ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Mustard gas exposure instigates retinal Müller cell gliosis.

Sulfur mustard (SM) is a chemical warfare agent (CWA) that causes severe eye pain, photophobia, excessive lacrimation, corneal and ocular surface defects, and blindness. However, SM's effects on retinal cells are relatively meager. This study investigated the role of SM toxicity on Müller glial cells responsible for cellular architecture, inner blood-retinal barrier maintenance, neurotransmitter recycling, neuronal survival, and retinal homeostasis. Müller glial cells (MIO-M1) were exposed to SM analog, nitrogen mustard (NM), at varying concentrations (50-500 µM) for 3 h, 24 h, and 72 h. Müller cell gliosis was evaluated using morphological, cellular, and biochemical methods. Real-time cellular integrity and morphological evaluation were performed using the xCELLigence real-time monitoring system. Cellular viability and toxicity were measured using TUNEL and PrestoBlue assays. Müller glia hyperactivity was calculated based on glial fibrillary acidic protein (GFAP) and vimentin immunostaining. Intracellular oxidative stress was measured using DCFDA and DHE cell-based assays. Inflammatory markers and antioxidant enzyme levels were determined by quantitative real-time PCR (qRT-PCR). AO/Br and DAPI staining further evaluated DNA damage, apoptosis, necrosis, and cell death. Inflammasome-associated Caspase-1, ASC, and NLRP3 were studied to identify mechanistic insights into NM toxicity in Müller glial cells. The cellular and morphological evaluation revealed the Müller glia hyperactivity after NM exposure in a dose- and time-dependent manner. NM exposure caused significant oxidative stress and enhanced cell death at 72 h. A significant increase in antioxidant indices was observed at the lower concentrations of NM. Mechanistically, we found that NM-treated MIO-M1 cells increased caspase-1 levels that activated NLRP3 inflammasome-induced production of IL-1ß and IL-18, and elevated Gasdermin D (GSDMD) expression, a crucial component actuating pyroptosis. In conclusion, NM-induced Müller cell gliosis via increased oxidative stress results in caspase-1-dependent activation of the NLRP3 inflammasome and cell death driven primarily by pyroptosis.

Damage-Associated Molecular Patterns (DAMPs) in Retinal Disorders.

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules released from the extracellular and intracellular space of damaged tissue or dead cells. Recent evidence indicates that DAMPs are associated with the sterile inflammation caused by aging, increased ocular pressure, high glucose, oxidative stress, ischemia, mechanical trauma, stress, or environmental conditions, in retinal diseases. DAMPs activate the innate immune system, suggesting their role to be protective, but may promote pathological inflammation and angiogenesis in response to the chronic insult or injury. DAMPs are recognized by specialized innate immune receptors, such as receptors for advanced glycation end products (RAGE), toll-like receptors (TLRs) and the NOD-like receptor family (NLRs), and purine receptor 7 (P2X7), in systemic diseases. However, studies describing the role of DAMPs in retinal disorders are meager. Here, we extensively reviewed the role of DAMPs in retinal disorders, including endophthalmitis, uveitis, glaucoma, ocular cancer, ischemic retinopathies, diabetic retinopathy, age-related macular degeneration, rhegmatogenous retinal detachment, proliferative vitreoretinopathy, and inherited retinal disorders. Finally, we discussed DAMPs as biomarkers, therapeutic targets, and therapeutic agents for retinal disorders.

Physicochemical properties of core-shell type nanoparticles govern their spatiotemporal biodistribution in the eye.

Due to the inherent barrier properties of eye tissues, a major challenge in treating eye diseases is to provide a therapeutic agent to the desired tissue in quantities and durations that are favorable. This study aimed at understanding the influence of physicochemical properties of nanoparticles on their spatiotemporal biodistribution in mouse eye. For this, core-shell nanoparticles with different properties were designed by varying either core or shell and administered as eye-drops to mice. The results demonstrated that all nanoparticles irrespective of type of core or shell followed the conjunctival-scleral pathway. The bioavailability of cores followed the order polylactide-co-glycolide≥polylactide≥polycaprolactone for all tissues and time-points. The bioavailability for all shell types was greater in conjunctiva, sclera, choroid and retina when compared to other eye tissues. Therefore, modulating physicochemical properties of nanoparticles can be used as a design strategy to devise drug carriers that target specific tissues of the eye.

Silk-gelatin hybrid hydrogel: a potential carrier for RNA therapeutics.

