Article pubs.acs.org/Biomac
Adhesion of Blood Plasma Proteins and Platelet-rich Plasma on L‑Valine-Based Poly(ester urea) Erin P. Childers,† Gregory I. Peterson,† Alex B. Ellenberger,† Karen Domino,† Gabrielle V. Seifert,† and Matthew L. Becker*,†,‡ †
Department of Polymer Science and ‡Department of Biomedical Engineering, The University of Akron, Akron, Ohio 44325, United States S Supporting Information *
ABSTRACT: The competitive absorption of blood plasma components including fibrinogen (FG), bovine serum albumin (BSA), and platelet-rich plasma (PRP) on L-valine-based poly(ester urea) (PEU) surfaces were investigated. Using four different PEU polymers, possessing compositionally dependent trends in thermal, mechanical, and critical surface tension measurements, water uptake studies were carried out to determine in vitro behavior of the materials. Quartz crystal microbalance (QCM) measurements were used to quantify the adsorption characteristics of PRP onto PEU thin films by coating the surfaces initially with FG or BSA. Pretreatment of the PEU surfaces with FG inhibited the adsorption of PRP and BSA decreased the absorption 4-fold. In vitro studies demonstrated that cells cultured on L-valine-based PEU thin films allowed attachment and spreading of rat aortic cells. These measurements will be critical toward efforts to use this new class of materials in blood-contacting biomaterials applications.
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INTRODUCTION Biomaterial implants that come in contact with blood must overcome adhesion of plasma proteins that can initiate a cascade of mechanisms that lead to thrombosis and intimal hyperplasia.1−3 Materials used for vascular grafts, vascular stents, heart valves, catheters, kidney dialyzers, blood oxygenators and many other implants all directly contact blood.1,4,5 Within seconds of implantation, plasma proteins adsorb to the surface of a material and proceed to activate a cellular response. Ideally, this response will not develop into a chronic inflammatory condition.6 While much of the literature has relied on empirical measurements of protein absorption, detailed computational and experimental studies from the Latour group have outlined how chemical groups present on a surface influence adsorbed protein conformation7 and concentration.8−10 Other studies have also stressed the importance in understanding surface chemistry or topography of implanted biomaterials as a way of controlling cellular response.2,11−13 Mitigating an adverse cellular response involves understanding the adsorption mechanisms of plasma proteins, how coagulation events are initiated, and how platelets are recruited to the site of implantation There are two primary pathways for coagulation: an intrinsic pathway involving the activation of proteins which come in contact with a foreign surface, and an extrinsic pathway, which is activated during cellular destruction.5 Both pathways lead to the adhesion of platelets at the site of activation and thrombin formation. Thrombin is produced from prothrombin, which acts as a serine protease and cleaves fibrinogen into fibrin, thereby eliciting clot formation.14 In a normal wound healing event clot formation is necessary, but at the surface of an implant this can lead to thrombosis, which obstructs blood flow locally. Additional © XXXX American Chemical Society
complications can occur if the clot breaks free and circulates, triggering blockage of vital blood vessels (embolism) in organs, which can causing massive injury or possibly death.15,16 In an effort to understand how to control, limit, or avoid this cascade, researchers have investigated the adsorption of the plasma proteins to implants under in vitro conditions.7,10 One important plasma protein, and the primary focus of this work, is fibrinogen.9,17 Fibrinogen (FG) in its native (nonadsorbed) state does not stimulate platelet activation and adhesion. Generally after adsorption onto a surface, FG