Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Anaerobic Lactate Production Is Associated With Decreased Microcirculatory Blood Flow and Decreased Mitochondrial Respiration Following Cardiovascular Surgery With Cardiopulmonary Bypass.

OBJECTIVES: Quantify the relationship between perioperative anaerobic lactate production, microcirculatory blood flow, and mitochondrial respiration in patients after cardiovascular surgery with cardiopulmonary bypass. DESIGN: Serial measurements of lactate-pyruvate ratio (LPR), microcirculatory blood flow, plasma tricarboxylic acid cycle cycle intermediates, and mitochondrial respiration were compared between patients with a normal peak lactate (≤ 2 mmol/L) and a high peak lactate (≥ 4 mmol/L) in the first 6 hours after surgery. Regression analysis was performed to quantify the relationship between clinically relevant hemodynamic variables, lactate, LPR, and microcirculatory blood flow. SETTING: This was a single-center, prospective observational study conducted in an academic cardiovascular ICU. PATIENTS: One hundred thirty-two patients undergoing elective cardiovascular surgery with cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients with a high postoperative lactate were found to have a higher LPR compared with patients with a normal postoperative lactate (14.4 ± 2.5 vs. 11.7 ± 3.4; p = 0.005). Linear regression analysis found a significant, negative relationship between LPR and microcirculatory flow index ( r = -0.225; ß = -0.037; p = 0.001 and proportion of perfused vessels: r = -0.17; ß = -0.468; p = 0.009). There was not a significant relationship between absolute plasma lactate and microcirculation variables. Last, mitochondrial complex I and complex II oxidative phosphorylation were reduced in patients with high postoperative lactate levels compared with patients with normal lactate (22.6 ± 6.2 vs. 14.5 ± 7.4 pmol O 2 /s/10 6 cells; p = 0.002). CONCLUSIONS: Increased anaerobic lactate production, estimated by LPR, has a negative relationship with microcirculatory blood flow after cardiovascular surgery. This relationship does not persist when measuring lactate alone. In addition, decreased mitochondrial respiration is associated with increased lactate after cardiovascular surgery. These findings suggest that high lactate levels after cardiovascular surgery, even in the setting of normal hemodynamics, are not simply a type B phenomenon as previously suggested.

Low postoperative perfused vessel density is associated with increased soluble endothelial cell adhesion molecules during circulatory shock after cardiac surgery.

INTRODUCTION: Microcirculatory dysfunction after cardiovascular surgery is associated with significant morbidity and worse clinical outcomes. Abnormal capillary blood flow can occur from multiple causes, including cytokine-mediated vascular endothelial injury, microthrombosis, and an inadequate balance between vasoconstriction and vasodilation. In response to proinflammatory cytokines, endothelial cells produce cellular adhesion molecules (CAMs) which regulate leukocyte adhesion, vascular permeability, and thus can mediate tissue injury. The relationship between changes in microcirculatory flow during circulatory shock and circulating adhesion molecules is unclear. The objective of this study was to compare changes in plasma soluble endothelial cell adhesion molecules (VCAM-1, ICAM-1, and E-Selectin) in patients with functional derangements in microcirculatory blood flow after cardiovascular surgery. METHODS: Adult patients undergoing elective cardiac surgery requiring cardiopulmonary bypass who exhibited postoperative shock were enrolled in the study. Sublingual microcirculation imaging was performed prior to surgery and within 2 h of ICU admission. Blood samples were taken at the time of microcirculation imaging for biomarker analysis. Plasma soluble VCAM-1, ICAM-1, and E-selectin in addition to plasma cytokines (IL-6, IL-8, and IL-10) were measured by commercially available enzyme-linked immunoassay. RESULTS: Of 83 patients with postoperative shock who were evaluated, 40 patients with clinical shock had a postoperative perfused vessel density (PVD) >1 SD above (High PVD group = 28.5 ± 2.3 mm/mm2, n = 20) or below (Low PVD = 15.5 ± 2.0 mm/mm2, n = 20) the mean postoperative PVD and were included in the final analysis. Patient groups were well matched for comorbidities, surgical, and postoperative details. Overall, there was an increase in postoperative plasma VCAM-1 and E-Selectin compared to preoperative levels, but there was no difference between circulating ICAM-1. When grouped by postoperative microcirculation, patients with poor microcirculation were found to have increased circulating VCAM-1 (2413 ± 1144 vs. 844 ± 786 ng/mL; p < 0.0001) and E-Selectin (242 ± 119 vs. 87 ± 86 ng/mL; p < 0.0001) compared to patients with increased microcirculatory blood flow. Microcirculatory flow was not associated with a difference in plasma soluble ICAM-1 (394 ± 190 vs. 441 ± 256; p = 0.52). CONCLUSIONS: Poor postoperative microcirculatory blood flow in patients with circulatory shock after cardiac surgery is associated with increased plasma soluble VCAM-1 and E-Selectin, indicating increased endothelial injury and activation compared to patients with a high postoperative microcirculatory blood flow. Circulating endothelial cell adhesion molecules may be a useful plasma biomarker to identify abnormal microcirculatory blood flow in patients with shock.

