To screen a range of novel thermo- responsive polymers designed to be used as dual drug-eluting systems in coating stents. Specifically, to assess the ability of xemilofiban released in conjunction with fluvastatin, to prevent thrombus formation/platelet adhesion to fibrinogen using Cellix’s VenaFlux platform.
Stents are designed to line the inner walls of the blood vessel, holding the artery open and maintaining normal blood flow. Problems arise when scar tissue grows rapidly inside the stent, eventually narrowing the artery and causing re- blockage, or restenosis, in as many as 25% of patients. In addition to restenosis, in- stent thrombosis can occur following stent deployment. Until now, physicians have had few options for preventing restenosis and have used oral anti-platelet therapy to prevent in-stent thrombosis. New devices called drug-eluting stents have made a major contribution to reducing the incidence of restenosis. These devices consist of a metallic stent covered with a polymer coating, slowly releasing a medication that blocks inflammatory and proliferative processes in the vessel wall, thereby limiting the overgrowth of vascular tissue during the healing process. Cellix has optimised a microfluidic set-up to study the interaction of whole blood incubated with fractions eluted over time from different co-polymer combinations. The co-polymers used in this study are Thermo-responsive polymers which are attractive materials for both biomedical engineering and cardiovascular applications. Treated whole blood was subjected to flow over microfluidic biochips coated with fibrinogen to provide varying platelet adhesion profiles for each co-polymer.
Platelet adhesion, thermoresponsive, drug elution, xemilofiban, fluvastatin, Vena8 Fluoro+ biochips
Drug: xemilofiban (an antiplatelet agent that blocks the binding of fibrinogen to specific membrane GPIIb/IIIa integrin receptors and thus prevents platelet aggregation induced by any known platelet agonist).
Drug: fluvastatin (a cholesterol- lowering drug that reduces low- density lipoprotein (LDL) cholesterol and total cholesterol in the blood, helping to prevent heart disease and hardening of the arteries, conditions that can lead to heart attack, stroke, and vascular disease. Fluvastatin also possesses potent anti-proliferative activity, which prevents the overgrowth of vascular smooth muscle characteristic of restenosis).
The copolymers contain different w/w ratios of the hydrophilic, temperature- sensitive poly(N-isopropylacrylamide, pNiPAAm) and the more hydrophobic, non-temperature-sensitive poly(N- tButylacrylamide, pNtBAAm). In each mixture, a condensed form (microgels) is dispersed throughout a matrix. The composition of each microgel or matrix system is indicated in terms of ratios, e.g. 85/15 denotes a co-polymer of composition 85% pNiPAAm, 15% pNtBAAm. The 85/15 matrix shows the most hydrophilic properties while the 50/50 matrix shows the most hydrophobic properties. The 65/35 microgel composition shows intermediate properties.
65/35 microgels containing fluvastatin embedded in a hydrophilic 85/15 matrix containing xemilofiban
65/35 microgels containing fluvastatin embedded in a hydrophobic 50/50 matrix containing xemilofiban
65/35 microgels containing xemilofiban embedded in a hydrophilic 85/15 matrix containing fluvastatin
Biochip Coating Procedure
Vena8 Fluoro+ biochips were coated with fibrinogen (20 μg/mL) and placed at 4°C, overnight. Prior to experimentation, the coated channels were further coated with BSA (10 μg/mL) for 30 mins at room temperature and washed with JNL buffer. JNL buffer was also used to wash and prime the Mirus Evo Pump.
Blood was collected from healthy volunteers who were not taking any medication and were free from aspirin and other anti-platelet agents within the preceding 2 weeks. The blood was drawn by venepuncture into light-blue VacuetteTM tubes containing a 1:10 volume of 3.8% (wt/vol) tri-sodium citrate, which is a reversible anticoagulant, and gently mixed. Blood was incubated with drug eluates for 5 min at room temperature and subjected to a constant shear stress of 32 dyne/cm2 for 3 min, followed by JNL buffer for a further 3 min at similar shear stress.
Platelet adhesion profiles were recorded using Image Pro Premier software. Cell images were captured from three microscopic fields from each channel. Data were exported into Excel to allow further analysis.
Data obtained were normalized against whole blood. Statistical analysis was conducted using 1-way ANOVA from Graphpad Prism® 5 software.
System A and System B illustrate the adhesion profile of platelets treated with drug eluates over the course of 14 days. Day 1 suggests that there is a significant release of drug resulting in inhibition of platelet adhesion to fibrinogen. Day 3 shows a similar pattern (as shown by the median). It is noted that the drug is released at a slower rate after Day 9 and Day 14 resulting in more platelets adhering to fibrinogen (as shown by the median).
System A and System B result in a more rapid release of drug during the first 3-day period compared to System C.
System C results in a steady release of drug from the microgel/matrix composition from Day 3.
Cellix’s VenaFlux platform allows researchers to mimic physiological shear stresses. In comparison to static experiments, the VenaFlux platform allows for a more dynamic and sensitive detection method for platelet adhesion.
Under flow conditions 0.1 μM xemilofiban results in an almost complete inhibition of platelet adhesion, whereas static experiments show a decrease by approximately 50%.
Interestingly, under flow conditions platelet adhesion to fibrinogen is inhibited at a lower concentration of xemilofiban. Data from flow experiments show that 0.01 μM xemilofiban results in a statistically significant inhibition of platelet adhesion compared to static experiments where the 0.01 μM concentration shows no significant inhibitory effects.
Statins are known to have pleiotrophic effects on the body. In this study, 0.1 μM fluvastatin showed a 70% inhibitory effect on the adhesion of platelets to fibrinogen (data not shown).
Each drug-eluting co-polymer used in this study displayed effectiveness in lowering platelet adhesion, thus contributing to the prevention of in-stent thrombosis. This is achieved by the synergistic effects of both xemilofiban and fluvastatin.
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We thank Professor Alan Keenan and Ms. Jennifer Hickey, B.Sc., Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland for a fruitful collaboration.
McGillicuddy FC, Lynch I, Rochev YA, Burke M, Dawson KA, Gallagher WM, Keenan AK: Novel "plum pudding" gels as potential drug-eluting stent coatings: Controlled release of fluvastatin. Journal of Biomedical Materials Research Part A, 2006; 79A: 923–933.
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