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Title: Bespoke nanotopography for the selective capture of VWF from whole blood at arterial shear : a potential diagnostic platform
Author: Bishop, David
Awarding Body: Ulster University
Current Institution: Ulster University
Date of Award: 2017
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Abstract:
The concept of surface mediated protein manipulation is well utilised throughout many diagnostic and therapeutic applications. Tailoring of surface chemistry, wettability and topography can facilitate targeted adherence of specific proteins for desired biological outcomes, von Willebrand Factor (VWF) is a key hemostatic blood protein that modulates platelet response at high arterial shear rates following vascular damage. Defective interaction of VWF and platelets is a fundamental risk factor for both thrombogenic events and bleeding disorders, depending on the nature of the defect. However, there is currently no physiologically relevant diagnostic platform operable under such conditions. The work reported in this thesis concerns augmentation of an existing Dynamic Platelet Function Assay (DPFA) with substrate surface topography capable of entrapment of autologous VWF from whole blood flowing at typical arterial shear rate (1500s1). These novel substrates are employed as part of a microfluidic platform for real-time fluorescent imaging of platelets. The adhesion and activity of which are indicative of VWF entrapment/function and how this relates to a diseased state. The primary substrates of interest have been created via spin coating of PMMA/PS polymer demixed solutions in chloroform. Polymer concentration is the chief variable utilised in the optimisation of topographical conditions for VWF capture, with PMMA:PS ratio and molecular weight investigated also. Concentrations of 1 %, 3 % and 5% w/v were employed, with the middle state (3%) proving the best performer in terms of both rate and total amount of platelet surface coverage; an average of 40% in comparison with 27% and 19% for the 1% and 5% concentrations respectively. Interestingly, these substrates exhibit a lag time in initial platelet response, not present on the immobilised VWF controls. This is thought to be a consequence of the time taken to accumulate a sufficient quantity of captured VWF for inciting platelet interaction. Inhibition studies, with antibodies that target the VWF-platelet binding pathway, confirmed the presence and action of autologous VWF in the platelet response. VWF supplementation of blood at 40 (ig/ml resulted in an increase in coverage of 25% when compared to untreated controls (38% and 13% respectively). XPS analysis revealed a stable PMMA chemistry (P>0.05) across all surfaces. Water contact angle (WCA) analysis showed an average value of 69° for surfaces prepared using all three concentrations, which compares to 72° for pristine PMMA. Optical microscopy revealed a hugely changeable morphology across the concentrations, related to interconnectivity, whilst AFM revealed the presence of two discrete layers of topography; a larger mesotopography consisting of a pitted morphology with feature heights ranging from 90 to 502 nm, overlaid by a nanotopography with pillared features averaging in the 10 to 50 nm height range. It is the presence of this nanotopography, which is hypothesised to be the primary mechanism of VWF capture. NIL was employed for the replication of the nanotopography in a more reproducible fashion. A stamp template was designed using the AFM data and used to generate channels with av. feature heights in the range of 40-47 nm. A loading pressure of 7.5kN was found to produce well-rounded features with av. height of 47 nm. Similar topographical features were created via spin coating with a 3% 75:25 PMMA:PS demixed solution with av. feature heights of 45 nm, which triggered good platelet adhesion thereby confirming its ability for VWF entrapment. Finally, a preliminary investigation of collagen integration to the novel substrates is reported. This approach involves a novel plasma assisted printing methodology to create a highly physiologically relevant hemostatic diagnostic platform.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.714499  DOI: Not available
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