Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.

Allergic conjunctivitis is one of the most common external eye diseases and the prevalence has been increasing. The mainstay of treatment is topical eye drops. However, low bioavailability, low ocular drug penetration, transient resident time on the ocular surface due to tear turnover, frequent topical applications and dependence on patient compliance, are the main drawbacks associated with topical administration. Nanotechnology-based medicine has emerged to circumvent these limitations, by encapsulating the drugs and preventing them from degradation and therefore providing sustained and controlled release. Using a nanotechnology-based approach to load the drug is particularly useful for the delivery of hydrophobic drugs such as immunomodulatory agents, which are commonly used in allergic conjunctival diseases. In this review, different nanotechnology-based drug delivery systems, including nanoemulsions, liposomes, nanomicelles, nanosuspension, polymeric and lipid nanoparticles, and their potential ophthalmic applications, as well as advantages and disadvantages, are discussed. We also summarize the results of present studies on the loading of immunomodulators or nonsteroidal anti-inflammatory drugs to nano-scaled drug delivery systems. For future potential clinical use, research should focus on the optimization of drug delivery designs that provide adequate and effective doses with safe and satisfactory pharmacokinetic and pharmaco-toxic profiles.

Age-related macular degeneration (AMD) is a leading cause of blindness in the modern world. The standard treatment regimen for neovascular AMD is the monthly/bimonthly intravitreal injection of anti-VEGF agents such as ranibizumab or aflibercept. However, these repeated invasive injections can lead to sight-threatening complications. Sustained delivery by encapsulation of the drug in carriers is a way to reduce the frequency of these injections. Liposomes are biocompatible, non-toxic vesicular nanocarriers, which can be used to encapsulate therapeutic agents to provide sustained release. The protein encapsulation was performed by a modified dehydration-rehydration (DRV) method. The liposomes formed were characterized for size, zeta potential, encapsulation efficiency, stability, in vitro release, and ex vivo release profiles. In addition, the localization of the liposomes themselves was studied ex vivo. Entrapment-efficiency of ranibizumab into 100-nm liposomes varied from 14.7 to 57.0%. Negatively-charged liposomes prepared from DPPC-DPPG were found to have the slowest release with a low initial burst release compared to the rest of liposomal formulations. The ex vivo protein release was found to slower than the in vitro protein release for all samples. In conclusion, the DPPC-DPPG liposomes significantly improved the encapsulation and release profile of ranibizumab.

Valproic acid (VPA) is a histone deacetylase inhibitor used clinically for neurological disorders. It is also potentially useful as anti-fibrotic therapy as it reduced collagen deposition in the post-operative conjunctiva. In this study, we further evaluated the effects of VPA on post-operative inflammation using the mouse model of conjunctival scarring. VPA, injected into the subconjunctiva immediately after surgery, did not cause any adverse tissue response when examined by live microscopy and produced an apparent reduction of proinflammatory and proangiogenic markers in immunohistological examinations. In-depth analyses of the treated operated tissues revealed that VPA selectively inhibited the CD45^highF4/80^low macrophage subset as well as the production of specific proinflammatory cytokines/ chemokines, including CXCL1, IL-5, IL-6, and IL-10 which were reduced by ≥ 2.0-fold. VPA also specifically reduced tissue NF-кB2 p100 protein by mean 3.87-fold. On conjunctival fibroblasts, VPA treatment resulted in decreased secretion of specific cytokines, including CCL2, VEGF-A, and IL-15. In the presence of TNF-α, VPA inhibited the induction of specific cytokines/chemokines, notably CCL5 and VEGF-A, as well as NF-кB2 p100. In corroboration, VPA suppressed TNF-α stimulation of NF-кB reporter transcription by 1.51-fold. These data indicate that VPA can modulate both proinflammatory cellular and molecular targets in a selective manner and may therefore attenuate surgery-induced conjunctival inflammation. These and previous findings suggest that, by suppressing key mediators of both inflammation and fibrosis, VPA is a useful therapeutic for improving surgical outcome involving the conjunctiva. Key messages VPA inhibited recruitment of a CD45^highF4/80^low macrophage subset. VPA reduced chemokine and cytokine levels in treated tissues. VPA selectively suppressed tissue NF-кB2 p100 levels. VPA suppressed TNF-α induction of chemokines, cytokines and NF-кB2 p100 expression. VPA suppressed TNF-α stimulation of NF-кB reporter.

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.