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Red Blood Cell Hitchhiking: A Novel Approach for Vascular Delivery of Nanocarriers.

Red blood cell (RBC) hitchhiking is a method of drug delivery that can increase drug concentration in target organs by orders of magnitude. In RBC hitchhiking, drug-loaded nanoparticles (NPs) are adsorbed onto red blood cells and then injected intravascularly, which causes the NPs to transfer to cells of the capillaries in the downstream organ. RBC hitchhiking has been demonstrated in multiple species and multiple organs. For example, RBC-hitchhiking NPs localized at unprecedented levels in the brain when using intra-arterial catheters, such as those in place immediately after mechanical thrombectomy for acute ischemic stroke. RBC hitchhiking has been successfully employed in numerous preclinical models of disease, ranging from pulmonary embolism to cancer metastasis. In addition to summarizing the versatility of RBC hitchhiking, we also describe studies into the surprisingly complex mechanisms of RBC hitchhiking as well as outline future studies to further improve RBC hitchhiking's clinical utility.

Targeted In Vivo Loading of Red Blood Cells Markedly Prolongs Nanocarrier Circulation.

Engineering drug delivery systems for prolonged pharmacokinetics (PK) has been an ongoing pursuit for nearly 50 years. The gold standard for PK enhancement is the coating of nanoparticles with polymers, namely polyethylene glycol (PEGylation), which has been applied in several clinically used products. In the present work, we utilize the longest circulating and most abundant component of blood─the erythrocyte─to improve the PK behavior of liposomes. Antibody-mediated coupling of liposomes to erythrocytes was tested in vitro to identify a loading dose that did not adversely impact the carrier cells. Injection of erythrocyte targeting liposomes into mice resulted in a ∼2-fold improvement in the area under the blood concentration versus time profile versus PEGylated liposomes and a redistribution from the plasma into the cellular fraction of blood. These results suggest that in vivo targeting of erythrocytes is a viable strategy to improve liposome PK relative to current, clinically viable strategies.

Molecularly Engineered Nanobodies for Tunable Pharmacokinetics and Drug Delivery.

The use of single-domain antibody fragments, or nanobodies, has gained popularity in recent years as an alternative to traditional monoclonal antibody-based approaches. Relatively little is known, however, about the utility of nanobodies as targeting agents for delivery of therapeutic cargoes, particularly to vascular epitopes or in the setting of acute inflammatory conditions. We used a nanobody (VCAMelid) directed against mouse vascular cell adhesion molecule 1 (VCAM-1) and techniques for site-specific radiolabeling and bioconjugation to measure targeting to sites of constitutive and inducible antigen expression and investigate the impact of various characteristics (affinity, valence, circulation time) on nanobody biodistribution and pharmacokinetics. Engineering of VCAMelid for bivalent binding (BiVCAMelid) increased affinity by an order of magnitude and provided 2.8- and 3.6-fold enhancements in splenic and brain targeting in naive mice, with a further 2.6-fold increase in brain uptake in the setting of focal CNS inflammation. In contrast, introduction of an albumin-binding arm (VCAM/ALB8) did not affect binding affinity, but its prolonged circulation time resulted in 3.5-fold and 17.4-fold increases in splenic and brain uptake at 20 min post-dose and remarkable 40-, 25-, and 15-fold enhancements in overall exposure of blood, spleen, and brain, respectively, relative to both VCAMelid and BiVCAMelid. Both therapeutic protein (superoxide dismutase, SOD-1) and nanocarrier (liposome) delivery were enhanced by conjugation to VCAM-1 targeted nanobodies. The bispecific VCAM/ALB8 maintained its superiority over VCAMelid in enhancing both circulation time and organ targeting of SOD-1, but its advantages were largely blunted by conjugation to liposomes.

Target-mediated exposure enhancement: a previously unexplored limit of TMDD.

