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 mum) 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.
High-resolution images of oxygen distributions in microheterogeneous samples are obtained by twophoton 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.
Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125
Fabry disease due to deficiency of α-galactosidase A (α-Gal) causes lysosomal accumulation of globotriaosylceramide (Gb3) in multiple tissues and prominently in the vascular endothelium. Although enzyme replacement therapy (ERT) by injection of recombinant α-Gal improves the disease outcome, effects on the vasculopathy associated to life-threatening cerebrovascular, cardiac and renal complications are still limited. We designed a strategy to enhance delivery of α-Gal to organs and endothelial cells (ECs). We targeted α-Gal to intercellular adhesion molecule 1 (ICAM-1), a protein expressed on ECs throughout the vasculature, by loading this enzyme on nanocarriers coated with anti-ICAM (anti-ICAM/α-Gal NCs). In vitro radioisotope tracing showed efficient loading of α-Gal on anti-ICAM NCs, stability of this formulation under storage and in model physiological fluids, and enzyme release in response to lysosome environmental conditions. In mice, delivery of 125 I-α-Gal was markedly enhanced by anti-ICAM/ 125 I-α-Gal NCs in brain, kidney, heart, liver, lung, and spleen, and transmission electron microscopy showed anti-ICAM/α-Gal NCs attached to and internalized into the vascular endothelium. Fluorescence microscopy proved targeting, endocytosis and lysosomal transport of anti-ICAM/α-Gal NCs in macro-and micro-vascular ECs, and a marked enhancement of Gb3 degradation. Therefore, ICAM-1-targeting strategy may help improve the efficacy of therapeutic enzymes for Fabry disease.
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.
Drugs often use endocytosis to achieve intracellular delivery, either by passive uptake from the extracellular fluid or by active targeting of cell surface features such as endocytic receptors. An example is enzyme replacement therapy, a clinically practiced treatment for several lysosomal storage diseases where glycosylated recombinant enzymes naturally target the mannose-6-phosphate receptor and are internalized by clathrin mediated endocytosis (CME). However, lysosomal substrate accumulation, a hallmark of these diseases, has been indirectly linked to aberrant endocytic activity. These effects are poorly understood, creating an obstacle to therapeutic efficiency. Here we explored endocytic activity in fibroblasts from patients with type A Niemann–Pick disease, a lysosomal storage disease characterized by acid sphingomyelinase (ASM) deficiency. The uptake of fluid phase markers and clathrin-associated ligands, formation of endocytic structures, and recruitment of intracellular clathrin to ligand binding sites were all altered, demonstrating aberrant CME in these cells. Model polymer nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1), which are internalized by a clathrin-independent route, enhanced the intracellular delivery of recombinant ASM more than 10-fold compared to free enzyme. This strategy reduced substrate accumulation and restored clathrin endocytic activity to wild-type levels. There appears to be a relationship between lysosomal storage and diminished CME, and bypassing this pathway by targeting ICAM-1 may enhance future therapies for lysosomal storage diseases.
Coupling drug carriers to antibodies for targeting endothelial cells (ECs) may improve treatment of vascular and pulmonary diseases. Selecting antibodies that deliver carriers to the cell surface or intracellularly may further optimize specificity of interventions. We studied antibody-directed targeting of nanocarriers to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial glycoprotein containing 6 Ig-like extracellular domains. PECAM-1 antibodies bind to ECs without internalization, but ECs internalize by endocytosis nanocarriers carrying multiple copies of anti-PECAM (anti-PECAM/NCs). To determine whether binding and intracellular transport of anti-PECAM/NCs depend on the epitope engaged, we targeted five PECAM-1 epitopes: mAb35, mAb37 and mAb62 (membrane-distal Ig domain 1), mAbGi34 (Ig domains 2/3), and mAb4G6 (membrane-proximal Ig domain 6). The antibodies bound to ECs regardless of the epitope proximity to the plasmalemma, whereas 130 nm diameter nanocarriers only targeted effectively distal domains (mAb4G6/NCs did not bind to ECs). ECs internalized mAb35, mAb62, and mAbGi34 carriers regardless of their size (0.13 to 5 microm diameter), yet they did not internalize mAb37/NCs. After internalization, mAb62/NCs trafficked to lysosomes within 2-3 h, whereas mAb35/NCs had prolonged residence in pre-lysosomal vesicles. Therefore, endothelial binding, endocytosis, and intracellular transport of anti-PECAM/NCs are epitope-specific. This paradigm will guide the design of endothelial drug delivery systems providing specific cellular localizations.
Targeting lysosomal enzymes to receptors involved in transport into and across cells holds promise to enhance peripheral and brain delivery of enzyme replacement therapies for lysosomal storage disorders. Receptors being explored include those associated with clathrin-mediated pathways, yet other pathways seem also viable. Well characterized examples are that of transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1), involved in iron transport and leukocyte extravasation, respectively. TfR and ICAM-1 support ERT delivery via clathrin- vs. cell adhesion molecule-mediated mechanisms, displaying different valency and size restrictions. To comparatively assess this, we used antibodies vs. larger multivalent antibody-coated carriers and evaluated TfR vs. ICAM-1 binding and endocytosis in endothelial cells, as well as in vivo biodistribution and delivery of a model lysosomal enzyme required in peripheral organs and brain: acid sphingomyelinase (ASM), deficient in types A–B Niemann Pick disease. We found similar binding of antibodies to both receptors under control conditions, with enhanced binding to activated endothelium for ICAM-1, yet only anti-TfR induced endocytosis efficiently. Contrarily, antibody-coated carriers showed enhanced binding, engulfment, and endocytosis for ICAM-1. In mice, anti-TfR enhanced brain targeting over anti-ICAM, with an opposite outcome in the lungs, while carriers enhanced ICAM-1 targeting over TfR in both organs. Both targeted carriers enhanced ASM delivery to the brain and lungs vs. free ASM, with greater enhancement for anti-ICAM carriers. Therefore, targeting TfR or ICAM-1 improves lysosomal enzyme delivery. Yet, TfR targeting may be more efficient for smaller conjugates or fusion proteins, while ICAM-1 targeting seems superior for multivalent carrier formulations.
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