**3.2. Protein adsorption**

208 Polyurethane

**Figure 3.** pH variation of HBSS buffer in which autoclaved PU/HPC membranes were incubated: A, 24

As one can see from Figs. 2 and 3, a long-period of incubation of unsterilized and sterilized (by autoclaving at 121 oC and 1 atm) PU/HPC membranes in simulated biological fluid did not meaningfully modify the physiological range pH value of the incubation environment, while a pronounced decrease of the environment pH was observed for pure PU-PEGA sample (Fig. 2 A). Thus, one can say that HPC gives an important contribution to hydrolytic

For autoclaved samples, the variation of pH values of the environment for poly(etherurethane)s remains in the range of the physiological value, while PU-PEGA/HPC membrane induced a higher decrease of pH (Fig. 3), a normal result owing the higher thermal

Another property that was changed by modifying PU membranes with HPC was the floatability (see Fig. 2B). As the surface wettability and water uptake for PU-PEGA sample is similar to HPC modified one (see Table 1), the reason of these different behavior could reside in different morphologies, as seen from SEM images of membrane cross-sections (Fig. 4).

**Figure 4.** SEM image of PU-PEGA (A) and PU-PEGA/HPC (B) membrane cross-sections

h of incubation; B, 72 h of incubation; C, pH variation curves

stability of urethane and ester bonds of PU chains.

degradability of poly(ester-urethane)s (Guelcher, 2008).

There are many data concerning mechanisms of protein adsorption on different surfaces (Gray, 2004; Scott & Elbert, 2007; Van Tassel, 2006; Wilson et. al., 2005). It was clearly demonstrated that proteins have amphoteric properties, being able to adsorb on both negatively and positively charged surfaces (Michelsen et. al., 2000; Van Tassel, 2006). The amount of adsorbed proteins is depending on their isoelectric points as well as on surface chemistry and hydrophilicity (Keselowsky et. al., 2003; Wertz & Santore, 2001). Hydrophobic surfaces manly interact with hydrophobic protein core that leads to the modification of the protein physiological conformation and its functionality. Opposite to hydrophobicity, superficial water maintains native protein conformation and specific functionality (Keselowsky et. al., 2003; Noinville & Revault, 2006).

Many authors have reported protein adsorption behaviour on different surfaces using simulated solutions and highly sensible methods in which very low protein concentrations are detected. Thus, the adsorption of albumin solutions of different concentrations on pure silica or on silica modified with NH2 and CH3 terminated self-assembled monolayers (SAMs) (Noinville & Revault, 2006) and on silica-titanium surfaces (Kurrat et. al. 1997) was studied. Other authors reported the competitive adsorption of fibrinogen on mica (Gettens et. al., 2005; Tsapikouni & Missirlis, 2007). Surface adsorption of SA, FB, fibronectin (FN), immunoglobulins (IGs) and lysozyme were investigated to evaluate the surface biocompatibility (Bernsmann et. al., 2010; Pompe et. al., 2006; Rezwan et. al., 2005), each class of these proteins providing specific surface properties for targeted application. Thus, FN adsorption is relevant for the prediction of cell adhesion, lysozyme – for enzymatic degradability predisposition, IGs - for immune-specific interactions, while SA and FB adsorption have haemocompatibility predictive value.

In order to estimate protein adsorption (retention) capacity of materials at blood or tissues contact, simulated physiological environment, close to normal blood conditions is required. For example, Bajpai, 2005 followed SA adsorption capacity of biomaterials at SA bulk concentration from 1 to 6 mg/ml, while Alves et. al., 2010 used mix protein conditions, considering physiological value for each protein. The mix protein adsorption conditions are considered to better reflect the complex interactions that occur between different proteins (Latour, 2008).

PU/HPC membranes were previously demonstrated to possess good mechanical properties (elongation at break for dried/hydrated PU-PEGA/HPC = 71/84; for PU-PTHF/HPC = 72/159

and for PU-PPG/HPC = 53/55), appropriate for cardio-vascular applications (Macocinschi et. al., 2009). The physisorption of SA and FB is further highlighted as screening criteria for biocompatibility and, more specifically, haemocompatibility. Very short characteristics of SA and FB, important for protein-material interaction are given below.

