Papers by Vladimir Muzykantov
American Journal of Physiology-lung Cellular and Molecular Physiology, May 1, 2006
Bulletin of the American Physical Society, Mar 3, 2020
Blood, Nov 16, 2006
Coupling tissue plasminogen activator (tPA) to carrier red blood cells (RBC): i) restricts tPA’s ... more Coupling tissue plasminogen activator (tPA) to carrier red blood cells (RBC): i) restricts tPA’s permeation into tissues and pre-existing hemostatic clots, minimizing hemorrhage; ii) protects it from plasma inhibitors; and iii) prolongs its circulation, permitting incorporation into nascent clots and their lysis from within. These features support the thromboprophylactic utility of RBC coupled tPA (RBC/tPA). In this study we explored the utility of RBC/tPA in traumatic brain injury (TBI), a disorder in which both cerebral thrombosis leading to cerebral ischemia and intracerebral hemorrhage (ICH) have been implicated. Eleven male, Sprague-Dawley rats (340–400g) were subjected to a moderate (avg. peak pressure 2.6atm) lateral fluid percussion injury to the left hemisphere. Rats were given a single intravenous dose of RBC/tPA (0.05mg/Kg, n=5) or vehicle (n=4), 15 min post-injury. Animals were sacrificed 48h later for histopathology and staining for fibrin. The lesions in control animals occupied 8.3 ± 2.8% (mean±SD) of the hemispheric volume. Animals treated with RBC/tPA had a significant decrease in mean lesion volume (1.4±0.7%; p<0.001). Thrombotic burden was reduced from a mean of 10 clots in vehicle-treated to 1 per RBC/tPA-treated rats p<0.001). These data indicate that RBC/tPA attenuates post-traumatic thrombosis without aggravating hemorrhage induced by TBI. These observations support the safety of RBC/tPA in thromboprophylaxis even in the context of brain trauma, due to intravascular containment of RBC/tPA, among other factors. Durable lysis of post-traumatic thrombi by long-circulating RBC/tPA may alleviate secondary brain ischemia and resultant ICH. Correlation between reduction of thrombotic burden and lesion size implicate thrombosis in the pathophysiology of parenchymal brain damage after head trauma. Supported by PENN Research Foundation, HL66442, HL60169 and in part by NS38104.
Blood, Dec 3, 2015
* These authors contributed equally to this work The microvasculature plays a key role in the pat... more * These authors contributed equally to this work The microvasculature plays a key role in the pathogenesis of sepsis, ARDS, multiorgan dysfunction, and a variety of other human diseases characterized by a pro-coagulant, pro-adhesive endothelial phenotype. The complex interactions that occur at the interface of blood and activated endothelium are difficult to resolve in animal models and challenging to recreate in cell culture systems. While individual processes - e.g., leukocyte adhesion and transmigration - have been extensively studied, development of a well-characterized model integrating the full range of pathogenic processes (coagulation, barrier dysfunction, innate immune system activation, etc.) remains an important unmet goal, which could both elucidate mechanisms of disease and aid in the design and testing of putative therapeutics. To this end, we sought to model an inflamed vascular segment using a Fluxion Bioflux system. 3-dimensional confluent endothelial cell (EC) monolayers were established within fibronectin-coated laminar flow chambers. ECs were flow adapted, activated with TNFa, and then perfused with whole blood (WB) at a variety of shear stresses. Real time fluorescence microscopy allowed continuous monitoring of fibrin deposition and leukocyte and platelet adhesion. The multi-channel format, which allows simultaneous testing of multiple conditions with replicates, proved to be a critical asset, given substantial day-to-day variability. Both fibrin deposition and adhesive events showed dependence on dose (1 vs. 10 ng/mL) and duration (4 vs. 6hr) of TNF activation. Confocal microscopy revealed TNF-dependent, increases in EC expression of ICAM-1, VCAM-1, and tissue factor (TF), as well as suppression of endothelial thrombomodulin (TM). Activation of the coagulation system was completely abrogated by treatment of the EC monolayer with a TF-inhibiting antibody, suggesting a primary role for the extrinsic pathway. Hirudin also limited fibrin deposition when added to whole blood prior to perfusion, although "breakthrough clotting" was seen in some channels. Finally, the role of endothelial TM was investigated in several ways, including by the use of a blocking antibody, which prevents thrombin binding. Treatment of ECs with this antibody markedly increased fibrin deposition, whereas TM/R6.5 scFv, a novel targeted fusion protein therapeutic, which anchors recombinant TM to endothelial ICAM-1, inhibited fibrin deposition upon subsequent infusion of WB. Neither soluble TM (sTM) nor anti-ICAM-1 R6.5 scFv alone had any effect on coagulation when infused in this setting (i.e., prior to WB) and even addition of a large excess of sTM to whole blood was less effective in reducing TNF-dependent fibrin deposition than pre-treatment with the ICAM-targeted TM fusion protein, indicating potential importance of precision drug delivery on the microscale. In summary, the described microfluidic, "endothelialized", whole blood model of an inflamed microvessel may prove useful in interrogating specific aspects of a variety of vascular pathologies and in devising and improving therapeutic…
Bulletin of the American Physical Society, Nov 18, 2018
Journal of Biomedical Nanotechnology, Jun 1, 2017
PubMed, 2011
Delivery and effects of therapeutics remain suboptimal. Most drugs do not have affinity to their ... more Delivery and effects of therapeutics remain suboptimal. Most drugs do not have affinity to their targets. Biotherapeutics including enzymes and genetic materials require specific sub-cellular addressing not attainable naturally. Endothelium, lining the luminal surface of blood vessels, represents a key therapeutic target in many diseases. Studies in cell culture and animal models revealed that targeted delivery of therapeutics to, into and across endothelium can be achieved using carriers targeted to specific molecules expressed on the surface of the endothelial cells. For example, cell adhesion molecules represent attractive targets for drug delivery. Rational design of the drug delivery systems (e.g., selection of optimal geometry and affinity to specific epitopes) provides an unprecedented level of control of such parameters of drug delivery as pharmacokinetics, circulation in blood, binding to selected endothelial cell phenotypes, anchoring on cell surface or internalization into the endothelium, subsequent intracellular addressing and duration of the effects. We discusse here key aspects of design of endothelium-targeted drug delivery systems with potential for translation into the clinical domain.
Humana Press eBooks, Jun 15, 2004
Streptavidin-biotin conjugates of enzymes with carrier antibodies provide a versatile means for t... more Streptavidin-biotin conjugates of enzymes with carrier antibodies provide a versatile means for targeting selected cellular populations in cell cultures and in vivo. Both specific delivery to cells and proper subcellular addressing of enzyme cargoes are important parameters of targeting. This chapter describes methodologies for evaluating the binding and internalization of labeled conjugates directed to endothelial surface adhesion molecules in cell cultures using anti-intercellular adhesion molecule/catalase or antiplatelet endothelial cell adhesion molecule/catalase conjugates as examples. It also describes protocols for characterization of biodistribution and pulmonary targeting of radiolabeled conjugates in rats using anti-intercellular adhesion molecule/tPA conjugates as an example. The experimental procedures, results, and notes provided may help in investigations of vascular immunotargeting of reporter, experimental, diagnostic, or therapeutic enzymes to endothelial and, perhaps, other cell types, both in vitro and in vivo.
