these tissues but also mimic pathological conditions to test novel therapeutic strategies. The main advantage of 3D models is that they recapitulate the spatial architecture of tissues and thereby provide more physiologically relevant information. The eye is an extremely complex organ that comprises a large variety of highly heterogeneous tissues that are divided into two asymmetrical portions: the anterior and posterior segments. The anterior segment consists of the cornea, conjunctiva, iris, ciliary body, sclera, aqueous humor, and the lens. Different diseases in these tissues can have devastating effects. To study these pathologies and develop new treatments, the use of cell culture models is instrumental, and the better the model, the more relevant the results. Thus, the development of sophisticated 3D models of ocular tissues is a significant challenge with enormous potential. In this review, we present a comprehensive overview of the latest advances in the development of 3D in vitro models of the anterior segment of the eye, with a special focus on those that use human primary cells