New research into designer molecules which allow real-time vascular disease diagnosis and treatment
...Why not both?
A patient enters the emergency room with a suspected blood clot, ready to embolize and cause a fatality at any moment. Imagine being to administer one agent which would; stay dormant until identifying its target, release a payload of thrombolytics upon arrival, and, be visible to the clinician throughout the entire process.
Recent feats in protein engineering, combined with very creative thinking, have led to the rise of several designer molecules boasting these highly desirable properties. The first building block was the synthesis of single-chain antibodies designed to seek out platelet-rich blood clots. These small and highly specific antibodies were designed to recognise an epitope which is only exposed when platelets become activated. This tactic localises imaging and therapeutic delivery to the exact site required, which minimizes effects on normal hemostasis.
Using advanced bio-conjugation techniques, these clot-targeting antibodies have been labelled with radiotracers for PET imaging, microparticles of iron oxide for MRI, as well as microbubbles for ultrasound detection.
They have also been conjugated to nanocapsules containing concentrated anti-platelet and fibrinolytic payloads. Systemic administration of such agents has the potential to cause significant bleeding, which can also result in death. By targeting payloads directly to clots, the potential for unwanted bleeding side effects is greatly reduced.
Modifications involving the biological “trigger” for payload release have also been performed. The most recent innovations include hijacking the body’s own coagulation system, by creating a capsule cleavable by high concentrations of coagulation enzymes. Other capsules have been designed to be “shear-sensitive”, which mean they disintegrate upon exposure to high-stress vascular environments such as those which occur during vessel stenosis in cardiovascular disease. The single-chain antibodies themselves have also been tweaked to target other sites of disease, including atherosclerotic plaques.
Preclinical in vivo studies assessing the efficacy and viability of these designer molecules are indeed promising. However, much remains to be known regarding the biodegradability, elimination and safety profiles of these new agents before clinical trials begin. Regardless, the holistic theranostic approach undoubtedly represents a new age in biopharmaceutical design and medicine.
Abstract: Nanotechnology has begun to play a remarkable role in various fields of science and technology. In biomedical applications, nanoparticles have opened new horizons, especially for biosensing, targeted delivery of therapeutics, etc. Among drug delivery systems (DDSs), smart nanocarriers that can respond to specific stimuli in their local environment represent a growing field. Nanoplatforms that can be activated by an external application of light, can be used for a wide variety of photo-activated therapies, (especially light-triggered DDSs) relying on photo-isomerization, photo-crosslinking/uncrosslinking, photo-reduction, etc. In addition, light activation also has potential in photodynamic therapy (PDT), photothermal therapy (PTT), radiotherapy, protected delivery of bioactive moieties, anti-cancer drug delivery systems, theranostics (i.e. real-time monitoring and tracking combined with a therapeutic action to different diseases sites and organs). Combinations of these approaches can lead to enhanced and synergistic therapies, employing light either as a trigger for release or for activation. Non-linear absorption mechanisms using deeply penetrating near-infrared light, such as two-photon absorption, and photon upconversion have been employed in the design of light-responsive DDSs. The integration of a light stimulus into dual/multi-responsive nanocarriers can provide both spatiotemporal controlled delivery and release of therapeutic agents. Targeted and controlled nanosystems combining delivery of two or more agents, can provide on-demand release under specific conditions, etc. Overall, light activated nanomedicines and DDSs are expected to provide more effective therapies against serious diseases such as cancers, inflammation, infections, and cardiovascular disease, with reduced side effects, and will open new doors toward treatment of patients throughout the world.
Pub.: 14 Feb '17, Pinned: 14 Apr '17
Abstract: Thrombolytic therapy for acute thrombosis is limited by life-threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine-protease thrombin (HtPlg), fused to an activation-specific anti-glycoprotein IIb/IIIa single-chain antibody (SCE5), thereby hijacking the coagulation system to initiate thrombolysis.The resulting fusion protein named SCE5-HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/IIIa expressed on activated human platelets. Following thrombin formation, SCE5-HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE5-HtPlg (4 μg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride-induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P<0.01) and also reduced emboli formation in a model of pulmonary embolism (P<0.01 versus saline). Furthermore, at these effective therapeutic doses, the SCE5-HtPlg did not prolong bleeding time compared with saline (P=0.99).Our novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis.
Pub.: 06 Feb '17, Pinned: 14 Apr '17
Abstract: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component.To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation.The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries.This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.
Pub.: 28 Jun '11, Pinned: 14 Apr '17
Abstract: One of the major challenges in our contemporary society is to facilitate healthy life for all human beings. In this context, cancer has become one of the most deadly diseases around the world, and despite many advances in theranostics techniques the treatment of cancer still remains an important problem. With recent advances made in the field of nano-biotechnology, carbon-based nanostructured materials have drawn special attention because of their unique physicochemical properties, giving rise to great potential for the diagnosis and therapy of cancer. This review deals with four different types of carbon allotrope including carbon nanotubes, graphene, fullerenes and nanodiamonds and summarizes the results of recent studies that are likely to have implications in cancer theranostics. We discuss the applications of these carbon allotropes for cancer imaging and drug delivery, hyperthermia, photodynamic therapy and acoustic wave assisted theranostics. We focus on the results of different studies conducted on functionalized/conjugated carbon nanotubes, graphene, fullerenes and nanodiamond based nanostructured materials reported in the literature in the current decade. The emphasis has been placed on the synthesis strategies, structural design, properties and possible mechanisms that are perhaps responsible for their improved theranostic characteristics. Finally, we discuss the critical issues that may accelerate the development of carbon-based nanostructured materials for application in cancer theranostics.
Pub.: 13 Apr '17, Pinned: 14 Apr '17
Abstract: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis.LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111)In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2) vs. 17390 ± 7470 DLU/mm(2); P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111)In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111)In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01).Nuclear imaging with (111)In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application.
Pub.: 12 Apr '11, Pinned: 14 Apr '17
Abstract: Early and reliable detection of pulmonary embolism (PE) is critical for improving patient morbidity and mortality. The desire for low-threshold screening for pulmonary embolism is contradicted by unfavorable radiation of currently used computed tomography or nuclear techniques, while standard magnetic resonance imaging still struggles to provide sufficient diagnostic sensitivity in the lung. In this study we evaluate a molecular-targeted contrast agent against activated platelets for non-invasive detection of murine pulmonary thromboembolism using magnetic resonance imaging. By intravenous injection of human thrombin, pulmonary thromboembolism were consistently induced as confirmed by immunohistochemistry of the lung. Magnetic resonance imaging after thrombin injection showed local tissue edema in weighted images which co-localized with the histological presence of pulmonary thromboembolism. Furthermore, injection of a functionalized contrast agent targeting activated platelets provided sensitive evidence of focal accumulation of activated platelets within the edematous area, which, ex vivo, correlated well with the size of the pulmonary embolism. In summary, we here show delivery and specific binding of a functionalized molecular contrast agent against activated platelets for targeting pulmonary thromboembolism. Going forward, molecular imaging may provide new opportunities to increase sensitivity of magnetic resonance imaging for detection of pulmonary embolism.
Pub.: 04 May '16, Pinned: 14 Apr '17
Abstract: A reliable method for the diagnosis of minimal cardiac ischemia would meet a strong demand for the sensitive diagnosis of coronary artery disease in cardiac stress testing and risk stratification in patients with chest pain but unremarkable ECGs and biomarkers. We hypothesized that platelets accumulate early on in ischemic myocardium and a newly developed technology of non-invasive molecular PET imaging of activated platelets can thus detect minimal degrees of myocardial ischemia. To induce different degrees of minimal cardiac ischemia, the left anterior descending artery (LAD) was ligated for 10, 20 or 60 min. Mice were injected with a newly generated scFvanti-GPIIb/IIIa-(64)CuMeCOSar radiotracer, composed of a single-chain antibody that only binds to activated integrin GPIIb/IIIa (αIIbβIII) and thus to activated platelets, and a sarcophagine cage MeCOSar complexing the long half-life PET tracer copper-64. A single PET/CT scan was performed. Evans Blue/TTC staining to detect necrosis as well as classical serological biomarkers like Troponin I and heart-type fatty acid-binding protein (H-FABP) were negative, whereas PET imaging of activated platelets was able to detect small degrees of ischemia. Taken together, molecular PET imaging of activated platelets represents a unique and highly sensitive method to detect minimal cardiac ischemia.
Pub.: 03 Dec '16, Pinned: 14 Apr '17
Abstract: Myocardial infarction and stroke are leading causes of morbidity/mortality. The typical underlying pathology is the formation of thrombi/emboli and subsequent vessel occlusion. Systemically administered fibrinolytic drugs are the most effective pharmacological therapy. However, bleeding complications are relatively common and this risk as such limits their broader use. Furthermore, a rapid non-invasive imaging technology is not available. Thereby, many thrombotic events are missed or only diagnosed when ischemic damage has already occurred.Design and preclinical testing of a novel 'theranostic' technology for the rapid non-invasive diagnosis and effective, bleeding-free treatment of thrombosis.A newly created, innovative theranostic microbubble combines a recombinant fibrinolytic drug, an echo-enhancing microbubble and a recombinant thrombus-targeting device in form of an activated-platelet-specific single-chain antibody. After initial in vitro proof of functionality, we tested this theranostic microbubble both in ultrasound imaging and thrombolytic therapy using a mouse model of ferric-chloride-induced thrombosis in the carotid artery. We demonstrate the reliable highly sensitive detection of in vivo thrombi and the ability to monitor their size changes in real time. Furthermore, these theranostic microbubbles proofed to be as effective in thrombolysis as commercial urokinase but without the prolongation of bleeding time as seen with urokinase.We describe a novel theranostic technology enabling simultaneous diagnosis and treatment of thrombosis, as well as monitoring of success or failure of thrombolysis. This technology holds promise for major progress in rapid diagnosis and bleeding-free thrombolysis thereby potentially preventing the often devastating consequences of thrombotic disease in many patients.
Pub.: 30 Mar '16, Pinned: 14 Apr '17
Abstract: Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that their effective concentrations are limited due to bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations.Here, we have exploited a key biomechanical feature specific to thrombosis; significantly increased blood shear stress due to a reduction in the lumen of the vessel, to achieve site directed delivery of the clinically used antiplatelet agent eptifibatide using shear-sensitive phosphatidylcholine based nanocapsules.2.8x10(12) PC based nanocapsules with high dose encapsulated eptifibatide were introduced in microfluidic blood perfusion assays and in in vivo models of thrombosis and tail bleeding.Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above 1,000 s(-1) shear rate while leaving thrombus formation under physiological shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear sensitive antiplatelet nanocapsules did not display prolonged bleeding times.Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site specific antiplatelet potential and may form a basis for developing more potent and safer antiplatelet drugs. This article is protected by copyright. All rights reserved.
Pub.: 08 Mar '17, Pinned: 14 Apr '17
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