Sergei Zaitsev

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.

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

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. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

Nuclear translocation of urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle alpha-actin (alpha-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.

H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer

In this article we describe the chromatographic separation of acid nuclear protein fractions which have previously been shown to be active in DNA transfection experiments. By combining anionic and cationic ion exchangers, we were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic mobility than H1. We have also found that the presence of CaCl2 in a low concentration in the cell culture medium is an important requirement for transfection.

Cerebrovascular Thromboprophylaxis in Mice by Erythrocyte-Coupled Tissue-Type Plasminogen Activator

Background— Cerebrovascular thrombosis is a major source of morbidity and mortality after surgery, but thromboprophylaxis in this setting is limited because of the formidable risk of perioperative bleeding. Thrombolytics (eg, tissue-type plasminogen activator [tPA]) cannot be used prophylactically in this high-risk setting because of their short duration of action and risk of causing hemorrhage and central nervous system damage. We found that coupling tPA to carrier red blood cells (RBCs) prolongs and localizes tPA activity within the bloodstream and converts it into a thromboprophylactic agent, RBC/tPA. To evaluate the utility of this new approach for preventing cerebrovascular thrombosis, we examined the effect of RBC/tPA in animal models of cerebrovascular thromboembolism and ischemia. Methods and Results— Preformed fibrin microemboli were injected into the middle carotid artery of mice, occluding downstream perfusion and causing severe infarction and 50% mortality within 48 hours. Preinjected RBC/tPA rapidly lysed nascent cerebral thromboemboli, providing rapid, durable reperfusion and reducing morbidity and mortality. These beneficial effects were not achieved by preinjection of tPA, even at a 10-fold higher dose, which increased mortality from 50% to 90% by 10 hours after embolization. RBC/tPA injected 10 minutes after tail amputation to simulate postsurgical hemostasis did not cause bleeding from the wound, whereas soluble tPA caused profuse bleeding. RBC/tPA neither aggravated brain damage caused by focal ischemia in a filament model of middle carotid artery occlusion nor caused postthrombotic hemorrhage in hypertensive rats. Conclusions— These results suggest a potential RBC/tPA utility as thromboprophylaxis in patients who are at risk for acute cerebrovascular thromboembolism.

Targeting of a mutant plasminogen activator to circulating red blood cells for prophylactic fibrinolysis.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions.

AbstractPurpose. One of the drawbacks of polycationic and cationic liposomalgene transfer is its sensitivity to serum. Gene therapy requires thetransfectant-DNA complex to be resistant to serum as well as blood.Since Ca2+ has proved to be an efficient cofactor of polycationic genetransfer, we decided to investigate its effects on transfection in thepresence of serum. Methods. We studied transgene expression of luciferase gene (pCMVLuc) on ECV 304 human endothelial cells using H1 histone andDOSPER as transfectants in the presence of 0-100% fetal calf serum. Results. H1-and DOSPER-mediated transfection was found to beinhibited by serum above the concentration of 10%. If 2 mM Ca2+ or2 mM Ca2+/0.1 mM chloroquine was included in the culture mediumwhich replace the transfection mixture and was left on the cells for24 hours postincubation, the inhibiting effect of even 100% serumwas overcome. Conclusions. A high serum level does not interfere with binding anduptake of H1- and DOSPER-DNA complexes, but inhibits subsequentsteps such as endosomal escape. Ca2+ in the form of nascent calciumphosphate microprecipitates and other lysosomolytical agents facilitateendosomal/lysosomal release by their fusigenic and membranolyticactivity.

Microthrombosis after experimental subarachnoid hemorrhage: time course and effect of red blood cell-bound thrombin-activated pro-urokinase and clazosentan.

Delayed cerebral ischemia (DCI) is a significant cause of morbidity and mortality for patients surviving the rupture of an intracranial aneurysm. Despite an association between vasospasm and DCI, thrombosis and thromboembolism may also contribute to DCI. In this study we investigate the time course of intravascular microclot formation after experimental subarachnoid hemorrhage (SAH) and assess the effects of the following two drugs on microclot burden: mutant thrombin-activated urokinase-type plasminogen activator (scFv/uPA-T), which is bound to red blood cells for use as a thromboprophylactic agent, and clazosentan, an endothelin antagonist. In the first study, adult male C57BL/6 mice were sacrificed at 24 (n=5), 48 (n=6), 72 (n=8), and 96 (n=3) hours after SAH induced by filament perforation of the anterior cerebral artery. Sham animals (n=5) underwent filament insertion without puncture. In the second study, animals received scFv/uPA-T (n=5) 3 hours after hemorrhage, clazosentan (n=5) by bolus and subcutaneous pump after SAH just prior to skin closure, or a combination of scFv/uPA-T and clazosentan (n=4). Control (n=6) and sham (n=5) animals received saline alone. All animals were sacrificed at 48 hours and underwent intra-cardiac perfusion with 4% paraformaldehyde. The brains were then extracted and sliced coronally on a cryostat and processed for immunohistochemistry. An antibody recognizing thrombin-anti-thrombin complexes was used to detect microclots on coronal slices. Microclot burden was calculated for each animal and compared among groups. Following SAH, positive anti-thrombin staining was detected bilaterally in the following brain regions, in order of decreasing frequency: cortex; hippocampus; hypothalamus; basal ganglia. Few microclots were found in the shams. Microclot burden peaked at 48 hours and then decreased gradually. Animals receiving scFv/uPA-T and scFv/uPA-T+clazosentan had a lower microclot burden than controls, whereas animals receiving clazosentan alone had a higher microclot burden (p<0.005). The overall mortality rate in the time course study was 40%; mortality was highest among control animals in the second study. Intravascular microclots form in a delayed fashion after experimental SAH. Microclots may be safely reduced using a novel form of thromboprophylaxis provided by RBC-targeted scFv/uPA-T and represent a potential target for therapeutic intervention in the treatment of DCI.

Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis

Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombins substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

Signaling, delivery and age as emerging issues in the benefit/risk ratio outcome of tPA For treatment of CNS ischemic disorders.

J. Neurochem. (2010) 113, 303–312.