Silvia Muro

Endocytosis in endothelial cells (ECs) is important for many biomedical applications, including drug delivery by nano- and microscale carriers. However, little is known about how carrier geometry influences endothelial drug targeting, intracellular trafficking, and effects. We studied this using prototype polymer carriers of various sizes (0.1–10 μm) and shapes (spheres versus elliptical disks). Carriers were targeted to intercellular adhesion molecule 1 (ICAM-1), a transmembrane glycoprotein that is upregulated in many pathologies and used as a target for intraendothelial drug delivery. ECs internalized anti-ICAM-coated carriers of up to several microns in size via cell adhesion molecule–mediated endocytosis. This pathway is distinct from caveolar and clathrin endocytosis that operate for submicron-size objects. Carrier geometry was found to influence endothelial targeting in the vasculature, and the rate of endocytosis and lysosomal transport within ECs. Disks had longer half-lives in circulation and higher targeting specificity in mice, whereas spheres were endocytosed more rapidly. Micron-size carriers had prolonged residency in prelysosomal compartments, beneficial for endothelial antioxidant protection by delivered catalase. Submicron carriers trafficked to lysosomes more readily, optimizing effects of acid sphingomyelinase (ASM) enzyme replacement in a model of lysosomal storage disease. Therefore, rational design of carrier geometry will help optimize endothelium-targeted therapeutics. Endocytosis in endothelial cells (ECs) is important for many biomedical applications, including drug delivery by nano- and microscale carriers. However, little is known about how carrier geometry influences endothelial drug targeting, intracellular trafficking, and effects. We studied this using prototype polymer carriers of various sizes (0.1–10 μm) and shapes (spheres versus elliptical disks). Carriers were targeted to intercellular adhesion molecule 1 (ICAM-1), a transmembrane glycoprotein that is upregulated in many pathologies and used as a target for intraendothelial drug delivery. ECs internalized anti-ICAM-coated carriers of up to several microns in size via cell adhesion molecule–mediated endocytosis. This pathway is distinct from caveolar and clathrin endocytosis that operate for submicron-size objects. Carrier geometry was found to influence endothelial targeting in the vasculature, and the rate of endocytosis and lysosomal transport within ECs. Disks had longer half-lives in circulation and higher targeting specificity in mice, whereas spheres were endocytosed more rapidly. Micron-size carriers had prolonged residency in prelysosomal compartments, beneficial for endothelial antioxidant protection by delivered catalase. Submicron carriers trafficked to lysosomes more readily, optimizing effects of acid sphingomyelinase (ASM) enzyme replacement in a model of lysosomal storage disease. Therefore, rational design of carrier geometry will help optimize endothelium-targeted therapeutics.

Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.

High-resolution images of oxygen distributions in microheterogeneous samples are obtained by two-photon laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO(2) in cells.