Objective Engagement of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells (ECs) by ICAM-1-targeted carriers induces cell adhesion molecule (CAM)-mediated endocytosis, providing intra-endothelial delivery of therapeutics. This pathway differs from classical endocytic mechanisms and invokes aspects of endothelial signaling during inflammation. ICAM-1 interacts with Na+/H+ exchanger NHE1 during endocytosis, but it is unclear how this regulates plasmalemma and cytoskeletal changes. We studied such aspects in this work. Methods and Results We used fluorescence and electron microscopy, inhibitors and knockout tools, cell culture and mouse models. ICAM-1 engagement by anti-ICAM carriers induced sphingomyelin-enriched engulfment structures. Acid sphingomyelinase (ASM), an acidic enzyme that hydrolyzes sphingomyelin into ceramide (involved in plasmalemma deformability and cytoskeletal reorganization), redistributed to ICAM-1-engagement sites at ceramide-enriched areas. This induced actin stress fibers and carrier endocytosis. Inhibiting ASM impaired ceramide enrichment, engulfment structures, cytoskeletal reorganization, and carrier uptake, which was rescued by supplying this enzyme activity exogenously. Interfering with NHE1 rendered similar outcomes, suggesting that Na+/H+ exchange might provide an acidic microenvironment for ASM at the plasmalemma. Conclusions These findings are consistent with the ability of ECs to internalize relatively large ICAM-1-targeted drug carriers, and expand our knowledge on the regulation of the sphingomyelin/ceramide pathway by the vascular endothelium.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8. GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGFinduced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

Targeting of drug carriers to endocytic cell-receptors facilitates intracellular drug delivery. Carrier size and number of targeting moieties (valency) influence cell binding and uptake. However, how these parameters influence receptor-mediated cell-signaling (the link between binding and uptake) remains uncharacterized. We studied this using polymer carriers of different sizes and valencies, targeted to endothelial intercellular adhesion molecule-1 (ICAM-1), a marker overexpressed in many pathologies. Unexpectedly, induction of cell-signals (ceramide and PKC enrichment and activation) and uptake, were independent of: carrier avidity, total number of carriers bound per cell, cumulative cell-surface area occupied by carriers, number of targeting antibodies at the carrier-cell contact, and cumulative receptor engagement by all bound carriers. Instead, “valency density” (number of antibodies per carrier surface area) ruled signaling, and carrier size independently influenced uptake. These results are key to understanding the interplay between carrier design parameters and receptor-mediated signaling conducive to endocytosis, paramount for intracellular drug delivery.

Metipranolol is a β‐adrenergic receptor antagonist that is given orally for the treatment of hypertension and also applied topically to the cornea for treating glaucoma. It also inhibits nitrosative stress which has previously been shown to be the cause of cone photoreceptor death in retinitis pigmentosa. In this study, we tested the hypothesis that metipranolol protects photoreceptor structure and function in the mouse model rd10. At P35, compared with vehicle‐treated rd10 mice in which rod degeneration was nearly complete, rd10 mice given daily subcutaneous injections of 40 mg/kg of metipranolol had reduction in markers of nitrosative stress, fewer TUNEL‐positive cells, increased outer nuclear layer thickness, and substantially more staining for rhodopsin. This was accompanied by significantly higher mean scotopic and photopic electroretinogram b‐wave amplitudes indicating improved photoreceptor function. At P50, metipranolol‐treated rd10 mice had decreased 3‐nitrotyrosine staining in the retina, increased immunostaining for cone arrestin, a marker for cone photoreceptors, and significantly higher scotopic and photopic b‐wave amplitudes at the highest stimulus intensity compared with vehicle‐treated mice. At P65, cone density was significantly higher in metipranolol‐treated versus vehicle‐injected rd10 mice. Metipranolol applied as eye drops promoted cone photoreceptor function in retinas of rd10 mice greater than subcutaneously injected metipranolol. The reduced nitrosative damage and rescue of functional loss of photoreceptors in rd10 mice suggests that metipranolol, a drug with established ocular safety and tolerability, may have potential for treating patients with retinitis pigmentosa.