Suprachoroidal Delivery in Rats and Guinea Pigs Using a High-Precision Microneedle Injector.

Purpose: Methods of injection into the suprachoroidal space (SCS) have been developed for larger animals and humans, but reliable administration to the SCS of rodents remains challenging given their substantially smaller eyes. Here, we developed microneedle (MN)-based injectors for SCS delivery in rats and guinea pigs. Methods: We optimized key design features, including MN size and tip characteristics, MN hub design, and eye stabilization, to maximize injection reliability. Performance of the injection technique was characterized in rats (n = 13) and guinea pigs (n = 3) in vivo using fundoscopy and histological examinations to validate targeted SCS delivery. Results: To enable SCS injection across the thin rodent sclera, the injector featured an ultrasmall, hollow MN measuring 160 µm in length for rats and 260 µm for guinea pigs. To control MN interaction with the scleral surface, we incorporated a three-dimensional (3D) printed needle hub to restrict scleral deformation at the injection site. A MN tip outer diameter of 110 µm and bevel angle of 55° optimized insertion without leakage. Additionally, a 3D printed probe was used to secure the eye by applying gentle vacuum. Injection by this technique took 1 minute to perform, was conducted without an operating microscope, and yielded a 100% success rate (19 of 19) of SCS delivery determined by fundoscopy and histology. A 7-day safety study revealed no notable adverse ocular effects. Conclusions: We conclude that this simple, targeted, and minimally invasive injection technique can enable SCS injection in rats and guinea pigs. Translational Relevance: This MN injector for rats and guinea pigs will expand and expedite preclinical investigations involving SCS delivery.

Enhanced immune responses to vaccine antigens in the corneal stroma.

To examine the widely accepted dogma that the eye is an immune-privileged organ that can suppress antigen immunogenicity, we explored systemic immune responses to a model vaccine antigen (tetanus toxoid) delivered to six compartments of the rodent eye (ocular surface, corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, vitreous body). We discovered that antigens delivered to corneal stroma induced enhanced, rather than suppressed, antigen-specific immune responses, which were 18- to 30-fold greater than conventional intramuscular injection and comparable to intramuscular vaccination with alum adjuvant. Systemic immune responses to antigen delivered to the other ocular compartments were much weaker. The enhanced systemic immune responses after intrastromal injection were related to a sequence of events involving the formation of an antigen "depot" in the avascular stroma, infiltration of antigen-presenting cells, up-regulation of MHC class II and costimulatory molecules CD80/CD86, and induction of lymphangiogenesis in the corneal stroma facilitating sustained presentation of antigen to the lymphatic system. These enhanced immune responses in corneal stroma suggest new approaches to medical interventions for ocular immune diseases and vaccination methods.

Six-month sustained delivery of anti-VEGF from in-situ forming hydrogel in the suprachoroidal space.

Patients with wet age-related macular degeneration (AMD) require intravitreal injections of bevacizumab (Bev) or other drugs, often on a monthly basis, which is a burden on the healthcare system. Here, we developed an in-situ forming hydrogel comprised of Bev and hyaluronic acid (HA) crosslinked with poly(ethylene glycol) diacrylate for slow release of Bev after injection into the suprachoroidal space (SCS) of the eye using a microneedle. Liquid Bev formulations were cleared from SCS within 5 days, even when formulated with high viscosity, unless Bev was conjugated to a high molecular-weight HA (2.6 MDa), which delayed clearance until 1 month. To extend release to 6 months, we synthesized in-situ forming Bev-HA hydrogel initially as a low-viscosity mixture suitable for injection and flow in the SCS to cover a large area extending to the posterior pole of the eye where the macula is located in humans. Within 1 h after injection, Bev and HA were crosslinked, which retained Bev for slow release as the hydrogel biodegraded. In vivo studies in the rabbit eye reported Bev release for >6 months, depending on gel formulation and Bev assay. The in-situ forming Bev-HA hydrogel was well tolerated, as assessed by clinical exam, fundus imaging, histological analysis, and intraocular pressure measurement. We conclude that Bev released from an in-situ forming hydrogel may enable long-acting treatments of AMD and other posterior ocular indications.

Computational modeling of corneal and scleral collagen photocrosslinking.

Scleral photocrosslinking is increasingly investigated for treatment of myopia and glaucoma. In this study a computational model was developed to predict crosslinking efficiency of visible/near infrared photosensitizers in the sclera. Photocrosslinking was validated against riboflavin corneal crosslinking experimental studies and subsequently modeled for the sensitizer, methylene blue, administered by retrobulbar injection to the posterior sclera and irradiated with a transpupillary light beam. Optimal ranges were determined for treatment parameters including light intensity, methylene blue concentration, injection volume, and inspired oxygen concentration. Additionally, sensitivity of crosslinking to various parameters was quantified. The most sensitive parameters were oxygen concentration in the injection solution, scleral thickness, and injection reservoir thickness (i.e., injection volume).