Penetrative Ionic Organic Molecular Cage Nanozyme for the Targeted Treatment of Keratomycosis.

Keratomycosis, caused by pathogenic fungi, is an intractable blinding eye disease. Corneal penetration is an essential requirement for conventional antifungal medications to address keratomycosis. Due to the distinctive anatomical and physiological structure of the cornea, the therapeutic efficacy is hampered by the inadequate penetration capacity. Despite the emergence of diverse antifungal drug delivery systems and advanced antifungal nanomaterials, it has remained challenging to achieve corneal penetration over the past decade. This study fabricates a penetrative ionic organic molecular cage-based nanozyme (OMCzyme) for treating keratomycosis. The synthesis of OMCzyme involved two steps. Initially, the ionic OMC is synthesized by a [2+3] cycloimination reaction of triformylphloroglucinol and 2,3-diaminopropionic acid. Subsequently, OMCzyme is fabricated by coordination of Fe2⁺ with carboxyl anions and phenolic hydroxyls in the organic cage, and further deposition of silver nanoparticles on the surface of OMC-Fe complex. The as-prepared OMCzyme demonstrates excellent water dispersion, peroxidase-like activity, in vitro and in vivo biocompatibility, and corneal penetration. Notably, the nanozyme displays targeted antifungal activity, effectively combating Fusarium solani with negligible cytotoxicity toward human corneal epithelial cells. The hybrid mimic is further demonstrated to be effective in treating keratomycosis in mice, indicating the potential of OMCzyme for curing fungal infectious diseases.

Injectable hydrogels embedded with chitosan nanoparticles coated with hyaluronic acid for sequential release of dual drugs.

An effective treatment for some disease, such as the model disease acute retinal necrosis (ARN), requires a combination of different drugs which should be administered at a certain interval. The precise sequential and long-term drug release are the critical questions. In this work, the as-prepared chitosan nanoparticles (CS-NPs) coated with hyaluronic acid (HA) were embedded in the aldehyde ß-cyclodextrin (ACD)/aminated hyaluronic acid (NHA) hydrogels to synthesize injectable hydrogels loaded with dual drugs named DEX-CS-NPs/GCV-Gel and HA-DEX-CS-NPs/GCV-Gel. In the first 24 h and 48 h, the releases of DEX from DEX-CS-NPs/GCV-Gel were 128.5 % and 82.8 % faster than those from HA-DEX-CS-NPs/GCV-Gel, respectively. There was no DEX released from HA-DEX-CS-NPs/GCV-Gel at the first 7 h, which has never been reported before, although some hydrogel systems loaded with different drugs release different drugs simultaneously at different rate which have been well studied. This is a good start of a precise sequence release. The composite hydrogels possessed appropriate rheology, gel time, degradation performance, and ideal cytocompatibility. The injectable hydrogel loaded with dual drugs presenting a precise sequential and long-term release has great potential in the treatment of diseases requiring combinations of drugs being released sequentially at different treating stages.

An injectable hydrogel based on hyaluronic acid prepared by Schiff base for long-term controlled drug release.

Drug-loaded injectable hydrogels have been studied widely in biomedical technology while the stable long-term controlled drug release and cytotoxicity are challenges. In this work, an injectable hydrogel with good swelling resistance was in situ synthetized using aminated hyaluronic acid (NHA) and aldehyde ß-cyclodextrin (ACD) via Schiff base reaction. The composition, morphology and mechanical property were characterized with FTIR, 13C NMR, SEM and rheology test, respectively. Voriconazole (VCZ) and Endophthalmitis was selected as a model drug and disease, respectively. The drug release, cytotoxicity and antifungal properties were detected in vitro. The results showed a long-term (> 60 days) drug release was realized, the NHA/ACD2/VCZ presented a zero-order release in the later stage. The cytotoxicity of NHA/ACD was detected by live/dead staining assay and Cell Counting Kit-8 (CCK-8). The survival rate of adult retina pigment epithelial cell line-19 (ARPE-19) was over 100 % after 3 d, it indicated a good cytocompatibility. The antifungal experiment presented samples had antifungal property. Biocompatibility in vivo proved NHA/ACD2 had no adverse effects on ocular tissues. Consequently, the injectable hydrogel based on hyaluronic acid prepared by Schiff base reaction provides a new option for long-term controlled drug release in the course of disease treatment from a material perspective.

ECM-Like Adhesive Hydrogel for the Regeneration of Large Corneal Stromal Defects.

The repair of large-diameter corneal stroma defects is a major clinical problem. Although some studies have attempted to use hydrogels to repair corneal damage, most of these hydrogels can only be used for focal stromal defects that are ≤3.5 mm in diameter due to poor hydrogel adhesion. Here, a photocurable adhesive hydrogel that mimics the extracellular matrix (ECM) with regard to composition for repairing 6 mm-diameter corneal stromal defects in rabbits is investigated. This ECM-like adhesive can be rapidly cured after light exposure, with high light transmittance and good mechanical properties. More importantly, this hydrogel maintains the viability and adhesion of cornea-derived cells and promotes their migration in vitro in 2D and 3D culture environments. Proteomics analysis confirms that the hydrogel promotes cell proliferation and ECM synthesis. Furthermore, in rabbit corneal stromal defect repair experiments, it is proven by histological and proteomic analysis that this hydrogel can effectively promote corneal stroma repair, reduce scar formation, and increase corneal stromal-neural regeneration at the six months follow-up. This work demonstrates the great application of ECM-like adhesive hydrogels for the regeneration of large-diameter corneal defects.

Nicotinamide suppresses bevacizumab-induced epithelial-mesenchymal transition of ARPE-19 cells by attenuating oxidative stress.

AIM: To investigate the effects of nicotinamide (NAM) on bevacizumab (BEV)-induced epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial cells (ARPE-19) and the underling mechanisms. METHODS: ARPE-19 cells were treated with BEV for 24, 48, and 72h, and the variation degrees of EMT-related markers (fibronectin, α-SMA, vimentin, and ZO-1) were assessed by Western blotting to select the optimal treatment time point which exhibited the most obvious changes of EMT-related markers for the subsequent experiments. Furthermore, NAM was added to the medium, the mRNA and protein levels of the EMT-related markers were then measured. The accumulation of reactive oxygen species (ROS) and H2O2 and the total antioxidant capacity (TAC) of the cells were also measured to evaluate the level of oxidative stress. RESULTS: After being treated with BEV for 72h, the protein expression levels of EMT-related markers in ARPE-19 cells showed significant changes. Meanwhile the levels of ROS and H2O2 were obviously increased, and the TAC of ARPE-19 cells was decreased. Totally 72h was chosen to be the optimal treatment time point in subsequent experiments. Furthermore, NAM inhibited BEV-induced EMT by downregulating fibronectin, α-SMA, and vimentin and upregulating ZO-1, decreased the accumulation of ROS and H2O2, and enhanced TAC in BEV-treated ARPE-19 cells. CONCLUSION: This study demonstrates that NAM suppressed BEV-induced EMT in ARPE-19 cells by attenuating oxidative stress. Hence, NAM may be a potential therapeutic agent for alleviating neovascular fibrosis of the ocular fundus after anti-vascular endothelial growth factor therapy.

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4.

AIM: To investigate the effect of leucine-rich-alpha-2-glycoprotein 1 (LRG1) on epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, and to explore the role of NADPH oxidase 4 (NOX4). METHODS: RPE cells (ARPE-19 cell line) were treated with transforming growth factor-ß1 (TGF-ß1) to induce EMT. Changes of the mRNA and protein expression levels of LRG1 were tested in the TGF-ß1 treated cells. The recombinant human LRG1 protein (rLRG1) and siRNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively, and to detect EMT-related markers including fibronectin, α-smooth muscle actin (α-SMA) and zonula occludens-1 (ZO-1). The mRNA and protein expression level of NOX4 were measured according to the above treatments. VAS2870 was used as a NOX4 inhibitor in rLRG1-treated cells. EMT-related markers were detected to verify the effect of NOX4 in the process of EMT. RESULTS: TGF-ß1 promoted the expression of LRG1 at both the mRNA and protein levels during the process of EMT which showed the up-regulation of fibronectin and α-SMA, as well as the down-regulation of ZO-1. Furthermore, the rLRG1 promoted EMT of ARPE-19 cells, which manifested high levels of fibronectin and α-SMA and low level of ZO-1, whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin and α-SMA and increasing the expression of ZO-1 in ARPE-19 cells. Besides, the rLRG1 activated and LRG1 siRNA suppressed NOX4 expression. EMT was inhibited when VAS2870 was used in the rLRG1-treated cells. CONCLUSION: These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4, which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

Long-Term Efficacy of Intravitreal Conbercept Injection in the Treatment of Idiopathic Choroidal Neovascularization.

Purpose: To investigate the long-term efficacy of intravitreal conbercept injection in treating idiopathic choroidal neovascularization (CNV). Methods: In this longitudinal retrospective cohort study, 27 eyes (27 patients) with idiopathic CNV receiving conbercept intravitreously (0.5 mg/0.05 mL) with 1+PRN regimen were included. Best corrected visual acuity (BCVA) and central retinal thickness (CRT) at the baseline and the end of follow-up were compared, respectively. The number of injections and recurrence time were evaluated. Results: Of the 27 patients, 7 were men and 20 were women. Their mean age at diagnosis was 31.37 ± 7.35 (16-46) years. The follow-up period was 44.59 ± 8.60 (27-58) months. The mean initial injection number was 1.22 ± 0.42, and 1.67 ± 1.04 injections were administered throughout the follow-up. Seven patients experienced CNV relapse, with 1 episode in 4 patients, 2 episodes in 2 patients, and 5 episodes in 1 patient. LogMAR BCVA changed from 0.77 ± 0.19 at baseline to 0.09 ± 0.10 at the final visit, and CRT decreased from 343 ± 73.5 µm to 172.41 ± 30.82 µm. Both BCVA improvement and CRT reduction were statistically significant (P < 0.001). No ocular or systemic complications occurred. Conclusions: Intravitreal injection of conbercept shows favorable effectiveness in the treatment of idiopathic CNV during a long-term period of follow-up.

Focal choroidal excavation complicated by choroidal neovascularization.

PURPOSE: To evaluate the clinical findings of focal choroidal excavation (FCE) complicated by choroidal neovascularization (CNV). DESIGN: Retrospective, observational case series. PARTICIPANTS: Twelve patients (15 eyes) with FCE and CNV. METHODS: The medical records of the patients were reviewed. Clinical findings including age, sex, refraction, color photography, fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), and spectral-domain optical coherence tomography (OCT) were analyzed. MAIN OUTCOME MEASURES: Fundus fluorescein angiography, ICGA, and OCT findings. RESULTS: The 12 patients included 6 women and 6 men. The mean age was 46.8±13.4 years (range, 26-64 years). One half of the patients were emmetropic, and the others were myopic (-0.5 to -3.0 diopters). All subjects were Chinese. Before CNV development, normal appearance or nonspecific pigment disturbance could be seen around the choroidal excavation. Corresponding to the excavation, window defects were observed by FFA, whereas hypofluorescence was found on ICGA images. Choroidal neovascularization in all eyes was classic as revealed by FFA. The OCT images showed that all eyes had a single choroidal excavation. In 7 of the 15 eyes, the choroidal excavation was located subfoveally, and in the other 8 eyes, it was eccentric. All CNV lesions grew from the bottom or slope of the excavation. Three patients had bilateral involvement. Choroidal neovascularization occurred in both conforming and nonconforming type FCEs, regardless of whether the excavation was shallow or deep, subfoveal or eccentric. All CNV lesions responded well to intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents. After a single injection, CNV regressed in 13 of 15 eyes. Two eyes received an additional injection. Nonconforming FCE changed to the conforming type after successful treatment of CNV. CONCLUSIONS: Focal choroidal excavation is not always stable. Choroidal neovascularization commonly can be seen in patients with FCE and responds well to intravitreal anti-VEGF agents.