There is a strong need for innovative and efficient drug delivery systems for ocular therapy development. However, testing intravitreal drug delivery systems without using live animals is challenging. Ex vivo animal models offer an interesting alternative. We analyzed the potential of using fresh porcine eyes obtained from the local slaughterhouse as a model for testing the intravitreal biodistribution and retention of liposomes with or without polyethylene glycol (PEG) conjugation and with different surface charges. The histology of the eyes was analyzed to localize the liposomes, and it was found that liposomes with PEG absorbed rapidly on the retina (within 1 h), with positively charged and PEG-coated liposomes being retained for at least 24 h. In parallel, fluorophotometry was employed on intact eyes, to determine the pharmacokinetics of the fluorophore calcein, as a substitute for a small hydrophilic therapeutic compound. We found a 4.5-fold increase in the vitreous half-life of calcein loaded in liposomes, compared with the free solution. Retinal toxicity was addressed using murine-derived retinal explant cultures. Liposomes were non-toxic up to 500 µg/mL. Toxicity was observed at 5 mg/mL for anionic and cationic liposomes, with 2-fold and 2.5-fold increased photoreceptor cell death, respectively. Overall, we could show that important ocular drug delivery considerations such as pharmacokinetics and biodistribution can be estimated in ex vivo porcine eyes, and may guide subsequent in vivo experiments.

Cyclodextrins (CDs) have been widely used as pharmaceutical excipients for formulation purposes for different delivery systems. Recent studies have shown that CDs are able to form complexes with a variety of biomolecules, such as cholesterol. This has subsequently paved the way for the possibility of using CDs as drugs in certain retinal diseases, such as Stargardt disease and retinal artery occlusion, where CDs could absorb cholesterol lumps. However, studies on the retinal toxicity of CDs are limited. The purpose of this study was to examine the retinal toxicity of different beta-(β)CD derivatives and their localization within retinal tissues. To this end, we performed cytotoxicity studies with two different CDs—2-hydroxypropyl-βCD (HPβCD) and randomly methylated β-cyclodextrin (RMβCD)—using wild-type mouse retinal explants, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and fluorescence microscopy. RMβCD was found to be more toxic to retinal explants when compared to HPβCD, which the retina can safely tolerate at levels as high as 10 mM. Additionally, studies conducted with fluorescent forms of the same CDs showed that both CDs can penetrate deep into the inner nuclear layer of the retina, with some uptake by Müller cells. These results suggest that HPβCD is a safer option than RMβCD for retinal drug delivery and may advance the use of CDs in the development of drugs designed for intravitreal administration.

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and Rho^P23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques.