Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p \u3c 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling

Detection of small molecules using aptamers

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high affinity and selectivity. They can be selected by an in vitro procedure against a broad range of targets molecules including small molecules (molecular weight <1000 g/mol). Small molecules include toxins, antibiotics, molecular markers, drugs, and heavy metals ions. The detection of small molecules is important in different areas including public health, environmental monitoring, food safety, and antiterrorism. To meet the increasing demand for small molecule detection, methods are needed that are sensitive, reliable, rapid, cost effective and simple to use. In this context, aptamer-based detection platforms are becoming a promising alternative to conventional methods for small molecule detection. Concerning signal generation, mass-dependent detection methods, sandwich assay format and single-site binding assay formats are not always suitable for small molecule detection. The structural flexibility of aptamers enables the development of unique aptamer-based sensing platforms because aptamers fold into a well-defined three-dimensional structure upon binding to their target molecules. This specific property of aptamers allows to develop target-Induced dissociation (TID) of complementary oligonucleotide and target-induced structure switching (TISS)-based assays. In this PhD work, a novel aptamer-based assay (Apta-qPCR) was developed, which relies on TID for the detection of small molecules originating from biological, food and environmental samples. The Apta-qPCR assay was developed and optimized for the detection of ATP, ochratoxin A, and oxytetracycline. The assays are highly sensitive and selective for the target molecules. In addition, a rapid colorimetric assay was developed based on the TISS principle, which can detect ATP and ochratoxin A in 15 minutes.Nukleinsäure-Aptamere sind vielseitige molekulare Biorezeptoren, die mit hoher Affinität und Selektivität an ihre Zielmoleküle binden. Durch ein In-vitro-Verfahren können sie gegen eine breite Palette von Zielmolekülen, einschließlich kleiner Moleküle (Molekulargewicht <1000 g/mol), selektiert werden. Zu den kleinen Molekülen gehören unter anderem Toxine, Antibiotika, molekulare Marker, Wirkstoffe und Schwermetallionen. Der Nachweis von kleinen Molekülen findet unter anderem in den Bereichen der öffentlichen Gesundheit, Umweltüberwachung, Lebensmittelsicherheit und Antiterrorismus Anwendung. Um der steigenden Nachfrage zum Nachweis verschiedener kleiner Moleküle gerecht zu werden, werden Methoden benötigt, die empfindlich, zuverlässig, schnell, kostengünstig und einfach zu bedienen sind. In diesem Zusammenhang entwickeln sich Aptamer-basierte Detektionsplattformen zu einer vielversprechenden Alternative zu herkömmlichen Methoden zur Detektion von kleinen Molekülen. Für den Nachweis von kleinen Molekülen sind massenabhängige Nachweisverfahren, Sandwich-Assays und Single-Site Binding-Assays nicht immer geeignet. Bei der Signalerzeugung ermöglicht die strukturelle Flexibilität der Aptamere die Entwicklung einzigartiger Aptamer-basierter Sensorplattformen, da die Nukleinsäure-Aptamere sich bei der Bindung an ihre Zielmoleküle in eine spezifische dreidimensionale Struktur falten. Diese besondere Eigenschaft von Aptameren ermöglicht die Entwicklung von Nachweisverfahren, die die Aptamer-spezifischen Mechanismen einer Target-induzierten Dissoziation von komplementären Oligonukleotiden (TID) und die Target-induzierte Strukturänderung (Target-Induced Structure Switch, TISS) nutzen. In dieser Doktorarbeit wurde ein neuartiger Assay, die Apta-qPCR, basierend auf dem TID Mechanismus für den Nachweis von kleinen Molekülen aus biologischen Proben, Lebensmittel- und Umweltproben, entwickelt. Der Apta-qPCR Assay wurde für den Nachweis von ATP, Ochratoxin A und Oxytetracyclin entwickelt und optimiert. Dabei konnte eine hohe Empfindlichkeit und Selektivität für die Zielmoleküle ermittelt werden. Außerdem konnte ein kolorimetrischer Schnelltest nach dem TISS-Prinzip entwickelt werden, der ATP und Ochratoxin A innerhalb von 15 Minuten nachweisen kann

Aptamer-Modified Magnetic Beads in Biosensing

Magnetic beads (MBs) are versatile tools for the purification, detection, and quantitative analysis of analytes from complex matrices. The superparamagnetic property of magnetic beads qualifies them for various analytical applications. To provide specificity, MBs can be decorated with ligands like aptamers, antibodies and peptides. In this context, aptamers are emerging as particular promising ligands due to a number of advantages. Most importantly, the chemical synthesis of aptamers enables straightforward and controlled chemical modification with linker molecules and dyes. Moreover, aptamers facilitate novel sensing strategies based on their oligonucleotide nature that cannot be realized with conventional peptide-based ligands. Due to these benefits, the combination of aptamers and MBs was already used in various analytical applications which are summarized in this article

An update to dialysis-based drug release testing—data analysis and validation using the pharma test dispersion releaser

10.3390/pharmaceutics13122007Pharmaceutics1312200

Microparticle formulations alter the toxicity of fenofibrate to the zebrafish Danio rerio embryo

10.1016/j.aquatox.2021.105798Aquatic Toxicology23410579

Exploring the Interplay between Drug Release and Targeting of Lipid-Like Polymer Nanoparticles Loaded with Doxorubicin

Targeted delivery of doxorubicin still poses a challenge with regards to the quantities reaching the target site as well as the specificity of the uptake. In the present approach, two colloidal nanocarrier systems, NanoCore-6.4 and NanoCore-7.4, loaded with doxorubicin and characterized by different drug release behaviors were evaluated in vitro and in vivo. The nanoparticles utilize a specific surface design to modulate the lipid corona by attracting blood-borne apolipoproteins involved in the endogenous transport of chylomicrons across the blood–brain barrier. When applying this strategy, the fine balance between drug release and carrier accumulation is responsible for targeted delivery. Drug release experiments in an aqueous medium resulted in a difference in drug release of approximately 20%, while a 10% difference was found in human serum. This difference affected the partitioning of doxorubicin in human blood and was reflected by the outcome of the pharmacokinetic study in rats. For the fast-releasing formulation NanoCore-6.4, the AUC0→1h was significantly lower (2999.1 ng × h/mL) than the one of NanoCore-7.4 (3589.5 ng × h/mL). A compartmental analysis using the physiologically-based nanocarrier biopharmaceutics model indicated a significant difference in the release behavior and targeting capability. A fraction of approximately 7.310–7.615% of NanoCore-7.4 was available for drug targeting, while for NanoCore-6.4 only 5.740–6.057% of the injected doxorubicin was accumulated. Although the targeting capabilities indicate bioequivalent behavior, they provide evidence for the quality-by-design approach followed in formulation development

Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p < 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling