Engineering Matrix-Free Drug Protein Nanoparticles with Promising Penetration through Biobarriers for Treating Corneal Neovascularization

Protein drugs have been widely used in treating various clinical diseases because of their high specificity, fewer side effects, and favorable therapeutic effect, but they greatly suffer from their weak permeability through tissue barriers, high sensitivity to microenvironments, degradation by proteases, and rapid clearance by the immune system. Herein, we disrupted the standard protocol where protein drugs must be delivered as the cargo via a delivery system and innovatively developed a free entrapping matrix strategy by simply mixing bevacizumab (Beva) with zinc ions to generate Beva-NPs (Beva-Zn2+), where Beva is coordinatively cross-linked by zinc ions with a loading efficiency as high as 99.2% ± 0.41%. This strategy was universal to generating various protein NPs, with different metal ions (Cu2+, Fe3+, Mg2+, Sr2+). The synthetic conditions of Beva-NPs were optimized, and the generated mechanism was investigated in detail. The entrapment, releasing profile, and the bioactivities of released Beva were thoroughly studied. By using in situ doping of the fourth-generation polyamindoamine dendrimer (G4), the Beva-G4-NPs exhibited extended ocular retention and penetration through biobarriers in the anterior segment through transcellular and paracellular pathways, effectively inhibiting corneal neovascularization (CNV) from 91.6 ± 2.03% to 13.5 ± 1.87% in a rat model of CNV. This study contributes to engineering of protein NPs by using a facile strategy for overcoming the weaknesses of protein drugs and protein NPs, such as weak tissue barrier permeability, low encapsulation efficiency, poor loading capacity, and susceptibility to inactivation

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

Engineering Matrix-Free Drug Protein Nanoparticles with Promising Penetration through Biobarriers for Treating Corneal Neovascularization

Protein drugs have been widely used in treating various clinical diseases because of their high specificity, fewer side effects, and favorable therapeutic effect, but they greatly suffer from their weak permeability through tissue barriers, high sensitivity to microenvironments, degradation by proteases, and rapid clearance by the immune system. Herein, we disrupted the standard protocol where protein drugs must be delivered as the cargo via a delivery system and innovatively developed a free entrapping matrix strategy by simply mixing bevacizumab (Beva) with zinc ions to generate Beva-NPs (Beva-Zn2+), where Beva is coordinatively cross-linked by zinc ions with a loading efficiency as high as 99.2% ± 0.41%. This strategy was universal to generating various protein NPs, with different metal ions (Cu2+, Fe3+, Mg2+, Sr2+). The synthetic conditions of Beva-NPs were optimized, and the generated mechanism was investigated in detail. The entrapment, releasing profile, and the bioactivities of released Beva were thoroughly studied. By using in situ doping of the fourth-generation polyamindoamine dendrimer (G4), the Beva-G4-NPs exhibited extended ocular retention and penetration through biobarriers in the anterior segment through transcellular and paracellular pathways, effectively inhibiting corneal neovascularization (CNV) from 91.6 ± 2.03% to 13.5 ± 1.87% in a rat model of CNV. This study contributes to engineering of protein NPs by using a facile strategy for overcoming the weaknesses of protein drugs and protein NPs, such as weak tissue barrier permeability, low encapsulation efficiency, poor loading capacity, and susceptibility to inactivation

Epigenetic Activation of Circadian Clock Genes Elicits Inflammation in Experimental Murine Dry Eye

To explore whether circadian clock genes contribute to elicit inflammation in experimental dry eye (EDE). RNA sequencing analyzed mRNA expression patterns in EDE model. RT-qPCR and/or Western blot determined the expression of inflammatory factors and circadian genes during EDE. MethylTarget™ assays determined the promoter methylation levels of Per genes in vivo. Per2 or Per3 knockdown assessed their effects on inflammatory factors in vitro. We utilized an intelligently controlled environmental system (ICES) to establish a mouse EDE model. The significant upregulated genes were enriched for circadian rhythms. Therein lied oscillatory and time-dependent upregulation of PER2 and PER3, as well as their promoter hypomethylation during EDE. Silencing PER2 or PER3 significantly decreased inflammatory factor expression and also reversed such increased inflammatory response in azacitidine (AZA) treatment in vitro model. Our findings suggest that DNA methylation mediated the upregulation of PER2 and PER3, leading to inflammatory response in EDE.</p

Table_1_The Association Between Sleep Disorders and Incidence of Dry Eye Disease in Ningbo: Data From an Integrated Health Care Network.DOCX

PurposeTo investigate the association between sleep disorders and dry eye disease (DED) in Ningbo, China.MethodsOur data came from the Yinzhou Health Information System (HIS), including 257932 patients and was based on a 1:1 matching method (sleep disorder patients vs. patients without sleep disorders) during 2013–2020. Sleep disorders and DED were identified using ICD-10 codes. Cox proportional hazards regression was used to identify the association between sleep disorders and DED.ResultsThe eight-year incidence of DED was significantly higher in participants with diagnosis of sleep disorders (sleep disorders: 50.66%, no sleep disorders: 16.48%, P ConclusionsSleep disrder was associated with a three-time increased risk of DED. This association can be helpful in effective management of both sleep disorders and DED.</p

Diagram of different routes for subretinal injections.

<p>This diagram showed the routes of trans-corneal and trans-scleral subretinal injections including the different routes to adult and neonatal mice.</p

The b-wave amplitudes of standard combined ERG at 5 weeks post-injection.

<p>A. B-wave amplitudes of standard combined ERG signals from four groups of mice. B. Statistical analysis of the b-wave amplitudes. Rod and cone mixed responses were elicited at 0 log cd s/m² intensity. N = 5 for each group. B-wave amplitudes of the non-injected group were higher than those from the other group. *P < 0.05.</p

OCT images of mouse retinas.

<p>The OCT images of A1-A3 were from age-matched uninjected control C57BL/6J eyes. The OCT images showed retinal re-attachment at post-injection days 1 (B1), 2 (B2), and 3 (B3) in the pseudo-injection group; at post-injection days 1 (C1), 2 (C2), and 3 (C3) in the group with 50–70% coverage; and at post-injection days 1 (D1), 2 (D2), and 3 (D3) in the group with 80–100% coverage.</p

Histopathology of retinas by H&E staining from 4 groups of mice at 5 weeks post-injection.

<p>(A) Representative retinal images of H&E-stained retinal section from age-matched normal uninjected eyes (A1–A3; A2 and A3 are insets of A1 with yellow-frames). (B) Representative retinal image from the pseudo-injection group (B1–B3; B2 is inset of B1 with black-frame, B3 is inset of B1 with yellow-frame). (C) Representative retinal images from the group with 50–70% coverage (C1–C3; C2 is inset of C1 with black-frame, C3 is inset of C1 with yellow-frame). (D) Representative retinal images from the group with 80–100% coverage (D1-D3; D2 is inset of D1 with black-frame, D3 is inset of D1 with yellow-frame). The structure of retinas outside of injection sites were normal (B3, C3 and D3). ONL, outer nuclear layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment; red arrow, injection-related damages of outer segments.</p

statistical analysis of the b-wave amplitudes.

<p>statistical analysis of the b-wave amplitudes.</p