Abstract
Purpose :
We investigated the vitreous barrier function by examining the ocular distribution patterns of fluorescein molecules with varying molecular weights (MW) following intravitreal injection, in an attempt to mimic the distribution of drugs and therapeutic agents like anti-VEGF treatments and AAV-based gene therapy.
Methods :
We studied the distribution of anionic fluorescein dextran dyes with different MW (3 kDa, 40 kDa, 70 kDa, 500 kDa, 2 MDa) in a pig eye ex-vivo model. For all dye weights, injections were performed either temporally or nasally, and subsequently, eyes were placed in PBS on a slow-speed shaker for 24 or 48 hours. The eyes were then fixed in 2% glutaraldehyde for 48 h, bisected, and analyzed for fluorescence area and radius of distribution. In total, 160 images from 80 eyes (40 animals) were analyzed using in-house software, an AI program (Biodock), and statistical analyses such as Student's t-test and ANOVA.
Results :
The lightest fluorescein molecule (3 kDa) showed near-complete diffusion in the vitreous cavity. Within 24 h, it had reached over 98% (±1.4%) of the vitreous cavity area. Slightly heavier fluorescein (40 kDa) covered 83%(±2.1%) of the vitreous cavity area after 24 h and continued to diffuse in the next 24 hours, reaching up to 93%(±2.7%) by the 48 h time point. Fluorescein molecules of 70 kDa, 500 kDa, and 2 MDa showed a distribution of 80±1.56%, 45.3±4.7%; and 37±3.2%, respectively, at 24 h, with no significant change in their diffusion at 48 h. Heavier fluorescein molecules also had a smaller radius of fluorescein distribution: 3 kDa, 40 kDa, 70 kDa, 500 kDa, 2 MDa, respectively, had 11.5±0.18 mm, 10.3±0.16 mm, 10.2±0.15 mm, 7.5±0.36 mm, 6.8±0.27 mm spread at 24 h). Findings following nasal and temporal injection were comparable.
Conclusions :
This study confirms a barrier role for the vitreous. Interestingly, molecules of 40 kDa and 70 kDa MW showed diffusion extension up to, but not past, a crescent-shaped area on the opposite side even as late as 48 h post-injection. This phenomenon suggests the presence of anatomical structures acting as molecular barriers. These findings may have important implications for clinicians when choosing where to inject large MW drugs, such as anti-VEGF treatments and AAV-based gene therapy products.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.