Remote-controlled dexamethasone-duration on eye-surface with a micelle-magnetic nanoparticulate co-delivery system for dry eye disease.

Dexamethasone (DEX) is used to treat ocular surface diseases. However, regulating DEX duration in tears while preventing its absorption into the anterior chamber is critical for balancing its therapy effects and the side effects. In this study, a novel magnetic nanoparticle (MNP)-micelle (MC) co-delivery system (MMDS) was developed. The MC moiety in the MMDS served as the carrier for DEX and the MNP part endowed the MMDS with magnetic-responsive properties. To extend its residency, the MMDS was magnetically attracted by an external magnet after instilling, which acted as a precorneal drug-depot enabling a sustainable release of DEX in tears. With combination of magnet treatment, the topical instillation of MMDS@DEX significantly prolonged the DEX-retention in tears and increased the DEX-concentration in the cornea and conjunctiva, as well as concurrently reduced the DEX-level in the aqueous humor, when compared with the commercial DEX eye drop treatment. The combination of MMDS@DEX and magnet treatment exerted significantly better therapeutic effects against DED with smaller side effects than conventional treatments including DEX suspension, commercial DEX eye drops, as well as the MMDS@DEX treatment alone. The present work provided a new method for the effective delivery of DEX to ocular surface tissues while reducing its side effects, which will be beneficial to the treatments of a wide range of ocular surface diseases.

Short-Term Outcomes of Modified Boston Type-II Keratoprosthesis Implantation With Autologous Auricular Cartilage Reinforcement.

PURPOSE: To report the short-term visual outcomes and complications of a modified Boston Type-II keratoprosthesis (Kpro) procedure. DESIGN: Retrospective case series. METHODS: Thirty-seven eyes of 37 patients who had an implantation of autologous auricular cartilage-reinforced (AACR) Boston Type-II Kpro (BK2) were included in the current study. Preoperative and postoperative data were recorded and analyzed for each eye. Main outcome measures included best-corrected visual acuity, symptoms as assessed by questionnaires, complications associated with implantation, and retention of the implanted BK2 device. RESULTS: A total of 37 eyes, consisting of 19 with severe autoimmune dry eye (ADE) and 18 with burn injury, completed ≥12 months of follow-up. The median (interquartile range) best-corrected visual acuity at baseline, 1 month, 3 months, 6 months, 1 year, and 2 years of follow-up was hand motion (HM) 20/60 (20/100-20/40), 20/60 (20/200-20/40), 20/60 (20/200-20/40), 20/100 (20/200-20/40), and 20/100 (20/400-20/40), respectively. All eyes retained the initial device (37/37, 100%). Common postoperative complications included retroprosthetic membrane (n = 21), de novo glaucoma (n = 7), endophthalmitis (n = 1), and conjunctival erosion (n = 4). No ear complications were discovered during follow-up assessments. The ocular surface disease index score improved from baseline to a 2-year follow-up (median 57.5 vs 21.43). CONCLUSION: The modified AACR-BK2 procedure could be considered to restore vision in patients with end-stage corneal blindness.

Aim2 Deficiency Ameliorates Lacrimal Gland Destruction and Corneal Epithelium Defects in an Experimental Dry Eye Model.

Purpose: Dry eye disease (DED) is a multifactorial disease that is associated with inflammation. Excessive DNA is present in the tear fluid of patients with DED. Absent in melanoma 2 (AIM2) is a key DNA sensor. This study aimed to investigate the role of AIM2 in the pathogenesis of DED. Methods: DED was induced by injection of scopolamine (SCOP). Aberrant DNA was detected by cell-free DNA (cfDNA) ELISA and immunostaining. Corneal epithelial defects were assessed by corneal fluorescein staining, zonula occludens-1 immunostaining and TUNEL. Tear production was analyzed by phenol red thread test. Lacrimal gland (LG) histology was evaluated by hematoxylin and eosin staining, and transmission electron microscopy examination. Macrophage infiltration in LG was detected by immunohistochemistry for the macrophage marker F4/80. Gene expression was analyzed by RT-qPCR. Protein production was examined by immunoblot analysis or ELISA. Results: Aim2-/- mice displayed a normal structure and function of LG and cornea under normal conditions. In SCOP-induced DED, wild type (WT) mice showed increased cfDNA in tear fluid, and aberrant accumulations of dsDNA accompanied by increased AIM2 expression in the LG. In SCOP-induced DED, WT mice displayed damaged structures of LG, reduced tear production, and severe corneal epithelium defects, whereas Aim2-/- mice had a better preserved LG structure, less decreased tear production, and improved clinical signs of dry eye. Furthermore, genetic deletion of Aim2 suppressed the increased infiltration of macrophages and inhibited N-GSDMD and IL18 production in the LG of SCOP-induced DED. Conclusions: Aim2 deficiency alleviates ocular surface damage and LG inflammation in SCOP-induced DED.

Corneal Collagen Cross-Linking Pretreatment Mitigates Injury-Induced Inflammation, Hemangiogenesis and Lymphangiogenesis In Vivo.

Purpose: The purpose of this study was to evaluate if corneal collagen cross-linking (CXL) pretreatment dampens suture-induced hemangiogenesis and lymphangiogenesis driven by inflammation. Methods: Four weeks after CXL pretreatment, suture emplacement was performed in rats. The time dependent effects were compared of this procedure in three groups: (1) suture-induced neovascularization (SNV group); (2) CXL treatment prior to suture-induced neovascularization (CXL + SNV group); (3) Normal control (NC group). Serial morphometric measurements evaluated suture-induced hemangiogenesis and lymphangiogenesis. CD45 and CD68 immunofluorescent staining pattern changes determined immune cell activation, stromal leucocyte, and macrophage infiltration. The real-time quantitative polymerase chain reaction (RT-qPCR) determined angiogenic and lymphangiogenic gene expression level changes. Western blots evaluated protein expression levels of vascular endothelial cell CD31 and lymphatic vessel endothelial hyaluronan receptor (LYVE-1). Results: On days 7 and 14 after suture emplacement, the rises in angiogenesis, lymphangiogenesis, CD45+ and CD68+ cell infiltration were less in the CXL pretreated (CXL + SNV) group than in the untreated (SNV) group. Angiogenic and lymphangiogenic mRNA levels and CD31 and LYVE-1 protein and proinflammatory cytokines were also suppressed, confirming that CXL pretreatment improved the wound healing response. Conclusions: CXL pretreatment inhibits injury-induced angiogenesis and lymphangiogenesis. These reductions suggest that prior CXL therapy decrease ocular inflammation reactivated by secondary trauma. Translational Relevance: CXL pretreatment induces increases in stromal stiffness which in turn reduces trauma or microbial driven increases in inflammation, angiogenesis, and lymphangiogenesis. These beneficial effects suggest that this novel procedure may improve therapeutic management of trauma-induced corneal disease in a clinical setting.

Metagenomic Profiling of Ocular Surface Microbiome Changes in Meibomian Gland Dysfunction.

Purpose: Ocular surface microbiome changes can affect meibomian gland dysfunction (MGD) development. This study aimed to delineate differences among the microbiome of eyelid skin, conjunctiva, and meibum in healthy controls (HCs) and patients afflicted with MGD. Methods: Shotgun metagenomic analysis was used to determine if there are differences between the microbial communities in ocular sites surrounding the meibomian gland in healthy individuals and patients afflicted with MGD. Results: The meibum bacterial content of these microbiomes was dissimilar in these two different types of individuals. Almost all of the most significant taxonomic changes in the meibum microbiome of individuals with MGD were also present in their eyelid skin, but not in the conjunctiva. Such site-specific microbe pattern changes accompany increases in the gene expression levels controlling carbohydrate and lipid metabolism. Most of the microbiomes in patients with MGD possess a microbe population capable of metabolizing benzoate. Pathogens known to underlie ocular infection were evident in these individuals. MGD meibum contained an abundance of Campylobacter coli, Campylobacter jejuni, and Enterococcus faecium pathogens, which were almost absent from HCs. Functional annotation indicated that in the microbiomes of MGD meibum their capability to undergo chemotaxis, display immune evasive virulence, and mediate type IV secretion was different than that in the microbiomes of meibum isolated from HCs. Conclusions: MGD meibum contains distinct microbiota whose immune evasive virulence is much stronger than that in the HCs. Profiling differences between the meibum microbiome makeup in HCs and patients with MGD characterizes changes of microbial communities associated with the disease status.

Overcoming the Anatomical and Physiological Barriers in Topical Eye Surface Medication Using a Peptide-Decorated Polymeric Micelle.

The sealed anatomical features of the eye and its physiological activity that rapidly removes drugs are called anatomical and physiological barriers, which are the cause of more than 90% of drug loss. This aspect remains a critical issue in eye surface medication. Thus, promoting tissue permeability of drugs as well as prolonging their retention on the eye surface can improve their bioavailability and enhance their therapeutic effects. Thanks to the existence of a negatively charged mucin layer on the eye surface, several peptide-decorated polymeric micelles were prepared to enhance the interaction between the micelle and eye surface, thus prolonging the drug retention on the eye surface and promoting its tissue permeability. Tacrolimus (also known as FK506) is a hydrophobic macrolide immunosuppressant used to treat dry eye syndrome and other eye diseases. However, its hydrophobic nature makes its delivery as a topical eye surface medication difficult, with the risk of side effects due to overdoses. Therefore, the aim of this work is to evaluate the ability of FK506 micelles in promoting their permeability on the eye surface. Our results showed that the positively charged nanomicelles could significantly prolong FK506 retention on the eye surface and enhance its corneal permeability in ex vivo and in vivo conditions. FK506 nanomicelles exhibited superior curing effects against dry eye diseases than the FK506 suspension and a commercial FK506 formula. It exerted better inhibitory effects on eye surface inflammation and corneal epithelium apoptosis when examined by a slip lamp and a transferase-mediated dUTP nick end labeling assay, respectively. Further assays revealed the higher suppressive effects on the expression of several inflammation-related factors at an mRNA and protein level. Hence, our results suggested that these positively charged nanomicelles might be a good drug delivery system for ocular surface medication.