Target-mediated drug disposition (TMDD) is often observed for targeted therapeutics, and manifests as decreases in clearance and volume of distribution with increasing dose as a result of saturable, high affinity target binding. In the present work, we demonstrate that classically defined TMDD is just one of the characteristic features of the system. In fact, for molecules with rapid non-specific elimination relative to target-mediated elimination, binding to target may actually lead to improved exposure at sub-saturating doses. This feature, which we refer to as target-mediated exposure enhancement (TMEE), produces the opposite trend to classical TMDD, i.e., with increasing dose levels, clearance and volume of distribution will also increase. The general model of TMDD was able to well-characterize the pharmacokinetics of two molecules that display TMEE, ALX-0081 and linagliptin. Additional fittings using the commonly reported TMDD model approximations revealed that both the quasi-equilibrium and quasi-steady-state approximations were able to well-describe TMEE; however, the Michaelis-Menten approximation was unable to describe this behavior. With the development of next-generation therapeutics with high affinity for target and rapid non-specific elimination, such as antibody fragments and peptides, this previously unexplored limit of TMDD is anticipated to become increasingly relevant for describing pharmacokinetics of investigational therapeutics.

Pharmacokinetic and Pharmacodynamic Properties of Drug Delivery Systems.

The use of drug delivery systems (DDS) is an attractive approach to facilitate uptake of therapeutic agents at the desired site of action, particularly when free drug has poor pharmacokinetics/biodistribution (PK/BD) or significant off-site toxicities. Successful translation of DDS into the clinic is dependent on a thorough understanding of the in vivo behavior of the carrier, which has, for the most part, been an elusive goal. This is, at least in part, due to significant differences in the mechanisms controlling pharmacokinetics for classic drugs and DDSs. In this review, we summarize the key physiologic mechanisms controlling the in vivo behavior of DDS, compare and contrast this with classic drugs, and describe engineering strategies designed to improve DDS PK/BD. In addition, we describe quantitative approaches that could be useful for describing PK/BD of DDS, as well as critical steps between tissue uptake and pharmacologic effect.

Vascular Targeting of Radiolabeled Liposomes with Bio-Orthogonally Conjugated Ligands: Single Chain Fragments Provide Higher Specificity than Antibodies.

Liposomes are a proven, versatile, and clinically viable technology platform for vascular delivery of drugs and imaging probes. Although targeted liposomes have the potential to advance these applications, complex formulations and the need for optimal affinity ligands and conjugation strategies challenge their translation. Herein, we employed copper-free click chemistry functionalized liposomes to target platelet-endothelial cell adhesion molecule (PECAM-1) and intracellular adhesion molecule (ICAM-1) by conjugating clickable monoclonal antibodies (Ab) or their single chain variable fragments (scFv). For direct, quantitative tracing, liposomes were surface chelated with 111In to a >90% radiochemical yield and purity. Particle size and distribution, stability, ligand surface density, and specific binding to target cells were characterized in vitro. Biodistribution of liposomes after IV injection was characterized in mice using isotope detection in organs and by noninvasive imaging (single-photon emission computed tomography/computed tomography, SPECT/CT). As much as 20-25% of injected dose of liposomes carrying PECAM and ICAM ligands, but not control IgG accumulated in the pulmonary vasculature. The immunospecificity of pulmonary targeting of scFv/liposomes to PECAM-1 and ICAM-1, respectively, was 10-fold and 2.5-fold higher than of Ab/liposomes. Therefore, the combination of optimal ligands, benign conjugation, and labeling yields liposomal formulations that may be used for highly effective and specific vascular targeting.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging.

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.

Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.

Endothelial thrombomodulin (TM) regulates coagulation and inflammation via several mechanisms, including production of activated protein C (APC). Recombinant APC and soluble fragments of TM (sTM) have been tested in settings associated with insufficiency of the endogenous TM/APC pathway, such as sepsis. We previously designed a fusion protein of TM [single-chain variable fragment antibody (scFv)/TM] targeted to red blood cells (RBCs) to improve pharmacokinetics and antithrombotic effects without increasing bleeding. Here, scFv/TM was studied in mouse models of systemic inflammation and ischemia-reperfusion injury. Injected concomitantly with or before endotoxin, scFv/TM provided more potent protection against liver injury and release of pathological mediators than sTM, showing similar efficacy at up to 50-fold lower doses. scFv/TM provided protection when injected after endotoxin, whereas sTM did not, and augmented APC production by thrombin ∼50-fold more than sTM. However, scFv/TM injected after endotoxin did not reduce thrombin/antithrombin complexes; nor did antibodies that block APC anticoagulant activity suppress the prophylactic anti-inflammatory effect of scFv/TM. Therefore, similar to endogenous TM, RBC-anchored scFv/TM activates several protective pathways. Finally, scFv/TM was more effective at reducing cerebral infarct volume and alleviated neurological deficits than sTM after cerebral ischemia/reperfusion injury. These results indicate that RBC-targeted scFv/TM exerts multifaceted cytoprotective effects and may find utility in systemic and focal inflammatory and ischemic disorders.-Carnemolla, R., Villa, C. H., Greineder, C. F., Zaitseva, S., Patel, K. R., Kowalska, M. A., Atochin, D. N., Cines, D. B., Siegel, D. L., Esmon, C. T., Muzykantov, V. R. Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.