RNA-based therapeutics have exhibited remarkable potential in targeting genetic factors for disease intervention, exemplified by recent mRNA vaccines for COVID-19. Nevertheless, the intrinsic instability of RNA and challenges related to its translational efficiency remain significant obstacles to the development of RNA as therapeutics. This study introduces an innovative RNA delivery approach using a silk fibroin (SF) and positively charged gelatin (Gel) hydrogel matrix to enhance RNA stability for controlled release. As a proof of concept, whole-cell RNA was incorporated into the hydrogel to enhance interactions with RNA molecules. Additionally, molecular modeling studies were conducted to explore the interactions between SF, collagen, chitosan (Chi), and the various RNA species including ribosomal RNAs (28S, 18S, 8.5S, and 5S rRNAs), transfer RNAs (tRNA-ALA, tRNA-GLN, and tRNA-Leu), as well as messenger RNAs (mRNA-GAPDH, mRNA-ß actin, and mRNA-Nanog), shedding light on the RNA-polymer interaction and RNA stability; SF exhibits a more robust interaction with RNA compared to collagen/gel and chitosan. We confirmed the molecular interactions of SF and RNA by FTIR and Raman spectroscopy, which were further supported by AFM and contact angle measurement. This research introduces a novel RNA delivery platform and insights into biopolymer-RNA interactions, paving the way for tailored RNA delivery systems in therapeutics and biomedical applications.

3D bioprinting of stromal cells-laden artificial cornea based on visible light-crosslinkable bioinks forming multilength networks.

3D bioprinting has the potential for the rapid and precise engineering of hydrogel constructs that can mimic the structural and optical complexity of a healthy cornea. However, the use of existing light-activated bioinks for corneal printing is limited by their poor cytocompatibility, use of cytotoxic photoinitiators (PIs), low photo-crosslinking efficiency, and opaque/colored surface of the printed material. Herein, we report a fast-curable, non-cytotoxic, optically transparent bioprinting system using a new water-soluble benzoyl phosphinate-based PI and photocrosslinkable methacrylated hyaluronic acid (HAMA). Compared with commercially available PIs, the newly developed PI, lithium benzoyl (phenyl) phosphinate (BP), demonstrated increased photoinitiation efficiency under visible light and low cytotoxicity. Using a catalytic amount of BP, the HA-based bioinks quickly formed 3D hydrogel constructs under low-energy visible-light irradiation (405 nm, <1 J cm-2). The mechanical properties and printability of photocurable bioinks were further improved by blending low (10 kDa) and high (100 kDa) molecular weight (MW) HAMA by forming multilength networks. For potential applications as corneal scaffolds, stromal cell-laden dome-shaped constructs were fabricated using MW-blended HAMA/BP bioink and a digital light processing printer. The HA-based photocurable bioinks exhibited good cytocompatibility (80%-95%), fast curing kinetics (<5 s), and excellent optical transparency (>90% in the visible range), potentially making them suitable for corneal tissue engineering.

Next-Generation Nanomedicine Approaches for the Management of Retinal Diseases.

Retinal diseases are one of the leading causes of blindness globally. The mainstay treatments for these blinding diseases are laser photocoagulation, vitrectomy, and repeated intravitreal injections of anti-vascular endothelial growth factor (VEGF) or steroids. Unfortunately, these therapies are associated with ocular complications like inflammation, elevated intraocular pressure, retinal detachment, endophthalmitis, and vitreous hemorrhage. Recent advances in nanomedicine seek to curtail these limitations, overcoming ocular barriers by developing non-invasive or minimally invasive delivery modalities. These modalities include delivering therapeutics to specific cellular targets in the retina, providing sustained delivery of drugs to avoid repeated intravitreal injections, and acting as a scaffold for neural tissue regeneration. These next-generation nanomedicine approaches could potentially revolutionize the treatment landscape of retinal diseases. This review describes the availability and limitations of current treatment strategies and highlights insights into the advancement of future approaches using next-generation nanomedicines to manage retinal diseases.

Azithromycin Protects Retinal Glia Against Oxidative Stress-Induced Morphological Changes, Inflammation, and Cell Death.

