Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.
Background and Purpose-Our aim was to assess the spatiotemporal evolution of the cerebrovascular inflammation occurring after ischemic and hemorrhagic strokes using a recently developed, fast, and ultra-sensitive molecular MRI method. Methods-We first assessed longitudinally the cerebrovascular inflammation triggered by collagenase-induced hemorrhage and by permanent/transient middle cerebral artery occlusion in mice, using MRI after injection of microparticles of iron oxide targeted to vascular cell adhesion molecule-1 (MPIOs-αVCAM-1). Thereafter, we used this method to study the anti-inflammatory effects of celecoxib, atorvastatin, and dipyridamole after stroke. Results-Using multiparametric MRI, we demonstrated that the level and the kinetics of cerebrovascular VCAM-1 expression depend on several parameters, including stroke pathogenesis, the natural history of the disease, and the administration of inflammation-modulating drugs. Interestingly, in transient middle cerebral artery occlusion and intracranial hemorrhage models, VCAM-1 expression was maximal at 24 hours and almost returned to baseline 5 days after stroke onset. In contrast, after permanent middle cerebral artery occlusion, VCAM-1 overexpression was sustained between 24 hours and 5 days, and was particularly significant in the peri-infarct areas. Our results suggest that these perilesional areas expressing VCAM-1 constitute an inflammatory penumbra that is recruited by the ischemic core during the subacute phase. Using MPIOs-αVCAM-1-enhanced imaging, we also provided evidence that celecoxib and atorvastatin (but not dipyridamole) alleviate VCAM-1 overexpression after stroke and prevent formation of the inflammatory penumbra. Conclusions-MPIOs-αVCAM-1-enhanced imaging seems to be promising in the detection of individuals presenting with severe cerebrovascular responses after stroke, which could therefore benefit from anti-inflammatory treatments.
Molecular targeting of nanoparticle drug carriers promises maximized therapeutic impact to sites of disease or injury with minimized systemic effects. Precise targeting demands addressing to subcellular features. Caveolae, invaginations in cell membranes implicated in transcytosis and inflammatory signaling, are appealing subcellular targets. Caveolar geometry has been reported to impose a ≈50 nm size cutoff on nanocarrier access to plasmalemma vesicle associated protein (PLVAP), a marker found in caveolae in the lungs. The use of deformable nanocarriers to overcome that size cutoff is explored in this study. Lysozyme-dextran nanogels (NGs) are synthesized with ≈150 or ≈300 nm mean diameter. Atomic force microscopy indicates the NGs deform on complementary surfaces. Quartz crystal microbalance data indicate that NGs form softer monolayers (≈60 kPa) than polystyrene particles (≈8 MPa). NGs deform during flow through microfluidic channels, and modeling of NG extrusion through porous filters yields sieving diameters less than 25 nm for NGs with 150 and 300 nm hydrodynamic diameters. NGs of 150 and 300 nm diameter target PLVAP in mouse lungs while counterpart rigid polystyrene particles do not. The data in this study indicate a role for mechanical deformability in targeting large high-payload drug-delivery vehicles to sterically obscured targets like PLVAP.
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.
Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. IntroductionPlasminogen activators (PAs; tissue-type, tPA; urokinase, uPA) can provide urgent thrombolysis within a narrow therapeutic window of time in the setting of life-or limb-threatening thrombosis. 1,2 However, their efficacy is limited by plasma inhibitors (eg, PAI-1) and inadequate delivery into impermeable occlusive clots, a situation that is exacerbated by delays in initiating treatment. Endowing tPA derivatives with higher affinity for fibrin 3,4 further impairs clot permeation, 5 while increased dosing and constitutive lytic activity enhances the risk of bleeding and central nervous system (CNS) toxicity.Coupling tPA to carrier red blood cells (RBCs) followed by re-infusion of the RBC/tPA conjugates in animals provides protracted thromboprophylaxis in arteries and veins, including the vulnerable cerebrovascular circulation. 6-8 RBC carriage prolongs the half-life of tPAs in the circulation from minutes to many hours and prevents drug extravasation and access to hemostatic plugs, thereby reducing the risk of bleeding episodes. 7 In the prophylactic setting where tPAs lacked efficacy but caused bleeding and CNS toxicity, RBCs/tPAs mediated timely reperfusion, reducing morbidity and mortality. 8 Translation of RBC/PA thromboprophylaxis into the clinical domain could improve management of patients known to be at high risk of thrombosis or rethrombosis, including after acute myocardial infarction (AMI), transient ischemic attack, pulmonary embolism, angioplasty, and abdominal or other surgical procedures such as knee replacements, where the efficacy of thromboprophylaxis is low and/or the risk of bleeding is high. The goal of this study was to modify an existing prototypic approach (ex vivo coupling of PAs to RBCs followed by RBC/PA re-infusion) into a more clinically applicable approach to thromboprophylaxis. To anchor the injected PAs to circulating RBCs, we used a scFv fragment of the monoclonal antibody Ter-119 that recognizes mouse glycophorin A (GPA), an abundant RBC-specific surface molecule (ϳ 10 6 copies/ RBC, 9,10 similar to its human homologue 11 ). We fused scFv Ter-119 to a recombinant low molecular weight single chain uPA lacking the kringle and growth factor-like domains (lmw scuPA). Lmw scuPA is a zymogen that is naturally activated by plasmi...
Hyperfibrinolysis is a systemic condition occurring in various clinical disorders such as trauma, liver cirrhosis, and leukemia. Apart from increased bleeding tendency, the pathophysiological consequences of hyperfibrinolysis remain largely unknown. Our aim was to develop an experimental model of hyperfibrinolysis and to study its effects on the homeostasis of the blood-brain barrier (BBB). We induced a sustained hyperfibrinolytic state in mice by hydrodynamic transfection of a plasmid encoding for tissue-type plasminogen activator (tPA). As revealed by near-infrared fluorescence imaging, hyperfibrinolytic mice presented a significant increase in BBB permeability. Using a set of deletion variants of tPA and pharmacological approaches, we demonstrated that this effect was independent of N-methyl-D-aspartate receptor, low-density lipoprotein-related protein, protease-activated receptor-1, or matrix metalloproteinases. In contrast, we provide evidence that hyperfibrinolysis-induced BBB leakage is dependent on plasmin-mediated generation of bradykinin and subsequent activation of bradykinin B2 receptors. Accordingly, this effect was prevented by icatibant, a clinically available B2 receptor antagonist. In agreement with these preclinical data, bradykinin generation was also observed in humans in a context of acute pharmacological hyperfibrinolysis. Altogether, these results suggest that B2 receptor blockade may be a promising strategy to prevent the deleterious effects of hyperfibrinolysis on the homeostasis of the BBB.
This study shows that supramolecular arrangement of proteins in nanoparticle structure predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of n anoparticles with a gglutinated p rotein (NAPs), defined by arrangement of protein in or on the nanoparticles via; a) hydrophobic interactions; b) crosslinking; c) electrostatic interactions. Nanoparticles with symmetric protein arrangement ( e.g. , viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We; a) demonstrate diagnostic imaging of ALI with NAPs; b) show NAP tropism for inflamed human donor lungs; c) show NAPs can remediate pulmonary edema in ALI. This work demonstrates structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.
Key Points The binding of administered FVIIa to endogenous EPCR enhances its ability to bypass FVIII or FIX deficiency in vivo. EPCR modulation of function of pharmacologic FVIIa administration may be exploited in protein or gene-based FVIIa therapeutics.
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