Nanocarriers for vascular delivery of anti-inflammatory agents.

There is a need for improved treatment of acute vascular inflammation in conditions such as ischemia-reperfusion injury, acute lung injury, sepsis, and stroke. The vascular endothelium represents an important therapeutic target in these conditions. Furthermore, some anti-inflammatory agents (AIAs) (e.g., biotherapeutics) require precise delivery into subcellular compartments. In theory, optimized delivery to the desired site of action may improve the effects and enable new mechanisms of action of these AIAs. Diverse nanocarriers (NCs) and strategies for targeting them to endothelial cells have been designed and explored for this purpose. Studies in animal models suggest that delivery of AIAs using NCs may provide potent and specific molecular interventions in inflammatory pathways. However, the industrial development and clinical translation of complex NC-AIA formulations are challenging. Rigorous analysis of therapeutic/side effect and benefit/cost ratios is necessary to identify and optimize the approaches that may find clinical utility in the management of acute inflammation.

Mechanisms that determine nanocarrier targeting to healthy versus inflamed lung regions.

Inflamed organs display marked spatial heterogeneity of inflammation, with patches of inflamed tissue adjacent to healthy tissue. To investigate how nanocarriers (NCs) distribute between such patches, we created a mouse model that recapitulates the spatial heterogeneity of the inflammatory lung disease ARDS. NCs targeting the epitope PECAM strongly accumulated in the lungs, but were shunted away from inflamed lung regions due to hypoxic vasoconstriction (HVC). In contrast, ICAM-targeted NCs, which had lower whole-lung uptake than PECAM/NCs in inflamed lungs, displayed markedly higher NC levels in inflamed regions than PECAM/NCs, due to increased regional ICAM. Regional HVC, epitope expression, and capillary leak were sufficient to predict intra-organ of distribution of NCs, antibodies, and drugs. Importantly, these effects were not observable with traditional spatially-uniform models of ARDS, nor when examining only whole-organ uptake. This study underscores how examining NCs' intra-organ distribution in spatially heterogeneous animal models can guide rational NC design.

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles.

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.

Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect.

Anchoring pharmacologic agents to the vascular lumen has the potential to modulate critical processes at the blood-tissue interface, avoiding many of the off-target effects of systemically circulating agents. We report a novel strategy for endothelial dual targeting of therapeutics, which both enhances drug delivery and enables targeted agents to partner enzymatically to generate enhanced biologic effect. Based on the recent discovery that paired antibodies directed to adjacent epitopes of platelet endothelial cell adhesion molecule (PECAM)-1 stimulate each other's binding, we fused single-chain fragments (scFv) of paired anti-mouse PECAM-1 antibodies to recombinant murine thrombomodulin (TM) and endothelial protein C receptor (EPCR), endothelial membrane proteins that partner in activation of protein C (PC). scFv/TM and scFv/EPCR bound to mouse endothelial PECAM-1 with high affinity (EC50 1.5 and 3.8 nM, respectively), and codelivery induced a 5-fold increase in PC activation not seen when TM and EPCR are anchored to distinct cell adhesion molecules. In a mouse model of acute lung injury, dual targeting reduces both the expression of lung inflammatory markers and trans-endothelial protein leak by as much as 40%, as compared to either agent alone. These findings provide proof of principle for endothelial dual targeting, an approach with numerous potential biomedical applications.

Advanced drug delivery systems for antithrombotic agents.

Despite continued achievements in antithrombotic pharmacotherapy, difficulties remain in managing patients at high risk for both thrombosis and hemorrhage. Utility of antithrombotic agents (ATAs) in these settings is restricted by inadequate pharmacokinetics and narrow therapeutic indices. Use of advanced drug delivery systems (ADDSs) may help to circumvent these problems. Various nanocarriers, affinity ligands, and polymer coatings provide ADDSs that have the potential to help optimize ATA pharmacokinetics, target drug delivery to sites of thrombosis, and sense pathologic changes in the vascular microenvironment, such as altered hemodynamic forces, expression of inflammatory markers, and structural differences between mature hemostatic and growing pathological clots. Delivery of ATAs using biomimetic synthetic carriers, host blood cells, and recombinant fusion proteins that are activated preferentially at sites of thrombus development has shown promising outcomes in preclinical models. Further development and translation of ADDSs that spare hemostatic fibrin clots hold promise for extending the utility of ATAs in the management of acute thrombotic disorders through rapid, transient, and targeted thromboprophylaxis. If the potential benefit of this technology is to be realized, a systematic and concerted effort is required to develop clinical trials and translate the use of ADDSs to the clinical arena.