Molecular Pharmaceutics, Jun 17, 2014
Journal of Clinical Investigation, Dec 21, 2015
The Journal of Nuclear Medicine, Dec 12, 2007
Blood, Dec 2, 2016
Delivery of bio-therapeutics by red blood cells (RBCs) can greatly enhance pharmacokinetics and p... more Delivery of bio-therapeutics by red blood cells (RBCs) can greatly enhance pharmacokinetics and pharmacodynamics of the appended or loaded agents, and may even potentiate induction of immunologic tolerance. Our group and others have successfully used fusion proteins, antibodies, and peptides to couple therapeutics to murine, but not human, RBCs. It is known that extracellular ligands have the potential to induce marked, epitope-dependent changes in red cell physiology, including changes in deformability, phosphatidyl-serine (PS) exposure, and reactive oxygen species (ROS) production, particularly for agents targeted to glycophorin A and Band 3, two highly-expressed membrane proteins. To produce clinically translatable strategies for human RBCs, it is critical to identify optimal red cell target epitopes, understand their effects on red cell physiology, and create humanized or human-like ligands to minimize immunogenicity. We constructed single chain antibodies (scFv) against antigenic determinants on Band 3 protein (Wrb) and RHCE protein (Rh17/Hr0) on human erythrocytes using phage display libraries prepared from immunized cynamolgous macaques (Macacafascicularis). Both these antigens are present on essentially 100% of the human population. Unfused scFvs were produced in E.coli while fusions of scFv with the extracellular domain of human thrombomodulin (TM-scFv) were produced in Drosophila S2 cells. Binding of recombinant proteins to human RBCs was measured by radioimmunoassay and flow cytometry. Generation of activated protein (APC) by RBCs loaded with TM-scFv fusions was measured by colorimetric assay. RBCs pre-incubated with varying concentrations of anti-Band3 and anti-RHCE fusions were assessed for osmotic resistance and mechanical integrity by exposure to hypo-osmolar medium and rotation in the presence of glass beads, respectively. PS exposure was measured by annexin V binding, and ROS generation was measured by dihydrorhodamine-associated fluorescence. Effects on RBC rheology were measured by flowing through microfluidic channels under controlled shear rates. Efficacy of TM-scFv fusions in diseased micro-vessels was assessed using a TNF-alpha stimulated, endothelialized microfluidic model. Single-chain antibody fragments and TM fusion proteins targeted to conserved epitopes on Band 3 protein and RHCE protein bound to human, but not murine or porcine, RBCs with high specificity and affinity (~50 nM), and in numbers consistent with the expected level of target expression (105 and 106 copies/RBC for RHCE and Band3, respectively). Coating RBCs with proteins targeted to Band 3 lessened RBC hypo-osmolar hemolysis (20% reduction) but increased hemolysis (2-fold) under mechanical stress, changes compatible with decreased red cell deformability. Proteins targeted to RHCE did not induce significant changes in hemolysis of RBCs under either osmotic or mechanical stress. Targeting neither Band 3 nor RHCE induced significant exposure of PS or production of ROS. Target-dependent effects on RBC rheology were observed under varying shear stresses in a microfluidic system. Fusion proteins of TM targeted to both epitopes demonstrated dose- and surface-copy-number-dependent generation of APC in the presence of PC and thrombin. Both TM-scFv fusion proteins were efficacious in a microfluidic model of disseminated intravascular coagulation using whole human blood by demonstrating near complete abrogation of fibrin generation in response to endothelial activation with TNF-alpha. In summary, we designed human RBC-specific non-human primate single chain antibody fragments capable of fusion to therapeutic cargoes. The TM-scFv fusions maintained therapeutic activity when bound to human RBCs and showed effective thromboprophylaxis in a whole-blood model of vasculitic injury. These antibodies and fusion proteins bound to erythroid-specific epitopes, and demonstrated target-dependent effects on several aspects of red cell physiology. The non-human primate origin of the antibodies should minimize their potential immunogenicity and the findings provide a platform to translate red cell targeted drug delivery into the clinical realm. Disclosures No relevant conflicts of interest to declare.
Biotechnology & Genetic Engineering Reviews, 2006
Biomacromolecules, Apr 22, 2009
Methods in molecular biology, Nov 25, 2009
Current Drug Metabolism, 2012
Royal Society Open Science, Jun 1, 2016
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Papers by Vladimir Muzykantov