Rapid Complexation of Aptamers by Their Specific Antidotes.

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Simple clotting test to detect procoagulant abdominal swabs.

During surgical procedures, abdominal swabs are routinely used to adsorb blood from the operation field and for the retention of tissues and organs. Due to the material characteristics, abdominal swabs exhibit a slight procoagulant activity, which is usually desirable and mostly harmless. However, during cardiac surgery with heart-lung machine (HLM) support, abnormal clot formation may result in life-threatening thromboembolic complications. Therefore, a simple clotting test (SCT) allowing in vitro detection of abdominal swabs with elevated hypercoagulant potency in the presence of heparinized human blood was developed and validated. In order to establish a SCT, heparinized human blood from 100 donors was incubated with five different cotton abdominal swabs for 30 min at 37 °C and then macroscopically analyzed. In a second study, 10 other swabs were screened with the established SCT (n=11) to confirm its suitability. Scanning electron microscopy, measurements of activated clotting times and thrombin-antithrombin were further performed. In the SCT, the results are dichotomized as negative (no detectable blood clot) and positive (blood clot formation). In the first study, three of the five tested abdominal swabs exhibited hypercoagulant potency in at least 25% of the donors. Calculations using the binomial distribution showed that blood of 11 donors is needed for routine testing with the SCT, which was confirmed in the second study using another 10 swabs. The established SCT can be used for detection of abdominal swabs with an elevated procoagulant potency, thereby minimizing the risk of thromboembolic complications during cardiac surgery with HLM support.

In vivo tissue engineering: mimicry of homing factors for self-endothelialization of blood-contacting materials.

Thrombogenicity of foreign surfaces is the major obstacle in cardiovascular interventions. Despite enormous advances in biomaterials research, the hemocompatibility of blood-contacting materials is still not satisfactory and the native endothelium still represents the ideal surface for blood contact. Circulating adult endothelial progenitor cells (EPCs) in the human blood provide an excellent source of autologous stem cells for the in vivo self-endothelialization of blood-contacting materials. For this purpose, material surfaces can be coated with capture molecules mimicking natural homing factors to attract circulating EPCs. Hitherto, several ligands, such as aptamers, monoclonal antibodies, peptides, selectins and their ligands, or magnetic molecules, are used to biofunctionalize surfaces for the capturing of EPCs directly from patient's bloodstream onto blood-contacting materials. Subsequently, attracted EPCs can differentiate into endothelial cells and generate an autologous endothelium. The in vivo self-endothelialization of blood-contacting materials prevents the recognition of them as a foreign body; this opens up revolutionary new prospects for future clinical stem-cell and tissue engineering strategies.

A novel in vitro model for preclinical testing of the hemocompatibility of intravascular stents according to ISO 10993-4.

Subacute stent thrombosis, caused by undesired interactions between blood and the stent surface, is a major concern in the first few weeks following coronary artery stent implantation. The aim of this study was to establish a novel in vitro model for hemocompatibility testing of coronary artery stents according to ISO 10993-4. The model consists of a modified Chandler-Loop design with closed heparin-coated PVC Loops and a thermostated water bath. The tests were performed with anticoagulated human whole blood. After incubation in the loop, blood was analyzed for coagulation and inflammatory activation markers (TAT, ß-TG, sP-selectin, SC5b-9 and PMN-elastase). Three different stent types with varying thrombogenicity were tested; statistically significant differences were found between the three stent types in measures of coagulation and platelet activation. The new Chandler-Loop model can be used as an alternative to animal and current in vitro models, especially for the determination of early events after stent implantation.

NCO-sP(EO-stat-PO) coatings on gold sensors--a QCM study of hemocompatibility.

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide-polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), ß-thromboglobulin (ß-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.

Selection of aptamers against triple negative breast cancer cells using high throughput sequencing.

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with [Formula: see text] values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

Platelet aggregation monitoring with a newly developed quartz crystal microbalance system as an alternative to optical platelet aggregometry.

The objective of this study was to establish a new test system for the monitoring of platelet aggregation during extracorporeal circulation (ECC) procedures. Even though extensive progress has been made in improving the haemocompatibility of extracorporeal circulation devices, activation of blood coagulation, blood platelets and inflammatory responses are still undesired outcomes of cardiopulmonary bypass. This study deals with an approach towards a platelet aggregation measuring system using a newly developed quartz crystal microbalance (QCM) system. Since QCM is a rarely used technique in the field of blood analytics, the challenge was to transfer the well established methods of aggregometry to the new test system. In a QCM system, either bare gold or fibrinogen-coated sensors were incubated with ADP or arachidonic acid (AA) stimulated platelet rich plasma. For negative controls the GPIIb/IIIa inhibitory antibody abciximab (Reopro®) was used as an inhibitor of platelet aggregation. During incubation, the frequency shifts of the sensors were recorded. The results gained from the QCM system were compared to results gained by optical platelet aggregometry (born aggregometry). For additional visualization of platelet adhesion to the sensor surfaces, fluorescent microscopy and scanning electron microscopy were used. The QCM sensor was able to detect platelet aggregation in both uncoated and fibrinogen coated sensors. The measuring curves of aggregation measurements and controls were clearly distinguishable from each other in terms of frequency shifts and kinetics. For aggregation measurements and inhibited controls the therapeutic diagnosis of platelet function is identical between aggregometer and QCM data. In future, QCM based measuring devices may become an alternative to established point of care methods for rapid bedside testing of platelet aggregation.

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro.

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing alpha(v)beta(3) integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1 beta under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking beta(3) integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.

Hemostatic wound dressings: Predicting their effects by in vitro tests.

BACKGROUND: Application of controlled in vitro techniques can be used as a screening tool for the development of new hemostatic agents allowing quantitative assessment of overall hemostatic potential. MATERIALS AND METHODS: Several tests were selected to evaluate the efficacy of cotton gauze, collagen, and oxidized regenerated cellulose for enhancing blood clotting, coagulation, and platelet activation. RESULTS: Visual inspection of dressings after blood contact proved the formation of blood clots. Scanning electron microscopy demonstrated the adsorption of blood cells and plasma proteins. Significantly enhanced blood clot formation was observed for collagen together with ß-thromboglobulin increase and platelet count reduction. Oxidized regenerated cellulose demonstrated slower clotting rates not yielding any thrombin generation; yet, led to significantly increased thrombin-anti-thrombin-III complex levels compared to the other dressings. As hemostyptica ought to function without triggering any adverse events, induction of hemolysis, instigation of inflammatory reactions, and initiation of the innate complement system were also tested. Here, cotton gauze provoked high PMN elastase and elevated SC5b-9 concentrations. CONCLUSIONS: A range of tests for desired and undesired effects of materials need to be combined to gain some degree of predictability of the in vivo situation. Collagen-based dressings demonstrated the highest hemostyptic properties with lowest adverse reactions whereas gauze did not induce high coagulation activation but rather activated leukocytes and complement.

Inflammatory potential of cotton-based surgically invasive devices: Implications for cardiac surgery.

Cotton-based surgical invasive devices with their desired hemostyptic properties have been used for decades in the surgical field. However, in cardiac surgery using the heart-lung machine with direct retransfusion of suction blood, activated blood may re-enter the circulation without filtration and may trigger a cascade reaction leading to systemic inflammation and thrombosis. We therefore set out to evaluate the inflammatory potential of untreated and pyrogen-impregnated cotton-based surgical invasive medical devices. After incubation of the swabs with whole blood or PBMC, the cell-free supernatant was investigated for IL1ß and IL6. While the reaction of human whole blood toward cotton swabs could not be influenced by any sterilization technique, dry heat and gamma-irradiation were able to diminish the inflammatory reaction of PBMC toward the material and the used pyrogens. In conclusion, using PBMC in direct contact to cotton we are the first to establish a suitable test method for quantification of the pyrogenic/inflammatory activity of this material. The unaltered reaction of whole blood, however, suggests a crosstalk of cells and plasma proteins in the inflammation activation that is not prevented by sterilization of the swabs. This new in vitro testing methodology may help to better display the clinical situation during development of new materials. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1877-1888, 2019.

New strategies for in vivo tissue engineering by mimicry of homing factors for self-endothelialisation of blood contacting materials.

For years intensive research has been done to endothelialise vascular prostheses with autologous endothelial cells before implantation in patients. However, this procedure is extremely time-, labor- and cost-intensive and can be realized only in very few clinical cases. The discovery of circulating endothelial progenitor cells (EPCs) in 1997 brought new perspectives for the endothelialisation of blood contacting materials. Coating of synthetic graft surfaces with capture molecules for circulating EPCs mimics a pro-homing substrate for fishing out EPCs directly from the bloodstream after implantation. These cells with high proliferation potential can cover the graft with non-thrombogenic endothelium which maintains optimal haemostasis and minimize the risk of restenosis. In this review, different concepts are discussed to capture circulating EPCs on synthetic vascular grafts after implantation. We hypothesize that in vivo self-endothelialisation of blood contacting materials by homing factor-mimetic capture molecules for EPCs may bring revolutionary new perspectives towards future clinical application of stem cell and tissue engineering strategies.

Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.

OBJECTIVE: Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). METHODS AND RESULTS: The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin alphaMbeta2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C-dependent mechanism. CONCLUSIONS: Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

CELL-SELEX: Novel perspectives of aptamer-based therapeutics.

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility.

Hemocompatibility of blood-contacting biomaterials is one of the most important criteria for their successful in vivo applicability. Thus, extensive in vitro analyses according to ISO 10993-4 are required prior to clinical applications. In this review, we summarize essential aspects regarding the evaluation of the hemocompatibility of biomaterials and the required in vitro analyses for determining the blood compatibility. Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed. Furthermore, the surface of biomaterials are evaluated concerning attachment of blood cells, adsorption of proteins, and generation of thrombus and fibrin networks.

Critical temperature ranges of hypothermia-induced platelet activation: possible implications for cooling patients in cardiac surgery.

Cooling of the patient is routinely applied in cardiac surgery to protect organs against ischemia. Hypothermia induces activation of platelets, but the effects of temperatures such as used during cardiac surgery are not well described. To investigate this in an in-vitro study heparinized whole blood was incubated at different temperatures (37 degrees C, 34.5 degrees C, 32 degrees C, 29.5 degrees C, 27 degrees C, 24.5 degrees C, 22 degrees C, 19.5 degrees C and 17 degrees C). The effect of these temperatures on aggregation, P-selectin expression, GP IIb/IIIa activation and platelet microparticle (PMP) formation of unstimulated and ADP-stimulated platelets of 36 subjects was evaluated in flow cytometry. A four-parametric logistic model was fitted to depict the temperature effect on platelet parameters. Lower temperatures increased aggregates, P-selectin expression, and GP IIb/IIIa activation. The number of PMPs decreases with hypothermia. Additional experiments revealed a slight influence of heparin on platelet P-selectin expression but excluded an effect of this anticoagulant on the other evaluated parameters. Threshold temperatures, which mark 5% changes of platelet parameters compared to values at 37 degrees C, were calculated. On ADP-stimulated platelets the thresholds for P-selectin expression and GP IIb/IIa activation are 34.0 degrees C and 36.4 degrees C, respectively, and lie in the temperature range routinely applied in cardiac surgery. Hypothermia-induced platelet activation may develop in most patients undergoing cardiac surgery, possibly resulting in thromboembolic events, coagulation defects, and proinflammatory leukocyte bridging by P-selectin bearing platelets and PMPs. These findings suggest that pharmacological protection of platelets against hypothermia-induced damage may be beneficial during cardiac surgery.

The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.

To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.

Aptamer-based strategies for stem cell research.

Aptamers have been introduced to analytical applications, target validation, and drug discovery processes and, recently, applied directly as therapeutic agents. Aptamers can be generated by a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This is quite remarkable for such a young technology, which is only created in the early 1990s. This paper reviews recent new applications of aptamers in stem cell research and tissue engineering.

Effect of biopassive and bioactive surface-coatings on the hemocompatibility of membrane oxygenators.

Postoperative complications associated with cardiopulmonary bypass (CPB) surgery and extracorporeal circulation (ECC) procedures are still a major clinical issue. Improving the hemocompatibility of blood contacting devices used for ECC procedures may ameliorate various postpump syndromes. In a simulated CPB model using human blood, we investigated the hemocompatibility, fibrinogen adsorption, and platelet receptor (GPIIb-IIIa) binding capacity of surface-modified membrane oxygenators (Jostra Quadrox). Three groups were compared: (i) biopassive protein coatings (SafeLine), (ii) bioactive heparin coatings (BioLine), and (iii) noncoated controls. During the 2 h recirculation period, plasma concentrations of activation markers for platelets (beta-thromboglobulin), inflammation (elastase), complement (C5a), and coagulation (prothrombin fragment 1+2, thrombin-antithrombin III) were lower in the groups with biopassive and bioactive coatings compared to the noncoated group (p < 0.01). These parameters did not significantly differ between the two surface-coated groups, except for complement activation: C5a levels were higher in the biopassive group compared to the bioactive group (p < 0.01). Moreover, surface-coated oxygenators showed less fibrinogen adsorption, GPIIb-IIIa binding, and platelet/leukocyte adhesion (p < 0.01). We assume that fewer fibrinogen and platelet receptor molecules bound to the surface-coated oxygenator surfaces results in fewer platelet adhesion and activation, which will significantly contribute to the improved hemocompatibility of the biopassive and bioactive oxygenators. Our results suggest that the application of bioactive oxygenators (BioLine) during CPB surgery may reduce postoperative complications for the patient more effectively than biopassive oxygenators (SafeLine).

Photocoupling of fibronectin to titanium surfaces influences keratinocyte adhesion, pellicle formation and thrombogenicity.

OBJECTIVES: Coating of implant surfaces with biomolecules can influence basic host responses and enhance subsequent tissue integration. The biological factors have to be immobilized on the implant material. Human fibronectin (Fn) was used as a model protein and covalently coupled to titanium (Ti) surfaces via silanization and an anthraquinone linker. The impact on several aspects of initial host/biomaterial interactions (keratinocyte adhesion, platelet interactions and pellicle formation) was studied. METHODS: Coupling efficiency was characterized by immunological techniques. The effects of coupled Fn on initial host/biomaterial interactions were assessed. Cell adhesion and spreading were investigated by fluorescent staining, pellicle formation by an acoustic sensor system (quartz crystal microbalance with dissipation, QCM-D), and platelet adhesion as one parameter mediating the inflammatory response by scanning electron microscopy (SEM) and immunological assays. RESULTS: Coupling efficiency was related to irradiation time used for photochemical coupling of the UV-activated anthraquinone to the silanized Ti surface. With an optimized protocol, the amount of Fn coupled to the surface could be almost doubled compared to standard dip-coating methods. On the anthraquinone-coupled Fn coatings, cell adhesion and spreading of human keratinocytes was significantly enhanced. Online detection of pellicle formation revealed strong reversibility of saliva protein adhesion on Fn coated surfaces compared to the pure Ti surface. Furthermore, the Fn coated Ti showed a low thrombogenicity. SIGNIFICANCE: This study suggests that anthraquinone-coupled biological coatings may be useful for biofunctionalization of Ti dental implants by enhancement of soft tissue re-integration (restoration of the epithelial seal) combined with diminished pellicle formation.

Low-thrombogenic fibrin-heparin coating promotes in vitro endothelialization.

Long-term performance of implanted cardiovascular grafts can be ensured if living endothelium overgrows their surface. Surface modifications to implants are therefore being sought that can encourage endothelialization while preventing thrombus formation until the natural endothelium is formed. In the present study, heparin was covalently attached to a fibrin mesh grown from a polyvinyl chloride (PVC) substrate surface by the catalytic action of surface immobilized thrombin on a fibrinogen solution. The coating prevented platelet activation, thrombin generation and clot formation, and reduced inflammatory reactions when exposed to fresh human whole blood circulating in a Chandler loop model. In addition, in vitro seeded human umbilical vein and human saphenous vein endothelial cells showed considerably enhanced attachment and proliferation on the coating. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2995-3005, 2017.