activates platelet adhesion. The change in conformation (adsorbed onto an implant) and exposure of specific binding domains such as RGD (Arg-Gly-Asp) or other active amino acid domains mimics physiologically activated FG in a wound cascade. The exposed domains cause platelet activation and the recruitment of additional platelets necessary for clot formation.16 In a study to determine the importance of the RGD domain in FG-platelet interaction, platelet αVβIII integrins (RGD-binding integrin) were blocked prior to exposure of adsorbed FG. Disrupting FG-platelet RGD interaction exhibited a 50% decrease in platelet adhesion showing that additional receptors are present for platelet adhesion.9 Additionally, the concentration of FG played a role in its adsorption conformation on different surfaces. High concentrations of FG adsorbed to a surface exhibited a native FG conformation significantly decreasing the amount of adsorbed platelets compared to lower concentration regimes.9 The need for biomaterials which naturally control the Received: August 8, 2016 Revised: September 2, 2016
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DOI: 10.1021/acs.biomac.6b01195 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
(dd, 12H) 1.32 (s, 4H) 1.58 (m, 4H) 2.15 (m, 2H) 2.28 (s, 6H), 3.84 (s, 2H) 4.11−4.15 (m, 4H) 7.12−7.14 (d, 4H) 7.50−7.52 (d, 4H) 8.32 (s, 6H); 13C NMR (500 MHz, DMSO-d6): 17.89, 18.71, 21.22, 25.24, 28.23, 29.79, 57.86, 65.91, 125.95, 128.63, 138.99, 145.35, 169.10. FT-IR (cm−1): 1740 [−C(CO)−O−]. Synthesis of Di-p-toluene Sulfonic bis Acid Salt of [L-Valine]-1,8octyl Diester (m(1-VAL-8)). Octandiol (0.14 mol, 20.00 g, mol. eq. 1), L-valine (0.29 mol, 43.00 g, mol. eq. 2.1), p-toluenesulfonic acid, (0.31 mol, 60.00 g, mol. eq. 2.3) and toluene (600 mL), yield 95%: mp: 168.7−172.1 °C. 1H NMR (500 MHz, DMSO-d6): 0.96 (dd, 12H) 1.24 (s, 8H) 1.54 (m, 4H) 2.15 (m, 2H) 2.28 (s, 6H), 3.84 (m, 2H) 4.15 (d, 4H) 7.13 (d, 4H) 7.48 (d, 4H) 8.29 (s, 6H); 13C NMR (500 MHz, DMSO-d6): 17.89, 18.71, 21.22, 25.24, 28.23, 28.89, 29.79, 57.86, 65.91, 125.95, 128.63, 138.99, 145.35, 169.10. FT-IR (cm−1): 1740 [−C(CO)−O−]. Synthesis of Di-p-toluene Sulfonic bis Acid Salt of [L-Valine]-1,10decyl Diester (m(1-VAL-10)). Decandiol (0.11 mol, 20.00 g, mol. eq. 1), L-valine (0.24 mol, 38.00 g, mol. eq. 2.1), p-toluenesulfonic acid, (0.26 mol, 50.00 g, mol. eq. 2.3), and toluene (500 mL), yield 93%: mp: 185.3−186.7 °C. 1H NMR (500 MHz, DMSO-d6): 0.98 (dd, 12H) 1.24−1.33 (s, 12H) 1.59 (m, 4H) 2.14 (m, 2H) 2.26 (s, 6H), 3.87 (d, 2H) 4.13−4.16 (m, 4H) 7.12−7.13 (d, 4H) 7.50−52 (d, 4H) 8.32 (s, 6H); 13C NMR (500 MHz, DMSO-d6): 17.89, 18.71, 21.22, 25.71, 28.41, 29.00, 29.36 29.80, 57.86, 65.91, 125.95, 128.63, 138.99, 145.35, 169.10. FT-IR (cm−1): 1740 [−C(CO)−O−]. Synthesis of Di-p-toluene Sulfonic bis Acid Salt of [L-Valine]-1,12dodecyl Diester (m(1-VAL-12)). Dodecanediol (0.10 mol, 20.00 g, mol. eq. 1), L-valine (0.21 mol, 33.00 g, mol. eq. 2.1), p-toluenesulfonic acid, (0.23 mol, 43.00 g, mol. eq. 2.3), and toluene (430 mL), yield 90%: mp: 197.3−193.5 °C. 1H NMR (500 MHz, DMSO-d6): 0.93−0.98 (dd, 12H) 1.24−1.31 (s, 16H) 1.59 (m, 4H) 2.15 (m, 2H) 2.29 (s, 6H), 3.86 (d, 2H) 4.12−4.17 (m, 4H) 7.13 (d, 4H) 7.50 (d, 4H) 8.32 (s, 6H); 13 C NMR (500 MHz, DMSO-d6): 17.89, 18.71, 21.22, 25.71, 28.41, 29.00, 29.34, 29.80, 57.86, 65.91, 125.95, 128.63, 138.99, 145.35, 169.10. FT-IR (cm−1): 1740 [−C(CO)−O−]. General Procedure for Synthesis of L-Valine-Based PEUs Using Interfacial Polymerization. The PEUs were synthesized using an interfacial polymerization as outlined in Scheme 1. The valine-based monomer, sodium carbonate, and water were mixed in a three-necked round-bottom flask equipped with an overhead mechanical stirrer and a thermometer. The mixture was stirred in a warm water bath at 40−50 °C for 30 min or until the products were dissolved. The water bath was then replaced with a brine ice bath. When the solution temperature decreased to