The reactivity of retinal glia in response to oxidative stress has a significant effect on retinal pathobiology. The reactive glia change their morphology and secret cytokines and neurotoxic factors in response to oxidative stress associated with retinal neurovascular degeneration. Therefore, pharmacological intervention to protect glial health against oxidative stress is crucial for maintaining homeostasis and the normal function of the retina. In this study, we explored the effect of azithromycin, a macrolide antibiotic with antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective properties against oxidative stress-induced morphological changes, inflammation, and cell death in retinal microglia and Müller glia. Oxidative stress was induced by H2O2, and the intracellular oxidative stress was measured by DCFDA and DHE staining. The change in morphological characteristics such as the surface area, perimeter, and circularity was calculated using ImageJ software. Inflammation was measured by enzyme-linked immunosorbent assays for TNF-α, IL-1ß, and IL-6. Reactive gliosis was characterized by anti-GFAP immunostaining. Cell death was measured by MTT assay, acridine orange/propidium iodide, and trypan blue staining. Pretreatment of azithromycin inhibits H2O2-induced oxidative stress in microglial (BV-2) and Müller glial (MIO-M1) cells. We observed that azithromycin inhibits oxidative stress-induced morphological changes, including the cell surface area, circularity, and perimeter in BV-2 and MIO-M1 cells. It also inhibits inflammation and cell death in both the glial cells. Azithromycin could be used as a pharmacological intervention on maintaining retinal glial health during oxidative stress.

A non-invasive nanoparticle-based sustained dual-drug delivery system as an eyedrop for endophthalmitis.

Endophthalmitis is an infectious disease that affects the entire eye spreading to the internal retinal layers and the vitreous and causes severe sight-threatening conditions. Current treatment strategies rely on intraocular injections of antibiotics that are invasive, may lead to procedural complications and, ultimately, blindness. In this study, we developed a non-invasive strategy as an eyedrop containing nanoparticle-based dual-drug delivery system in which the hydrophobic poly-L-lactide core was loaded with azithromycin or triamcinolone acetonide, and the hydrophilic shell was made of chitosan. The developed nanoparticles were ~200-250 nm in size, spherical in shape, moderately hydrophilic, lysozyme tolerant, cytocompatible, and hemocompatible. Application of these chitosan-coated nanoparticles as eye drops to C57BL/6 mice showed higher bioavailability in choroid and retina when compared to the uncoated nanoparticles. The delivery system showed sustained release of drug for 300 h and exhibited antimicrobial effects against Gram-positive and Gram-negative bacteria and anti-inflammatory effects on activated microglial cells. Interestingly, the combination of the nanoparticles loaded with azithromycin and the nanoparticles loaded with triamcinolone acetonide acted synergistically as compared to either of the nanoparticles/drugs alone. Overall, the developed dual-drug delivery system is non-invasive, has antimicrobial and anti-inflammatory effects, and shows potential as an eye drop formulation against endophthalmitis.

Aged Nrf2-Null Mice Develop All Major Types of Age-Related Cataracts.

Purpose: Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 -/-). Methods: Three independent cohorts of Nrf2 -/- and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results: Nrf2 -/- mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 -/- mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions: Nrf2 -/- mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.

Fate of GdF3 nanoparticles-loaded PEGylated carbon capsules inside mice model: a step toward clinical application.

The successful translation of nanostructure-based bioimaging and/or drug delivery system needs extensive in vitro and in vivo studies on biocompatibility, biodistribution, clearance, and toxicity for its diagnostic applications. Herein, we have investigated the in vitro cyto-hemocompatibility, in vivo biodistribution, clearance, and toxicity in mice after systemic administration of GdF3 nanoparticles loaded PEGylated mesoporous carbon capsule (GdF3-PMCC)-based theranostic system. In vitro cyto-hemocompatibility study showed a very good biocompatibility up to concentration of 500 µg/ml. Biodistribution studies carried out from 1 h to 8 days showed that GdF3-PMCC was found in major organs, such as liver, kidney, spleen, and muscle till 4th day and it was negligible in any tissue after 8th day. The clearance study was carried out for a period of 8 days and it was observed that the urinary system is the main route of excretion of GdF3-PMCC. The tissue toxicity study was done for 15 days and histopathological analysis indicated that the GdF3-PMCC based theranostic system does not have any adverse effect in tissues. Thus, PMCCs are nontoxic and can be applied as theranostic agents in contrast to the other carbon-based systems (PEGylated carbon nanotubes and PEGylated graphene oxide) which showed significant toxicity.

A non-invasive nanoparticle mediated delivery of triamcinolone acetonide ameliorates diabetic retinopathy in rats.

