ORCID Profile
0000-0002-7114-4023
Current Organisation
Monash University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Nanobiotechnology | Bioinorganic chemistry | Nanomaterials | Biologically active molecules | Analytical Chemistry | Functional Materials | Nanotechnology | Analytical Spectrometry | Materials Engineering | Metals and Alloy Materials | Chemical Characterisation of Materials | Inorganic chemistry |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in Engineering | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in Technology
Related Links
Publications
Carbohydrates@MOFs
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C8MH01611A
Abstract: MOFs have demonstrated outstanding properties for the protection and controlled release of different bio-entities, from proteins to living cells.
Early Results from GLASS–JWST. XXI. Rapid Asembly of a Galaxy at z = 6.23 Revealed by Its C/O Abundance
Publisher: American Astronomical Society
Date: 07-2023
Abstract: The abundance of carbon relative to oxygen (C/O) is a promising probe of star formation history in the early universe, as the ratio changes with time due to production of these elements by different nucleosynthesis pathways. We present a measurement of log ( C / O ) = − 1.01 ± 0.12 (stat) ±0.15 (sys) in a z = 6.23 galaxy observed as part of the GLASS–JWST Early Release Science Program. Notably, we achieve good precision thanks to the detection of the rest-frame ultraviolet O iii ], C iii ], and C iv emission lines delivered by JWST/NIRSpec. The C/O abundance is ∼0.8 dex lower than the solar value and is consistent with the expected yield from core-collapse supernovae, indicating that longer-lived intermediate-mass stars have not fully contributed to carbon enrichment. This in turn implies rapid buildup of a young stellar population with age ≲100 Myr in a galaxy seen ∼900 Myr after the big bang. Our chemical abundance analysis is consistent with spectral energy distribution modeling of JWST/NIRCam photometric data, which indicates a current stellar mass log M * / M ☉ = 8.4 − 0.2 + 0.4 and specific star formation rate ≃20 Gyr −1 . These results showcase the value of chemical abundances and C/O in particular to study the earliest stages of galaxy assembly.
18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography
Publisher: MDPI AG
Date: 21-06-2022
DOI: 10.3390/IJMS23136886
Abstract: Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation.
Immuno-magnetoliposomes targeting activated platelets as a potentially human-compatible MRI contrast agent for targeting atherothrombosis
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.BIOMATERIALS.2015.02.088
Abstract: To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5 mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (∼86%, ∼0.22 mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2 = 422 s(-1) mM(-1) and r2(∗) = 452 s(-1) mM(-1)) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor ex vivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets.
Magnetic fibrinolysis: putting the therapeutic wheels in a corkscrew motion
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1111/JTH.13969
Triggered and Tunable Hydrogen Sulfide Release from Photogenerated Thiobenzaldehydes
Publisher: Wiley
Date: 27-07-2017
Abstract: Hydrogen sulfide (H
Enzyme-Mediated Site-Specific Bioconjugation of Metal Complexes to Proteins: Sortase-Mediated Coupling of Copper-64 to a Single-Chain Antibody
Publisher: Wiley
Date: 28-04-2014
Abstract: The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins.
Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons
Publisher: Wiley
Date: 20-09-2002
DOI: 10.1002/CNE.10371
Abstract: In the hippoc us, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippoc us proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippoc al layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippoc us was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippoc us of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippoc al principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.
Testosterone Metabolism In Rat Brain Is Differentially Enhanced By Phenytoin‐Inducible Cytochrome P450 Isoforms
Publisher: Wiley
Date: 08-1999
DOI: 10.1046/J.1365-2826.1999.00371.X
Abstract: Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti-epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin-induced animals was examined by use of high performance liquid chromatography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6alpha-hydroxytestosterone (OHT) as the main metabolite and 6beta-OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2alpha-, 6beta-, 16alpha-, 16beta-OHT and androstenedione, whereby 16alpha-OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16alpha-OHT, 16beta-OHT and androstenedione. Western blots revealed the phenytoin-inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6beta-hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin-induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin-dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.
Single-chain antibodies as new antithrombotic drugs
Publisher: Georg Thieme Verlag KG
Date: 03-2007
Abstract: Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in erse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.
Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2007
DOI: 10.1161/ATVBAHA.106.138875
Abstract: Objective— Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. Methods and Results— Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv anti-LIBS ) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv anti-LIBS -TAP was proven in flow cytometry anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride–induced carotid artery thrombosis model in mice. ScFv anti-LIBS -TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv anti-LIBS -TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv anti-LIBS -TAP. Conclusions— The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.
Shear‐Responsive Drug Delivery Systems in Medical Devices: Focus on Thrombosis and Bleeding
Publisher: Wiley
Date: 04-06-2023
Abstract: Cardiovascular disease is the leading cause of death worldwide. Blood‐contacting medical devices provide critical support for patients with severe respiratory and/or cardiac failure. High shear stress zones and non‐biocompatible circuit can increase the risk of thrombus formation in patients. These thrombi restrict blood flow through the circuits and travel to the lungs and brain, creating significant consequences. Antithrombotic drugs are used to lower this risk, but they also raise the incidence of bleeding problems. This article explores the delicate balance between thrombosis and bleeding in medical devices by examining platelet activation and thrombosis formation under shear stress. The feasibility of a shear‐responsive nanomedicine‐based drug delivery system has been explored as a potential approach for targeted administration of antithrombotic medications to lower systemic drug levels and reduce the bleeding risk. Furthermore, the lack of in vitro platforms to investigate the biological behavior of antithrombotic nanoparticles is impeding their clinical translation. As a result, microfluidic technology offers a platform for investigating nanoparticle behavior in vitro and linking it to their performance in vivo. Finally, the challenges and factors that affect the functionality, stability, and circulation time of liposomal drugs are investigated to improve their efficacy for targeted drug administration in medical devices.
Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel.
Publisher: MyJove Corporation
Date: 29-06-2015
DOI: 10.3791/52838
Engineering of Nebulized Metal–Phenolic Capsules for Controlled Pulmonary Deposition
Publisher: Wiley
Date: 10-01-2020
Detection of activated platelets in a mouse model of carotid artery thrombosis with 18 F-labeled single-chain antibodies.
Publisher: Elsevier BV
Date: 03-2014
DOI: 10.1016/J.NUCMEDBIO.2013.12.006
Abstract: Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel (18)F PET radiotracer based on this antibody. ScFv(anti-LIBS) and control antibody mut-scFv were reacted with N-succinimidyl-4-[(18)F]fluorobenzoate (S[(18)F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl(3) induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. After incubation with increasing concentrations of (18)F-scFv(anti-LIBS) clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled (18)F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6). Our results show that the novel antibody radiotracer (18)F-scFv(anti-LIBS) is useful for the sensitive detection of activated platelets and thrombosis. We describe the first (18)F variant of a scFv(anti-LIBS) against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.
Targeting the platelet integrin GPIIb/IIIa
Publisher: Bentham Science Publishers Ltd.
Date: 12-2010
DOI: 10.2174/138161210794519255
Abstract: The platelet integrin GPIIb/IIIa plays an essential role in thrombus formation through interactions with adhesive ligands and has emerged as a primary target for the development of anti-thrombotic agents. Receptor activation is under strict control, with activators, inhibitors, and signalling mechanisms controlling its conformation. Structural biology research has produced high-resolution images defining the ligand binding site at the atomic level. Successful blockade of this ligand binding has validated GPIIb/IIIa as a therapeutic target in cardiovascular medicine. GPIIb/IIIa inhibitors were the first rationally designed anti-platelet agents and have been used effectively in a wide variety of clinical scenarios including unstable angina, myocardial infarction, and high risk percutaneous coronary interventions with and without intracoronary stenting. Three inhibitors (abciximab, eptifibatide, and tirofiban) are currently licensed for human use. Surprisingly, oral GPIIb/IIIa antagonists have not been successful and there is an unmet need for effective anti-GPIIb/IIIa drugs that cause less bleeding problems and that can be orally applied. Here we review our current knowledge about GPIIb/IIIa structure, signalling pathways and receptor function, the benefits and limitations of current GPIIb/IIIa blockers and we take a look forward how the lessons learned from the mixture of success and failure of GPIIb/IIIa blocker development can be transformed in new and better GPIIb/IIIa blockers.
A novel ex vivo approach for measuring plasminogen activation upon established plasma clots
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1002/RTH2.12771
Bio-click chemistry: enzymatic functionalization of PEGylated capsules for targeting applications
Publisher: Wiley
Date: 28-06-2012
Abstract: All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.
Polymer Capsules for Plaque-Targeted In Vivo Delivery
Publisher: Wiley
Date: 30-06-2016
Abstract: Targeted polymer capsules can selectively bind to unstable plaques in mice after intravenous injection. Different formulations of the capsules are explored with a synthetic/biopolymer hybrid capsule showing the best stability and small-molecule drug retention. The synthetic polymer is composed of pH-sensitive blocks (PDPA), low-binding blocks (PEG), and click-groups for postfunctionalization with targeting peptides specific to plaques.
Self-Assembled Nanoparticles from Phenolic Derivatives for Cancer Therapy
Publisher: Wiley
Date: 16-05-2017
Abstract: Therapeutic nanoparticles hold clinical promise for cancer treatment by avoiding limitations of conventional pharmaceuticals. Herein, a facile and rapid method is introduced to assemble poly(ethylene glycol) (PEG)-modified Pt prodrug nanocomplexes through metal-polyphenol complexation and combined with emulsification, which results in ≈100 nm diameter nanoparticles (PtP NPs) that exhibit high drug loading (0.15 fg Pt per nanoparticle) and low fouling properties. The PtP NPs are characterized for potential use as cancer therapeutics. Mass cytometry is used to quantify uptake of the nanoparticles and the drug concentration in in idual cells in vitro. The PtP NPs have long circulation times, with an elimination half-life of ≈18 h in healthy mice. The in vivo antitumor activity of the PtP NPs is systematically investigated in a human prostate cancer xenograft mouse model. Mice treated with the PtP NPs demonstrate four times better inhibition of tumor growth than either free prodrug or cisplatin. This study presents a promising strategy to prepare therapeutic nanoparticles for biomedical applications.
High-density lipoprotein delivered after myocardial infarction increases cardiac glucose uptake and function in mice.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 11-10-2017
DOI: 10.1126/SCITRANSLMED.AAM6084
Abstract: Protecting the heart after an acute coronary syndrome is a key therapeutic goal to support cardiac recovery and prevent progression to heart failure. A potential strategy is to target cardiac glucose metabolism at the early stages after ischemia when glycolysis is critical for myocyte survival. Building on our discovery that high-density lipoprotein (HDL) modulates skeletal muscle glucose metabolism, we now demonstrate that a single dose of reconstituted HDL (rHDL) delivered after myocardial ischemia increases cardiac glucose uptake, reduces infarct size, and improves cardiac remodeling in association with enhanced functional recovery in mice. These findings applied equally to metabolically normal and insulin-resistant mice. We further establish direct effects of HDL on cardiomyocyte glucose uptake, glycolysis, and glucose oxidation via the Akt signaling pathway within 15 min of reperfusion. These data support the use of infusible HDL preparations for management of acute coronary syndromes in the setting of primary percutaneous interventions.
Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy
Publisher: Springer Science and Business Media LLC
Date: 10-03-2022
DOI: 10.1038/S41598-022-07892-Z
Abstract: Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core–shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose–response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community.
Boronate–Phenolic Network Capsules with Dual Response to Acidic pH and cis‐Diols
Publisher: Wiley
Date: 18-06-2015
Abstract: Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of phenylborate and phenolic materials. The BPN capsules are stable in the presence of competing carbohydrates, but dissociate at acidic pH or in the presence of competing cis-diols at physiological pH. This engineered capsule system provides a platform for a wide range of biological and biomedical applications.
Collagen-Targeted Theranostic Nanosponges for Delivery of the Matrix Metalloproteinase 14 Inhibitor Naphthofluorescein
Publisher: American Chemical Society (ACS)
Date: 14-04-2020
Towards Effective and Safe Thrombolysis and Thromboprophylaxis
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 28-03-2014
DOI: 10.1161/CIRCRESAHA.114.302514
Abstract: 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. We developed a novel targeted fibrinolytic drug that is directed against activated platelets. We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFv SCE5 ), 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 scFv SCE5 –scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline ( P .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 scFv SCE5 –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 scFv SCE5 –scuPA as compared with the same dose of a mutated, nontargeting scFv–scuPA or vehicle. The ability of scFv SCE5 –scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. 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.
Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage
Publisher: Wiley
Date: 2010
DOI: 10.1111/J.1582-4934.2008.00409.X
Abstract: The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti‐atherosclerotic therapies. Vascular cell adhesion molecule‐1 (VCAM‐1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM‐1 as a target for gene therapy. As a proof of concept, integrin α IIb β 3 ‐mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of β 3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM‐1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM‐1. Indeed, VCAM‐1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM‐1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM‐1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
An Activation-Specific Platelet Inhibitor That Can Be Turned On/Off by Medically Used Hypothermia
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2011
DOI: 10.1161/ATVBAHA.111.226241
Abstract: Therapeutic hypothermia is successfully used, for ex le, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a β-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.
Psammomys obesus: a Natural Diet-Controlled Model for Diabetes and Cardiovascular Diseases
Publisher: Springer Science and Business Media LLC
Date: 17-07-2018
Multifunctional Thrombin‐Activatable Polymer Capsules for Specific Targeting to Activated Platelets
Publisher: Wiley
Date: 03-08-2015
Abstract: Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis.
Targeted Antithrombotic Protein Micelles
Publisher: Wiley
Date: 10-12-2014
A novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries using gene and microRNA expression profiling
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 19-07-2013
DOI: 10.1161/CIRCRESAHA.113.301562
Abstract: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited by the lack of animal models that reproduce plaque instability observed in humans. Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis. On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E–deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques. The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.
Platelet-targeted thrombolysis for treatment of acute ischemic stroke.
Publisher: American Society of Hematology
Date: 20-02-2022
DOI: 10.1182/BLOODADVANCES.2021006691
Abstract: Thrombolysis with tissue-type plasminogen activator (tPA) is still the main treatment for acute ischemic stroke. Nevertheless, tPA intervention is limited by a short therapeutic window, low recanalization rates and a risk of intracranial haemorrhage (ICH), raising a clinical demand for improved thrombolytic drugs. We examined a novel thrombolytic agent termed 'SCE5-scuPA', comprising a single-chain urokinase plasminogen activator (scuPA) fused with a single-chain antibody (SCE5) that targets the activated GPIIb/IIIa platelet receptor, for its effects in experimental stroke. SCE5-scuPA was first tested in whole blood clot degradation assay to demonstrate the benefit of platelet-targeted thrombolysis. The tail bleeding time, blood clearance and biodistribution were then determined to inform the use of SCE5-scuPA in mouse models of photothrombotic stroke and Middle Cerebral Artery occlusion (MCAo) against tenecteplase (TNK-tPA). The impacts of SCE5-scuPA on motor function, ICH, blood-brain barrier (BBB) integrity and immunosuppression were evaluated. Infarct size was measured by Computed Tomography and Magnetic Resonance Imaging. SCE5-scuPA enhanced clot degradation ex vivo compared to its non-platelet-targeting control. The maximal SCE5-scuPA dose that maintained hemostasis and a rapid blood clearance were determined. SCE5-scuPA administration both prior and 2h after photothrombotic stroke reduced the infarct volume. SCE5-scuPA also improved neurological deficit, decreased intracerebral blood deposits, preserved the BBB and alleviated immunosuppression post-stroke. In MCAo, SCE5-scuPA did not worsen stroke outcomes or caused ICH, and protected the BBB. Our findings support the ongoing development of platelet-targeted thrombolysis with SCE5-scuPA as a novel emergency treatment for AIS with a promising safety profile.
GPVI and GPIBα mediate Staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors
Publisher: Public Library of Science (PLoS)
Date: 28-04-2011
Self‐Assembly of Oriented Antibody‐Decorated Metal–Organic Framework Nanocrystals for Active‐Targeting Applications
Publisher: Wiley
Date: 02-01-2022
Abstract: Antibody (Ab)‐targeted nanoparticles are becoming increasingly important for precision medicine. By controlling the Ab orientation, targeting properties can be enhanced however, to afford such an ordered configuration, cumbersome chemical functionalization protocols are usually required. This aspect limits the progress of Abs‐nanoparticles toward nanomedicine translation. Herein, a novel one‐step synthesis of oriented monoclonal Ab‐decorated metal–organic framework (MOF) nanocrystals is presented. The crystallization of a zinc‐based MOF, Zn 2 (mIM) 2 (CO 3 ), from a solution of Zn 2+ and 2‐methylimidazole (mIM), is triggered by the fragment crystallizable (Fc) region of the Ab. This selective growth yields biocomposites with oriented Abs on the MOF nanocrystals (MOF*Ab): the Fc regions are partially inserted within the MOF surface and the antibody‐binding regions protrude from the MOF surface toward the target. This ordered configuration imparts antibody–antigen recognition properties to the biocomposite and shows preserved target binding when compared to the parental antibodies. Next, the biosensing performance of the system is tested by loading MOF*Ab with luminescent quantum dots (QD). The targeting efficiency of the QD‐containing MOF*Ab is again, fully preserved. The present work represents a simple self‐assembly approach for the fabrication of antibody‐decorated MOF nanocrystals with broad potential for sensing, diagnostic imaging, and targeted drug delivery.
Identification, induction and localization of cytochrome P450s of the 3A-subfamily in mouse brain
Publisher: Springer Science and Business Media LLC
Date: 07-2001
DOI: 10.1007/BF03033195
Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?
Publisher: American Society of Hematology
Date: 18-04-2013
DOI: 10.1182/BLOOD-2012-08-449694
Abstract: Targeting of an anticlotting drug to activated platelets promises effective blood clot prevention without bleeding side effects.
HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi
Publisher: Georg Thieme Verlag KG
Date: 2015
DOI: 10.1160/TH14-12-1073
Abstract: High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p 0.05) but failed to show binding to platelets from RAGE-/- mice (p 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.
Magnetic Resonance Imaging Contrast Agent Targeted Toward Activated Platelets Allows In Vivo Detection of Thrombosis and Monitoring of Thrombolysis
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 15-07-2008
DOI: 10.1161/CIRCULATIONAHA.107.753657
Abstract: Background— Platelets are the key to thrombus formation and play a role in the development of atherosclerosis. Noninvasive imaging of activated platelets would be of great clinical interest. Here, we evaluate the ability of a magnetic resonance imaging (MRI) contrast agent consisting of microparticles of iron oxide (MPIOs) and a single-chain antibody targeting ligand-induced binding sites (LIBS) on activated glycoprotein IIb/IIIa to image carotid artery thrombi and atherosclerotic plaques. Methods and Results— Anti-LIBS antibody or control antibody was conjugated to 1-μm MPIOs (LIBS MPIO/control MPIO). Nonocclusive mural thrombi were induced in mice with 6% ferric chloride. MRI (at 9.4 T) was performed once before and repeatedly in 12-minute-long sequences after LIBS MPIO/control MPIO injection. After 36 minutes, a significant signal void, corresponding to MPIO accumulation, was observed with LIBS MPIOs but not control MPIOs ( P .05). After thrombolysis, in LIBS MPIO-injected mice, the signal void subsided, indicating successful thrombolysis. On histology, the MPIO content of the thrombus, as well as thrombus size, correlated significantly with LIBS MPIO-induced signal void (both P .01). After ex vivo incubation of symptomatic human carotid plaques, MRI and histology confirmed binding to areas of platelet adhesion/aggregation for LIBS MPIOs but not for control MPIOs. Conclusions— LIBS MPIOs allow in vivo MRI of activated platelets with excellent contrast properties and monitoring of thrombolytic therapy. Furthermore, activated platelets were detected on the surface of symptomatic human carotid plaques by ex vivo MRI. This approach represents a novel noninvasive technique allowing the detection and quantification of platelet-containing thrombi.
Novel Single-Chain Antibody-Targeted Microbubbles for Molecular Ultrasound Imaging of Thrombosis
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 26-06-2012
DOI: 10.1161/CIRCULATIONAHA.111.030312
Abstract: Molecular imaging is a fast emerging technology allowing noninvasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Lipid-shell based gas-filled MBs were conjugated to either a single-chain antibody specific for activated glycoprotein IIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MBs) or a nonspecific single-chain antibody (control MBs). Successful conjugation was assessed in flow cytometry and immunofluorescence double staining. LIBS-MBs but not control MBs strongly adhered to both immobilized activated platelets and microthrombi under flow. Thrombi induced in carotid arteries of C57Bl6 mice in vivo by ferric chloride injury were then assessed with ultrasound before and 20 minutes after MB injection through the use of gray-scale area intensity measurement. Gray-scale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control MB injection (9.55±1.7 versus 1.46±1.3 dB P .01). Furthermore, after thrombolysis with urokinase, LIBS-MB ultrasound imaging allows monitoring of the reduction of thrombus size ( P .001). We demonstrate that glycoprotein IIb/IIIa–targeted MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis and monitoring of the success or failure of pharmacological thrombolysis in vivo.
Concordant up‐regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment
Publisher: Wiley
Date: 18-03-2009
DOI: 10.1111/J.1471-4159.2009.05994.X
Abstract: Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug-hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2alpha-, 2beta-, 6beta-, 16alpha- and 16beta-hydroxytestosterones, while 6alpha- and 15alpha-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chlor henicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippoc us, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug-hormone crosstalk.
Recent advances in molecular imaging of atherosclerotic plaques and thrombosis
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0NR00599A
Abstract: Recent advances in emerging molecular imaging techniques bring new opportunities in the diagnosis of atherosclerosis plaques and thrombosis.
Microlyse: a thrombolytic agent that targets VWF for clearance of microvascular thrombosis
Publisher: American Society of Hematology
Date: 27-01-2022
Abstract: Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13−/− mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.
Construction and characterization of a recombinant plasminogen activator composed of an anti‐fibrin single‐chain antibody and low‐molecular‐weight urokinase
Publisher: Elsevier BV
Date: 05-2004
A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic αIIbβ3(GPIIb/IIIa) Antagonists
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2007
DOI: 10.1161/01.ATV.0000255307.65939.59
Abstract: Objective— Integrins are attractive therapeutic targets. Inhibition of integrin α IIb β 3 effectively blocks platelet aggregation. However, limitations with intravenous α IIb β 3 antagonists and failure of oral α IIb β 3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results— Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic α IIb β 3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of α IIb β 3 , (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic α IIb β 3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic α IIb β 3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca 2+ -signaling. Blockade of the ADP receptor P2Y 12 by AR-C69931MX and clopidogrel inhibits α IIb β 3 antagonist-induced platelet activation. Conclusion— These findings can explain limitations of ligand-mimetic anti-α IIb β 3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.
Highly Sensitive Detection of Minimal Cardiac Ischemia using Positron Emission Tomography Imaging of Activated Platelets
Publisher: Springer Science and Business Media LLC
Date: 02-12-2016
DOI: 10.1038/SREP38161
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 scFv anti-GPIIb/IIIa - 64 CuMeCOSar radiotracer, composed of a single-chain antibody that only binds to activated integrin GPIIb/IIIa (α IIb β III ) 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.
Low-Fouling and Biodegradable Protein-Based Particles for Thrombus Imaging
Publisher: American Chemical Society (ACS)
Date: 07-06-2018
High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting
Publisher: Public Library of Science (PLoS)
Date: 27-08-2015
Enzymatic Antibody Tagging: Toward a Universal Biocompatible Targeting Tool
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.TCM.2012.07.004
Abstract: Targeted delivery or "smart delivery" of pharmaceutical or imaging agents and even entire cells such as stem cells is an emerging trend in modern biotechnology. A binding ligand such as an antibody that can specifically bind to receptors expressed at a disease site is an essential component of such constructs. Different chemical methods have been widely used to apply antibodies for delivery systems however, they typically result in impairment or loss of antibody functionality. Enzyme-mediated conjugation approaches have been developed to overcome this major disadvantage of conventional chemical methods. Sortase, an enzyme derived from Staphylococcus aureus, is able to provide a biochemically robust, highly reproducible, and site-specific coupling method for the conjugation of antibodies to pharmaceutical agents, nanoparticles, and cells for drug delivery, molecular imaging, and cell homing. Here, we review the use of sortase and other enzyme-based methods as bioconjugation tools with a focus on cardiovascular applications.
Template-Assisted Antibody Assembly: A Versatile Approach for Engineering Functional Antibody Nanoparticles
Publisher: American Chemical Society (ACS)
Date: 07-04-2022
Molecular imaging of activated platelets via antibody-targeted ultra-small iron oxide nanoparticles displaying unique dual MRI contrast
Publisher: Elsevier BV
Date: 07-2017
Oxidative Hydrolysis of Scoparone by Cytochrome P450 CYP2C29 Reveals a Novel Metabolite
Publisher: Elsevier BV
Date: 07-2001
Predominantly neuronal expression of cytochrome P450 isoforms cyp3a11 and cyp3a13 in mouse brain
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0306-4522(02)00955-7
Abstract: Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippoc us) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain's plasticity.
Ex-vivo thrombolytic gene therapy for vein graft patency: The frontier for development of selective, localised therapeutic approaches
Publisher: Georg Thieme Verlag KG
Date: 2009
DOI: 10.1160/TH09-06-0343
Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use
Publisher: Springer Science and Business Media LLC
Date: 11-12-2015
Abstract: Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.
A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry
Publisher: Wiley
Date: 11-05-2015
Abstract: A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.
Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1111/JTH.13666
Abstract: Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 10
Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 17-07-2009
DOI: 10.1161/CIRCRESAHA.108.190611
Abstract: C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis monocyte activation, as determined by conformational change of integrin Mac-1 generation of reactive oxygen species and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H 2 O 2 -treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects.
Polymer Capsules for Plaque‐Targeted In Vivo Delivery
Publisher: Wiley
Date: 09-2016
Development and validation of a high throughput whole blood thrombolysis plate assay.
Publisher: Springer Science and Business Media LLC
Date: 24-05-2017
DOI: 10.1038/S41598-017-02498-2
Abstract: The objective of this work was to develop a high throughput assay for testing in vitro the thrombolytic activity using citrated whole blood s les, and to overcome the limitations of currently available techniques. We successfully developed a method that involves forming halo shaped, tissue factor induced, whole blood clots in 96 well plates, and then precisely measuring the thrombolysis process with a spectrophotometer plate reader. We here describe the implementation of this novel method, which we refer to as halo assay, and its validation with plasmin, urokinase and tissue plasminogen activator at different doses. The resulting data is a highly detailed thrombolysis profile, allowing comparison of different fibrinolytic agents. The time point analysis allows kinetic data to be collected and calculated to determine key parameters such as the activation time and the rate of fibrinolysis. We also assessed the capacity of the model to study the effect of clot maturation time on the fibrinolytic rate, an aspect of thrombosis rather unexplored with currently available methods, but of increasing importance in drug development. This novel thrombolysis assay could be an extremely useful research tool to study the complex process of thrombolysis, and a valuable translational clinical tool as a screening device to rapidly identify hypo- or hyper-fibrinolysis.
Single-Chain Antibody Conjugated to a Cage Amine Chelator and Labeled with Positron-Emitting Copper-64 for Diagnostic Imaging of Activated Platelets
Publisher: American Chemical Society (ACS)
Date: 14-07-2014
DOI: 10.1021/MP500209A
Abstract: Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.
The potential impact of nanomedicine on COVID-19-induced thrombosis
Publisher: Springer Science and Business Media LLC
Date: 19-12-2023
Molecular Imaging of Activated Platelets Allows the Detection of Pulmonary Embolism with Magnetic Resonance Imaging
Publisher: Springer Science and Business Media LLC
Date: 03-05-2016
DOI: 10.1038/SREP25044
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 "Equation missing" 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.
Functional Brush Poly(2‐ethyl‐2‐oxazine)s: Synthesis by CROP and RAFT, Thermoresponsiveness and Grafting onto Iron Oxide Nanoparticles
Publisher: Wiley
Date: 12-02-2019
Abstract: Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for ex le, in nanomedicine. Here, the synthesis of functional phosphonate-ester-bearing brush polymers based on poly(2-oxazine)s is reported through a combination of cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazine and reversible addition-fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well-defined (Đ ≤ 1.17) poly(oligo(2-ethyl-2-oxazine) methacrylate) P(OEtOzMA)
Polyphenol-Functionalized Cubosomes as Thrombolytic Drug Carriers.
Publisher: Wiley
Date: 13-09-2022
Abstract: The safe administration of thrombolytic agents is a challenge for the treatment of acute thrombosis. Lipid-based nanoparticle drug delivery technologies present opportunities to overcome the existing clinical limitations and deliver thrombolytic therapy with enhanced therapeutic outcomes and safety. Herein, lipid cubosomes are examined as nanocarriers for the encapsulation of thrombolytic drugs. The lipid cubosomes are loaded with the thrombolytic drug urokinase-type plasminogen activator (uPA) and coated with a low-fouling peptide that is incorporated within a metal-phenolic network (MPN). The peptide-containing MPN (pep-MPN) coating inhibits the direct contact of uPA with the surrounding environment, as assessed by an in vitro plasminogen activation assay and an ex vivo whole blood clot degradation assay. The pep-MPN-coated cubosomes prepared with 22 wt% peptide demonstrate a cell membrane-dependent thrombolytic activity, which is attributed to their fusogenic lipid behavior. Moreover, compared with the uncoated lipid cubosomes, the uPA-loaded pep-MPN-coated cubosomes demonstrate significantly reduced nonspecific cell association (<10% of the uncoated cubosomes) in the whole blood assay, a prolonged circulating half-life, and reduced splenic uPA accumulation in mice. These studies confirm the preserved bioactivity and cell membrane-dependent release of uPA within pep-MPN-coated lipid cubosomes, highlighting their potential as a delivery vehicle for thrombolytic drugs.
Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting
Publisher: Wiley
Date: 18-01-2023
Abstract: A highly effective 2‐step system for site‐specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine‐independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox‐activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide‐alkyne cycloaddition ′click′ conjugation. The versatility of this 2‐step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine‐modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine‐mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.
An Engineered Nanosugar Enables Rapid and Sustained Glucose‐Responsive Insulin Delivery in Diabetic Mice
Publisher: Wiley
Date: 12-03-2023
Abstract: Glucose‐responsive insulin‐delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge‐switchable phytoglycogen nanoparticles capable of glucose‐stimulated insulin release are engineered. The nanoparticles are “nanosugars” bearing glucose‐sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL –1 ) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose‐regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose‐responsive insulin delivery system based on a natural and biodegradable nanosugar.
Novel Thrombolytic Drug Based on Thrombin Cleavable Microplasminogen Coupled to a Single-Chain Antibody Specific for Activated GPIIb/IIIa
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-02-2017
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/ III a single‐chain antibody ( SCE 5), thereby hijacking the coagulation system to initiate thrombolysis. The resulting fusion protein named SCE 5‐HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/ III a expressed on activated human platelets. Following thrombin formation, SCE 5‐HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE 5‐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 .01) and also reduced emboli formation in a model of pulmonary embolism ( P .01 versus saline). Furthermore, at these effective therapeutic doses, the SCE 5‐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.
Mopping up crystals to keep the blood flowing
Publisher: Elsevier BV
Date: 2022
Engineering Poly(ethylene glycol) Particles for Improved Biodistribution
Publisher: American Chemical Society (ACS)
Date: 21-01-2015
DOI: 10.1021/NN5061578
Abstract: We report the engineering of poly(ethylene glycol) (PEG) hydrogel particles using a mesoporous silica (MS) templating method via tuning the PEG molecular weight, particle size, and the presence or absence of the template and investigate the cell association and biodistribution of these particles. An ex vivo assay based on human whole blood that is more sensitive and relevant than traditional cell-line based assays for predicting in vivo circulation behavior is introduced. The association of MS@PEG particles (template present) with granulocytes and monocytes is higher compared with PEG particles (template absent). Increasing the PEG molecular weight (from 10 to 40 kDa) or decreasing the PEG particle size (from 1400 to 150 nm) reduces phagocytic blood cell association of the PEG particles. Mice biodistribution studies show that the PEG particles exhibit extended circulation times (>12 h) compared with the MS@PEG particles and that the retention of smaller PEG particles (150 nm) in blood, when compared with larger PEG particles (>400 nm), is increased at least 4-fold at 12 h after injection. Our findings highlight the influence of unique aspects of polymer hydrogel particles on biological interactions. The reported PEG hydrogel particles represent a new class of polymer carriers with potential biomedical applications.
Poly(ethylene glycol) Cross-Linked Antibody Nanoparticles for Tunable Biointeractions
Publisher: American Chemical Society (ACS)
Date: 21-06-2023
Anti‐epileptic drug phenytoin enhances androgen metabolism and androgen receptor expression in murine hippocampus
Publisher: Wiley
Date: 29-11-2005
DOI: 10.1111/J.1471-4159.2005.03555.X
Abstract: Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hippoc us. Previous studies of brain tissue have demonstrated that long-term administration of the anti-epileptic drug (AED) phenytoin leads to enhanced metabolism of testosterone mediated by cytochrome P450 (CYP) isoforms. Thus, we speculate that AEDs affect androgen signalling in the hippoc us. In the present study, we investigated how the AED phenytoin influences the levels of testosterone, 17beta-oestradiol, and androgen receptor (AR) in the hippoc us of male C57Bl/6J mice. Phenytoin administration led to a 61.24% decreased hippoc al testosterone level as compared with controls, while serum levels were slightly enhanced. 17beta-Oestradiol serum level was elevated 2.6-fold. Concomitantly, the testosterone metabolizing CYP isoforms CYP3A11 and CYP19 (aromatase) have been found to be induced 2.4- and 4.2-fold, respectively. CYP3A-mediated depletion of testosterone-forming 2beta-, and 6beta-hydroxytestosterone was significantly enhanced. Additionally, AR expression was increased 2-fold (mRNA) and 1.8-fold (protein), predominantly in the CA1 region. AR was shown to concentrate in nuclei of CA1 pyramidal neurons. We conclude that phenytoin affects testosterone metabolism via induction of CYP isoforms. The increased metabolism of testosterone leading to augmented androgen metabolite formation most likely led to enhanced expression of CYP19 and AR in hippoc us. Phenytoin obviously modulates the androgen signalling in the hippoc us.
Nanoporous Metal–Phenolic Particles as Ultrasound Imaging Probes for Hydrogen Peroxide
Publisher: Wiley
Date: 09-2015
Abstract: Nanoporous metal-phenolic particles are fabricated through the nanostructural replication of dense Fe
Engineering Multifunctional Capsules through the Assembly of Metal-Phenolic Networks
Publisher: Wiley
Date: 02-04-2014
Abstract: Metal-organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli-responsiveness. Previous studies have primarily focused on the assembly aspects of metal-coordination capsules however, the engineering of metal-specific functionality for capsule design has not been explored. A library of functional metal-phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a erse range of functional materials can be generated.
Destination known: Targeted drug delivery in atherosclerosis and thrombosis
Publisher: Wiley
Date: 21-10-2013
DOI: 10.1002/DDR.21103
MRI of inducible P-selectin expression in human activated platelets involved in the early stages of atherosclerosis
Publisher: Wiley
Date: 29-12-2010
DOI: 10.1002/NBM.1606
Abstract: The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet-endothelial cell interactions in atherosclerosis-prone arteries at early stages, and the prominent role of P-selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P-selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti-human P-selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T(2) and T(1) values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10-VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo- and hyper-signals in the platelet pellet on T(2) - and T(1) -weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo-signal at 4.7 T, as a result of the accumulation of VH10-VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10-VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody-conjugated contrast agent used.
Thrombus-Targeted Theranostic Microbubbles: A New Technology towards Concurrent Rapid Ultrasound Diagnosis and Bleeding-free Fibrinolytic Treatment of Thrombosis
Publisher: Ivyspring International Publisher
Date: 2016
DOI: 10.7150/THNO.14514
Single-chain antibodies as diagnostic tools and therapeutic agents
Publisher: Georg Thieme Verlag KG
Date: 2009
DOI: 10.1160/TH08-12-0816
Abstract: Over three decades after the generation of the first mouse monoclonal antibodies by Kohler and Milstein, recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. Technology improvements have provided several approaches to manufacturing human antibodies with high affinity for biologically relevant targets. Approximately 300 development programs for therapeutic antibodies have been reported in industrial and academic laboratories, and this clearly demonstrates the expectations towards antibody technology. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as one of the smallest possible format for recombinant antibodies and their use as diagnostic tools and therapeutic agents. We describe the structure, selection and production of single-chain antibodies. Furthermore, we review current applications of antibody fragments focusing on thrombus targeting using fibrin- and platelet-specific single-chain antibodies as well as describing novel noninvasive imaging approaches for the diagnosis of thrombosis and inflammation.
Platelet-Targeted Delivery of Peripheral Blood Mononuclear Cells to the Ischemic Heart Restores Cardiac Function after Ischemia-Reperfusion Injury
Publisher: Ivyspring International Publisher
Date: 2017
DOI: 10.7150/THNO.19698
Versatile Loading of Diverse Cargo into Functional Polymer Capsules
Publisher: Wiley
Date: 29-01-2015
Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody
Publisher: Public Library of Science (PLoS)
Date: 30-03-2011
Sensor manufacturer, temperature, and cyanobacteria morphology affect phycocyanin fluorescence measurements
Publisher: Springer Science and Business Media LLC
Date: 27-10-2017
DOI: 10.1007/S11356-017-0473-5
Abstract: Sensors to measure phycocyanin fluorescence in situ are becoming widely used as they may provide useful proxies for cyanobacterial biomass. In this study, we assessed five phycocyanin sensors from three different manufacturers. A combination of culture-based experiments and a 30-s le field study was used to examine the effect of temperature and cyanobacteria morphology on phycocyanin fluorescence. Phycocyanin fluorescence increased with decrease in temperature, although this varied with manufacturer and cyanobacterial density. Phycocyanin fluorescence and cyanobacterial biovolume were strongly correlated (R
Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain
Publisher: Wiley
Date: 2001
DOI: 10.1046/J.1471-4159.2001.00011.X
Abstract: Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippoc us) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.
7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver
Publisher: Elsevier BV
Date: 03-2010
Abstract: The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. Because the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug-CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by testing other alkoxyresorufins (7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin) in mice and correlation of the data with testosterone 6beta-hydroxylation and a plethora of isoform-specific chemical inhibitors (orphenadrine, chlor henicol, nifedipine, ketoconazole, and sulfaphenazole). Isoform-specific expression and induction of CYP3A11 in mouse liver was tested by RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblot. With the BROD assay, we could clearly dissect CYP3A11 from other P450s induced by phenytoin-like CYP2C29, CYP2B9, CYP1A1, and CYP4A. We conclude that the BROD assay is a specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system.
Conformation-Specific Blockade of the Integrin GPIIb/IIIa
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-07-2006
DOI: 10.1161/01.RES.0000232317.84122.0C
Abstract: Platelet activation causes conformational changes of integrin GPIIb/IIIa (α IIb β 3 ), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.
Self-Assembled Metal–Phenolic Networks on Emulsions as Low-Fouling and pH-Responsive Particles
Publisher: Wiley
Date: 29-08-2018
Abstract: Interfacial self-assembly is a powerful organizational force for fabricating functional nanomaterials, including nanocarriers, for imaging and drug delivery. Herein, the interfacial self-assembly of pH-responsive metal-phenolic networks (MPNs) on the liquid-liquid interface of oil-in-water emulsions is reported. Oleic acid emulsions of 100-250 nm in diameter are generated by ultrasonication, to which poly(ethylene glycol) (PEG)-based polyphenolic ligands are assembled with simultaneous crosslinking by metal ions, thus forming an interfacial MPN. PEG provides a protective barrier on the emulsion phase and renders the emulsion low fouling. The MPN-coated emulsions have a similar size and dispersity, but an enhanced stability when compared with the uncoated emulsions, and exhibit a low cell association in vitro, a blood circulation half-life of ≈50 min in vivo, and are nontoxic to healthy mice. Furthermore, a model anticancer drug, doxorubicin, can be encapsulated within the emulsion phase at a high loading capacity (≈5 fg of doxorubicin per emulsion particle). The MPN coating imparts pH-responsiveness to the drug-loaded emulsions, leading to drug release at cell internalization pH and a potent cell cytotoxicity. The results highlight a straightforward strategy for the interfacial nanofabrication of pH-responsive emulsion-MPN systems with potential use in biomedical applications.
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