Synthesis of biomimetic segmented polyurethanes as antifouling biomaterials

Abstract

Controlling the non-specific adsorption of proteins, cells and bacteria onto biomaterial surfaces is of crucial importance for the development of medical devices with specific levels of performance. Among the strategies pursued to control the interactions between material surfaces and biological tissues, the immobilization of non-fouling polymers on biomaterial surfaces as well as the synthesis of the so-called biomimetic polymers are considered promising approaches to elicit specific cellular responses. In this study, in order to obtain materials able to prevent infectious and thrombotic complications related to the use of blood-contacting medical devices, heparin-mimetic segmented polyurethanes were synthesized and fully characterized. Specifically, sulfate or sulfamate groups, known to be responsible for the biological activity of heparin, were introduced into the side chain of a carboxylated polyurethane. Due to the introduction of these groups, the obtained polymers possessed a higher hard/soft phase segregation (lower glass transition temperatures) and a greater hydrophilicity than the pristine polymer. In addition, the synthesized polymers were able to significantly delay the activated partial thromboplastin time, this increased hemocompatibility being related both to polymer hydrophilicity and to the presence of the –SO₃H groups. This last feature was also responsible for the ability of these biomimetic polymers to prevent the adhesion of a strain of Staphylococcus epidermidis.

Introduction

Segmented polyurethanes are an important class of thermoplastic elastomers, being widely employed in the medical field for the manufacture of medical devices such as vascular grafts, catheters, artificial blood vessels and heart valves. Their successful employment in clinics is mainly related to their good blood compatibility and suitable mechanical properties arising from their structure of hard and soft segments [1].

One of the main drawbacks related with the use of blood-contacting medical devices is the high risk of infections and thrombosis associated with their implantation [2], [3], these two complications being closely interrelated. In fact, upon exposure to the biological environment, plasma proteins, including fibrin, albumin and fibrinogen, adsorb on the device’s surface, allowing the adhesion and activation of platelets and leukocytes. This process, known as biofouling, is usually the first stage of a cascade of biological events which leads to blood clot formation and promotes bacterial adhesion and biofilm formation on device surfaces. The resulting infectious and thrombotic complications can impair the function of the device and lead to implant failure and life-threatening consequences, as in the case of vascular grafts.

A common approach pursued to reduce biofouling is the immobilization of non-fouling polymers on biomaterial surfaces. This strategy is particularly interesting since it avoids the use of drugs (either by systemic administration or by local release from medicated devices) that, when administered over long periods of time, may be associated with undesired side effects.

On the basis of the empirical criteria recently proposed by Ostuni and colleagues [4], non-fouling polymers should be hydrophilic, electrically neutral and possess hydrogen-bond acceptors. Accordingly, several polymer classes have been explored [5], including polyacrylates [6], polyzwitterions [7], [8] and poly(ethylene glycol) (PEG) derivates [9], [10]. Of these, PEG, which has the ability to impart protein resistance, believed to be related to both hydration and steric effects [11], is the most widely studied non-fouling polymer. It has been grafted onto the surface of a series of materials, including glass [12], gold [13], poly(ethylene terephthalate) [14] and polyurethanes [15] with variable degrees of success, depending on the PEG’s molecular weight, degree of branching and surface packing density. Although PEG possesses unique properties of non-toxicity and biocompatibility, a number of limitations have been associated with PEG grafting, including stability (autoxidation) and poor functionality [16]. Therefore, research in this field is still focused on the development of a more efficacious approach to obtain surfaces resistant to fouling by proteins, cells and bacteria.

In this regard, the synthesis of biomimetic polymers [17], [18], [19], [20], i.e. materials able to mime the biological environment, has lately emerged as a promising alternative strategy since these materials may be able to actively participate in the regulation of protein and cell adhesion. Heparin, a highly sulfated glycosaminoglycan, has attracted particular attention in the field of hemocompatible biomaterials since it possesses a number of biological functions, such as anticoagulant activity, cell growth stimulation and antivirus ability. It is in fact able to reversibly bind to many biofunctional proteins, such as antithrombin III (ATIII) and platelet factor 4 [21]. Moreover, it contains distinct recognition sites for growth factors, including basic fibroblast growth factor and vascular endothelial growth factor [21]. The physical adsorption or chemical grafting of heparin to artificial surfaces has been shown to be a successful strategy to improve device hemocompatibility [22], [23], [24]. Our group has developed different hydrophilic polyurethanes able to bind significant amounts of heparin ionically [25] or covalently [26]. More recently, heparinization of surfaces has also been shown to contribute to a reduction in bacterial colonization, as demonstrated by a randomized-controlled clinical trial of heparin-coated and uncoated non-tunnelled central venous catheters [27] and a retrospective comparative analysis of heparin-coated and uncoated tunnelled dialysis catheters [28]. However, a limitation of heparin adsorption is its lack of stability in immobilization [21]. On the other hand, covalent binding of heparin to surfaces may cause a decrease in activity with respect to the raw material [21].

In this regard, in the last few decades several heparin-like materials, including sulfated polyurethanes [29], [30], polyacrylates [31], hyaluronic acid [32] and dextrans [33], have been developed and assayed in their anticoagulant and antiplatelet properties. However, only few studies [34], [35], [36] have been carried out to evaluate the role of these sulfated matrices in the control of bacterial adhesion.

In this work, we report the synthesis of novel segmented polyurethanes containing in the side chain sulfate or sulfamate groups, which are known to be responsible for the biological activity of heparin. In particular, a carboxylated polyurethane, already employed by our group to bind bioactive molecules, including antibiotics [37], [38], [39], enzymes [40] and antibacterial silver ions [41], [42], was first amidated with different functional amines and then reacted with pyridine–SO₃ or DMF–SO₃ adducts. The main advantage of our approach relies on the easy introduction of side chains bearing amino or hydroxyl groups in different concentrations, thanks to the use of a carboxylated segmented polyurethane. This feature could allow the physical properties of the resulting polymers to be tuned in terms of phase segregation, surface charge and hydrophilicity/hydrophobicity ratio, all these factors being extremely important in determining the biological properties of the obtained polymer. Usually, in the development of heparin-like materials the improvement of polymer hydrophilicity is obtained by surface grafting or coating with sulfated hydrophilic macromolecules (e.g. PEG or hyaluronic acid). In contrast, in our case, we planned to increase the polymer surface hydrophilicity by bulk reactions with small molecules, which should bring about a high phase segregation with a consequent migration of the hydrophilic groups to the surface.

The obtained polymers were further characterized by ¹H nuclear magnetic resonance (NMR), thermal and mechanical analysis, water swelling and contact angle measurements.