Diabetic retinopathy (DR) is a multifactorial manifestation associated with microvascular complications and is the fourth leading cause of visual impairment and blindness world-wide. Current day treatment of DR relies heavily on invasive techniques such as intravitreal injections of therapeutic agents. Unfortunately, intravitreal injections are associated with various complications such as intraocular bleeding, endophthalmitis, pain and discomfort resulting in poor patient compliance. To date, there has been no non-invasive drug delivery system reported for DR treatment. To address this, we developed a core-shell nanoparticle-based delivery system consisting of a hydrophobic polycaprolactone core and a hydrophilic Pluronic® F68 shell, loaded with triamcinolone acetonide and evaluated its efficacy in a DR rat model. After being administered as eye drops, the drug loaded nanoparticles significantly improved structural (retinal thickness and vascular health) and functional activity (rod and cone function) of retina as compared to DR controls that were treated with the drug alone or placebo nanoparticles. Furthermore, drug loaded nanoparticles reduced retinal inflammation as evidenced by a decrease in NF-κB, ICAM-1 and TNFα expression after 20 days of treatment. Similarly, a reduction in glial cell hyperplasia as evidenced by reduced GFAP expression, and a decrease in microvascular complications as evidenced by a decrease in VEGF secretion and microvascular tuft formation were observed in rat retinas after 40 days of treatment. The combined reduction in retinal inflammation and vascular abnormalities, both hallmarks of DR, demonstrates the potential of the nanoparticulate delivery system for use as a topical formulation for treating DR.

Understanding the influence of surface properties of nanoparticles and penetration enhancers for improving bioavailability in eye tissues in vivo.

Nanoparticulate drug delivery systems, mucoadhesive polymers and penetration enhancers have been used individually to overcome ocular barriers and increase bioavailability to eye tissues. However the combined influence of mucoadhesive polymer coating and penetration enhancers on NP permeability has not been investigated. Hence, in this study, three types of core-shell nanoparticles with same hydrophobic core but different hydrophilic mucoadhesive shells were developed. Initially the influence of a single penetration enhancer (PE) [benzalkonium chloride (BAC)] on ocular permeability of all the three core-shell nanoparticles was studied. Then ocular permeability of a single nanoparticle system [polycaprolactone-pluronicF68 (PCL-PF68)] in presence of different types of PEs namely BAC, capric acid (CA), EDTA, sodium glycocolate (SG) and sodium taurocholate (ST) was studied. The results indicated that BAC differentially enhanced ocular permeability of nanoparticles depending on their surface properties. All the PEs except EDTA enhanced ocular permeability of PCL-PF68 nanoparticles to anterior part of the eye. BAC and CA showed increased bioavailability of PCL-PF68 nanoparticles in conjunctiva, SG in cornea, iris and ciliary body, and ST in cornea. Overall, the combination of PEs and surface properties of nanoparticles can differentially influence ocular permeability and bioavailability and can be advantageously used to develop improved ocular drug delivery systems.

PEGylated Carbon Nanocapsule: A Universal Reactor and Carrier for In Vivo Delivery of Hydrophobic and Hydrophilic Nanoparticles.

We have developed PEGylated mesoporous carbon nanocapsule as a universal nanoreactor and carrier for the delivery of highly crystalline hydrophobic/hydrophilic nanoparticles (NPs) which shows superior biocompatibility, dispersion in body fluids, good biodistribution and NPs independent cellular uptake mechanism. The hydrophobic/hydrophilic NPs without surface modification were synthesized in situ inside the cavities of mesoporous carbon capsules (200-850 nm). Stable and inert nature of carbon capsules in a wide range of reaction conditions like high temperature and harsh solvents, make it suitable for being used as nano/microreactors for the syntheses of a variety of NPs for bioimaging applications, such as NaYF4:Eu(3+)(5%), LaVO4:Eu(3+)(10%), GdVO4:Eu(3+)(10%), Y2O3:Eu(3+)(5%), GdF3:Tb(3+)(10%), Mo, Pt, Pd, Au, and Ag. Multiple types of NPs (Y2O3:Eu(3+)(5%) (hydrophobic) and GdF3:Tb(3+)(10%) (hydrophilic)) were coloaded inside the carbon capsules to create a multimodal agent for magneto-fluorescence imaging. Our in vivo study clearly suggests that carbon capsules have biodistribution in many organs including liver, heart, spleen, lungs, blood pool, and muscles.