Fabrication of chitosan based nanocomposite with legumain sensitive properties using charge driven self-assembly strategy.

Chitosan (CS) based nanoparticles (NPs) have several advantages in delivering drugs. They are usually prepared in a micro-emulsion solvent system but this route can leave significant levels of potentially harmful organic solvent residue in the NPs. In this study, we prepared CS based nanocomposites using charge driven self-assembly in an aqueous buffer, thus avoiding the use of organic solvents. Doxorubicin (DOX) was covalently attached to positive charged CS with a legumain substrate peptide to confer targeted drug release property, since legumain is often overexpressed in tumors or tumor associated micro environments. This DOX prodrug solution interacted with negative charged methoxyl poly (ethylene glycol)-block-poly (glutamic acid) copolymer (PEG-PGA) in an aqueous buffer forming nanocomposite with a regular morphology. The particle size and zeta potential of these NPs was regulated by the addition of different PEG-PGA concentrations into the DOX prodrug solution. Due to its potential for legumain triggered release, this DOX NP exhibited enhanced cytotoxicity against choroidal melanoma cell line (Mum-2C) and reduced cytotoxicity on normal human corneal epithelial cells (HCEC), suggesting a good potential for enhanced targeted delivery of chemotherapeutic agents. A chitosan based nanocomposite with legumain sensitive properties are rapidly controllable prepared in aqueous buffer by charge driven self-assembly strategy, without using micro-emulsion solvent system and cross-linking agents.

Hyperosmotic Stress-Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells.

Purpose: The purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1ß secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs). Methods: Real-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1ß mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H2O2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 µM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown. Results: In the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1ß gene and protein expression levels rose after 4 hours (P ≤ 0.043), whereas ACA preincubation suppressed these rises (P ≤ 0.044). Similarly, H2O2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1ß secretion (P ≤ 0.036), whereas ACA pretreatment suppressed these effects (P ≤ 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P = 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1ß gene (P ≤ 0.028) and protein expression (P ≤ 0.037). Conclusions: TRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1ß expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.

Corneal Collagen Cross-Linking With Riboflavin and UVA Regulates Hemangiogenesis and Lymphangiogenesis in Rats.

Purpose: The purpose of this study was to determine whether corneal collagen crosslinking (CXL) inhibits hemangiogenesis and lymphangiogenesis during acute corneal inflammation in an in vivo rat model. Methods: Inflammatory corneal neovascularization was induced by suture placement into a rat cornea. At day 3 after suture, a CXL protocol using riboflavin and UVA was administered after mechanical epithelial debridement. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically. CD45 and CD68 immunostaining evaluated corneal leucocyte and macrophage immune cell infiltration, respectively. A TUNEL assay detected stromal cell apoptosis. Quantitative RT-PCR analysis identified angiogenic and lymphangiogenic genes as well as proinflammatory cytokine expression. Western blot analysis characterized vascular endothelial cell CD31 and lymphatic vessel endothelial hyaluronan receptor (LYVE-1) protein expression. Results: CXL treatment significantly reduced corneal pathologic suture-induced hemangiogenesis and lymphangiogenesis 7 days after suture emplacement, but this procedure failed to affect hemangiogenesis and lymphangiogenesis 14 days after suture. Increased cell apoptosis and reduced CD45+ and CD68+ cell infiltration were evident in CXL-treated rats on days 7 and 14 after suture emplacement. CXL treatment significantly decreased angiogenic and lymphangiogenic mRNA expression levels and both CD31 and LYVE-1 protein expression levels, whereas it increased proinflammatory cytokine levels on day 7 after suture emplacement. However, on day 14 after corneal neovascularization, angiogenic and lymphangiogenic mRNA gene expression levels were upregulated along with hematic CD31 and lymphatic LYVE-1 protein expression. Conclusions: CXL treatment only temporarily inhibits corneal inflammatory-associated hemangiogenesis and lymphangiogenesis in vivo. Such insight suggests that future studies are warranted to develop novel CXL strategies with longer-lasting effectiveness in attenuating hemantic- and lymphatic-related corneal diseases.