Silvia Muro

Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers.

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.

A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1

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.

Endothelial Targeting of High-Affinity Multivalent Polymer Nanocarriers Directed to Intercellular Adhesion Molecule 1

Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (∼200 nm diameter spheres carrying ∼200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 ∼5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax ∼350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd ∼80 pM versus ∼8 nM) in cell culture and, probably because of this factor, higher value (185.3 ± 24.2 versus 50.5 ± 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 ± 10.9 versus 2.1 ± 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

Oxygen Microscopy by Two‐Photon‐Excited Phosphorescence

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.

Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelial cells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endothelium alleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conjugated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vasculature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalase to donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ameliorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improves the function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immunotargeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy include improving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.

Challenges in design and characterization of ligand-targeted drug delivery systems.

Targeting of therapeutic agents to molecular markers expressed on the surface of cells requiring clinical intervention holds promise to improve specificity of delivery, enhancing therapeutic effects while decreasing potential damage to healthy tissues. Drug targeting to cellular receptors involved in endocytic transport facilitates intracellular delivery, a requirement for a number of therapeutic goals. However, after several decades of experimental design, there is still considerable controversy on the practical outcome of drug targeting strategies. The plethora of factors contributing to the relative efficacy of targeting makes the success of these approaches hardly predictable. Lack of fully specific targets, along with selection of targets with spatial and temporal expression well aligned to interventional requirements, pose difficulties to this process. Selection of adequate sub-molecular target epitopes determines accessibility for anchoring of drug conjugates and bulkier drug carriers, as well as proper signaling for uptake within the cell. Targeting design must adapt to physiological variables of blood flow, disease status, and tissue architecture by accommodating physicochemical parameters such as carrier composition, functionalization, geometry, and avidity. In many cases, opposite features need to meet a balance, e.g., sustained circulation versus efficient targeting, penetration through tissues versus uptake within cells, internalization within endocytic compartment to avoid efflux pumps versus accessibility to molecular targets within the cytosol, etc. Detailed characterization of these complex physiological factors and design parameters, along with a deep understanding of the mechanisms governing the interaction of targeted drugs and carriers with the biological environment, are necessary steps toward achieving efficient drug targeting systems.

Endothelial Endocytic Pathways: Gates for Vascular Drug Delivery

Vascular endothelium plays strategic roles in many drug delivery paradigms, both as an important therapeutic target itself and as a barrier for reaching tissues beyond the vascular wall. Diverse means are being developed to improve vascular drug delivery including stealth liposomes and polymer carriers. Affinity carriers including antibodies or peptides that specifically bind to endothelial surface determinants, either constitutive or pathological, enhance targeting of drugs to endothelial cells (EC) in diverse vascular areas. In many cases, binding to endothelial surface determinants facilitates internalization of the drug/carrier complex. There are several main endocytic pathways in EC, including clathrin- and caveoli-mediated endocytosis, phagocytosis and macropinocytosis (these two are less characteristic of generic EC) and the recently described Cell Adhesion Molecule (CAM)-mediated endocytosis. The latter may be of interest for intracellular drug delivery to EC involved in inflammation or thrombosis. The metabolism and effects of internalized drugs largely depend on the routes of intracellular trafficking, which may lead to degrading lysosomal compartments or other organelles, recycling to the plasma membrane or transcytosis to the basal surface of endothelium. The latter route, characteristic of caveoli-mediated endocytosis, may serve for trans-endothelial drug delivery. Paracellular trafficking, which can be enhanced under pathological conditions or by auxiliary agents, represents an alternative for transcytosis. Endothelial surface determinants involved in endocytosis, mechanisms of the latter and trafficking pathways, as well as specific characteristics of EC in different vascular areas, are discussed in detail in the context of modern paradigms of vascular drug delivery.

Delivery of Acid Sphingomyelinase in Normal and Niemann-Pick Disease Mice Using Intercellular Adhesion Molecule-1-Targeted Polymer Nanocarriers

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 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

In Vivo Imaging of 64Cu-Labeled Polymer Nanoparticles Targeted to the Lung Endothelium

Nanoparticles (NPs) targeting the intercellular adhesion molecule 1 (ICAM-1) hold promise as a mean of delivering therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases. As these new materials become available, strategies are needed to understand their behavior in vivo. We have evaluated the use of 64Cu and PET to noninvasively image the lung uptake and distribution of NPs coated with an anti-ICAM antibody. Methods: Model fluorescent NPs were coated with a mixture of an anti-ICAM antibody (or nonspecific IgG) and 64Cu-DOTA-IgG (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Biodistribution and small-animal PET and CT studies were performed in healthy mice and in mice pretreated with lipopolysaccharides (LPSs). Metabolism studies were also performed to evaluate the stability of 64Cu-labeled NPs in lungs in vivo. Results: The lungs of mice administered anti-ICAM NPs labeled with 64Cu were clearly imaged by small-animal PET 1, 4, and 24 h after administration. Both biodistribution and small-animal imaging showed a 3- to 4-fold higher uptake in the lungs of mice injected with ICAM-targeted NPs relative to that of the control group. Lung uptake was further enhanced by pretreating the mice with LPS, presumably because of ICAM-1 upregulation. However, an approximately 2-fold decrease in lung signal was observed in each experimental group over 24 h. Metabolism studies in lung tissues harvested from mice injected with 64Cu-labeled anti-ICAM NPs showed considerable release of a small 64Cu-radiometabolite from the NPs beginning as early as 1 h after injection. A decrease in lung fluorescence was also observed, most likely reflecting partial release of NPs from the lungs in vivo. Conclusion: The use of small-animal PET to track 64Cu-labeled nanostructures in vivo shows potential as a strategy for the preclinical screening of new NP drug delivery agents targeting the lung endothelium and other tissues. Future design optimization to prolong the stability of the radiolabel in vivo will further improve this promising approach.

Endothelial targeting of antibody-decorated polymeric filomicelles.

The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers.