An Ultrasound-Responsive Theranostic Cyclodextrin-Loaded Nanoparticle for Multimodal Imaging and Therapy for Atherosclerosis.

Atherosclerosis is a major cause of mortality and morbidity worldwide. Left undiagnosed and untreated, atherosclerotic plaques can rupture and cause cardiovascular complications such as myocardial infarction and stroke. Atherosclerotic plaques are composed of lipids, including oxidized low-density lipoproteins and cholesterol crystals, and immune cells, including macrophages. 2-Hydroxypropyl-beta-cyclodextrin (CD) is FDA-approved for capturing, solubilizing, and delivering lipophilic drugs in humans. It is also known to dissolve cholesterol crystals and decrease atherosclerotic plaque size. However, its low retention time necessitates high dosages for successful therapy. This study reports CD delivery via air-trapped polybutylcyanoacrylate nanoparticles (with diameters of 388 ± 34 nm) loaded with CD (CDNPs). The multimodal contrast ability of these nanoparticles after being loaded with IR780 dye in mice is demonstrated using ultrasound and near-infrared imaging. It is shown that CDNPs enhance the cellular uptake of CD in murine cells. In an ApoE-/- mouse model of atherosclerosis, treatment with CDNPs significantly improves the anti-atherosclerotic efficacy of CD. Ultrasound triggering further improves CD uptake, highlighting that CDNPs can be used for ultrasound imaging and ultrasound-responsive CD delivery. Thus, CDNPs represent a theranostic nanocarrier for potential application in patients with atherosclerosis.

Near-infrared light-responsive liposomes for protein delivery: Towards bleeding-free photothermally-assisted thrombolysis.

Smart drug delivery systems represent state-of-the-art approaches for targeted therapy of life-threatening diseases such as cancer and cardiovascular diseases. Stimuli-responsive on-demand release of therapeutic agents at the diseased site can significantly limit serious adverse effects. In this study, we engineered a near-infrared (NIR) light-responsive liposomal gold nanorod-containing platform for on-demand delivery of proteins using a hybrid formulation of ultrasmall gold nanorods (AuNRs), thermosensitive phospholipid (DPPC) and non-ionic surfactant (Brij58). In light-triggered release optimization studies, 55.6% (± 4.8) of a FITC-labelled model protein, ovalbumin (MW 45 kDa) was released in 15 min upon NIR irradiation (785 nm, 1.35 W/cm2 for 5 min). This platform was then utilized to test on-demand delivery of urokinase-plasminogen activator (uPA) for bleeding-free photothermally-assisted thrombolysis, where the photothermal effect of AuNRs would synergize with the released uPA in clot lysis. Urokinase light-responsive liposomes showed 80.7% (± 4.5) lysis of an in vitro halo-clot model in 30 min following NIR irradiation (785 nm, 1.35 W/cm2 for 5 min) compared to 36.3% (± 4.4) and 15.5% (± 5.5) clot lysis from equivalent free uPA and non-irradiated liposomes respectively. These results show the potential of low-dose, site-specific thrombolysis via the combination of light-triggered delivery/release of uPA from liposomes combined with photothermal thrombolytic effects from gold nanorods. In conclusion, newly engineered, gold nanorod-based, NIR light-responsive liposomes represent a promising drug delivery system for site-directed, photothermally-stimulated therapeutic protein release.

Antithrombotic Therapy in Myocardial Infarction: Historic Perils and Current Challenges-A 70-Year Journey.

There have been numerous and intriguing advancements in antithrombotic therapy for myocardial infarction since it was described in the earliest issues of Thrombosis and Haemostasis. In this article, we revisit historical breakthroughs and describe the four most challenging contemporary themes relating to antithrombotic therapy in myocardial infarction. In all four, the challenge is to find the best balance of reducing specific levels of ischaemic risks without increasing bleeding risk. The first is the question of the optimal duration of dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI). This includes discussion of monotherapy after a period of DAPT. The second relates to the role of genotype and phenotype-guided individualisation of antiplatelet therapy. There is emerging evidence for a role of pheno/genotyping in identifying individuals at high risk for recurrent ischaemic events or in guiding the timing of cardiac surgery for patients on DAPT. The third addresses the increasing evidence for dual pathway inhibition, for example, with rivaroxaban in addition to aspirin in patients where high ischaemic and low bleeding risk is demonstrated. Finally the fourth highlights the challenge of the most appropriate combination of antiplatelet and anticoagulation therapy for patients with known atrial fibrillation after PCI. In most individuals, oral P2Y12 inhibitor therapy combined with a direct acting oral anticoagulant appears to be the best strategy based on the available evidence. Overall, the progress in antithrombotic therapy achieved over the last seven decades is remarkable, however, there are important issues to address and progress still to be made.

Dual-Targeted Theranostic Delivery of miRs Arrests Abdominal Aortic Aneurysm Development.

Abdominal aortic aneurysm (AAA) is an often deadly disease without medical, non-invasive treatment options. The upregulation of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium provides an early target epitope for a novel biotechnological theranostic approach. MicroRNA-126 was used as a therapeutic agent, based on its capability to downregulate VCAM-1 expression in endothelial cells and thereby reduces leukocyte adhesion and exerts anti-inflammatory effects. Ultrasound microbubbles were chosen as carriers, allowing both molecular imaging as well as targeted therapy of AAA. Microbubbles were coupled with a VCAM-1-targeted single-chain antibody (scFvmVCAM-1) and a microRNA-126 mimic (M126) constituting theranostic microbubbles (TargMB-M126). TargMB-M126 downregulates VCAM-1 expression in vitro and in an in vivo acute inflammatory murine model. Most importantly, using TargMB-M126 and ultrasound-guided burst delivery of M126, the development of AAA in an angiotensin-II-induced mouse model can be prevented. Overall, we describe a unique biotechnological theranostic approach with the potential for early diagnosis and long-sought-after medical therapy of AAA.

A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology.

Progress in pharmaceutical development is highly-dependent on preclinical in vivo animal studies. Small animal imaging is invaluable for the identification of new disease markers and the evaluation of drug efficacy. Here, we report for the first time the use of a three-dimensional fluorescence bioimager called FLuorescence Emission Computed Tomography (FLECT) for the detection of a novel recombinant fluoroprobe that is safe, easily prepared on a large scale and stably stored prior to scan. This novel fluoroprobe (Targ-Cy7) comprises a single-chain antibody-fragment (scFvTarg), which binds exclusively to activated-platelets, conjugated to a near-infrared (NIR) dye, Cy7, for detection. Upon mouse carotid artery injury, the injected fluoroprobe circulates and binds within the platelet-rich thrombus. This specific in vivo binding of the fluoroprobe to the thrombus, compared to its non-targeting control-fluoroprobe, is detected by the FLECT imager. The analyzed FLECT image quantifies the NIR signal and localizes it to the site of vascular injury. The detected fluorescence is further verified using a two-dimensional IVIS® Lumina scanner, where significant NIR fluorescence is detected in vivo at the thrombotic site, and ex vivo, at the injured carotid artery. Furthermore, fluorescence levels in various organs have also been quantified for biodistribution, with the highest fluoroprobe uptake shown to be in the injured artery. Subsequently, this live animal imaging technique is successfully employed to monitor the response of the induced thrombus to treatment over time. This demonstrates the potential of using longitudinal FLECT scanning to examine the efficacy of candidate drugs in preclinical settings. Besides intravascular thrombosis, we have shown that this non-invasive FLECT-imaging can also detect in vivo pulmonary embolism. Overall, this report describes a novel fluorescence-based preclinical imaging modality that uses an easy-to-prepare and non-radioactive recombinant fluoroprobe. This represents a unique tool to study mechanisms of thromboembolic diseases and it will strongly facilitate the in vivo testing of antithrombotic drugs. Furthermore, the non-radiation nature, low-cost, high sensitivity, and the rapid advancement of optical scanning technologies make this fluorescence imaging an attractive development for future clinical applications.

Molecular imaging of activated platelets via antibody-targeted ultra-small iron oxide nanoparticles displaying unique dual MRI contrast.

Magnetic resonance imaging (MRI) is a powerful and indispensable tool in medical research, clinical diagnosis, and patient care due to its high spatial resolution and non-limited penetration depth. The simultaneous use of positive and negative MRI imaging that employs the same contrast agents will significantly improve detection accuracy. Here we report the development of functional multimodal iron oxide nanoparticles for targeted MRI of atherothrombosis using a combination of chemical and biological conjugation techniques. Monodisperse, water-soluble and biocompatible ultra-small magnetic dual contrast iron oxide nanoparticles (DCIONs) were generated using a high-temperature co-precipitation route and appeared to be efficient positive and negative dual contrast agents for magnetic resonance imaging. Using a unique chemo-enzymatic approach involving copper-free click chemistry and Staphylococcus aureus sortase A enzyme conjugation, DCIONs were functionalized with single-chain antibodies (scFv) directed against activated platelets for targeting purposes. The DCIONs were also labelled with fluorescent molecules to allow for optical imaging. The antigen binding activity of the scFv was retained and resulted in the successful targeting of contrast agents to thrombosis as demonstrated in a range of in vitro and in vivo experiments. T1- and T2-weighted MRI of thrombi was recorded and demonstrated the great potential of dual T1/T2 contrast iron oxide particles in imaging of cardiovascular disease.

Thrombus-Targeted Theranostic Microbubbles: A New Technology towards Concurrent Rapid Ultrasound Diagnosis and Bleeding-free Fibrinolytic Treatment of Thrombosis.

RATIONALE: 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. OBJECTIVE: Design and preclinical testing of a novel 'theranostic' technology for the rapid non-invasive diagnosis and effective, bleeding-free treatment of thrombosis. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.

RATIONALE: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.