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of the Improved Polyurethane Membranes for Medical Applications 211

**Figure 5.** Amount of adsorbed BSA (left) and FB (right) from individual protein solutions and in coadsorptive environment (mixed protein solution) of physiological concentrations, i.e., 3 mg/ml for FB

Figure 6 shows the ratios of adsorbed BSA and FB from mixed protein solutions and from blood plasma. In both studied conditions and for all membranes, the amount of adsorbed SA is higher than that of adsorbed FB, a normal result considering the lower concentration of FB in solutions. The total amount of the adsorbed SA and FB proteins from blood plasma is lower as compared to that adsorbed from mixed solutions due to the competitive adsorption of some other blood plasma proteins. Moreover, the ratio between adsorbed FB

**Figure 6.** Total amount of adsorbed FB and BSA from: A – mixed protein solution at 3 mg/ml FB and 45

As conclusion, comparing PU-PEGA and PU-PEGA/HPC membranes one can observe that small amount of polysaccharide rich in functional substituents can bio-stabilize PU

mg/ml BS physiological concentration; B - human blood plasma with 2.98 mg/ml initial FB

and SA is lower in blood plasma than in mixed solutions.

concentration and 45.3 mg/ml initial SA concentration

and 45 mg/ml for BSA

**SA** is a protein belonging to the so called "soft" class of proteins, with a molar mass of about 65 kD for BSA and 67 kD for human SA (HSA). This protein represents about 60% of the blood proteins. Normal blood concentration of HSA is 35 – 50 mg/ml. This protein is involved in many physiological phenomena as carrier protein for fatty acids, metals, cholesterol, bile pigment, hormones and drugs. SA is also characterised by antioxidant properties (Bourdon et. al. 1999; Kouoh et. al., 1999; H. Lee et. al., 2000) that is higher in alkaline pH, up to 8 (H. Lee et. al., 2000). SA is preponderantly negatively charged, its isoelectric point being close to 4.8 (Carter & Ho 1994; Noinville & Revault, 2006). Approximately 67% of the secondary SA structure is represented by the α-helix. It was demonstrated that the stability of SA secondary structure strictly depends on pH (Freeman, 2006) that influence the protein conformation. Thus, at pH = 5, SA takes almost spherical, native, unfolded shape that forms a thick layer on the adsorptive surfaces. At pH = 7 (close to physiological pH), due to molecular spreading, SA forms an extended contact area with adsorptive surfaces. This behavior can be influenced by surface charge, surface functionality and functionality distribution, surface morphology or wettability conditions (Wilson et. al., 2005). The role of adsorbed SA on biomaterial biocompatibility is still ambiguously described in the literature. While some authors have demonstrated biocompatibility improvement of material with increased adsorption of SA (Eberhart et.al. 1987; Marconi et. al., 1996; Randrasana et. al., 1994), others demonstrated a better biocompatibility of SAresistant surfaces (Ostuni et. al., 2001; Wan et. al., 2006).

**FB** is a high molecular weight (340 kD) complex glycoprotein that has 2 molecular domains, each of them consisting of three polypeptide chains called Aα, Bβ and γ. Molecular updated analysis of FB can be found in recent reports (Cardinali et.al, 2010). FB is an important factor of haemostasis. Through fibrin network formation as first cell scaffold, FB is involved in wound healing and tissue regeneration. Its normal blood concentration varies from 2 to 4 mg/ml. In inflammations or in other pathological statuses - as cardiovascular diseases - FB can reach up to 7 mg/ml, therefore adsorption properties of biomaterials for this protein should be carefully analysed, especially for those targeted for blood contact applications.

The results obtained in adsorption experiments of SA and FB from both individual and mixed solutions on PU/HPC membranes are presented in Fig. 5.

No significant differences between adsorption behavior of both proteins in their pure and mixed solutions were registered, except a small tendency to decrease adsorbed BSA from mixed solution as compared to individual solution, especially on PU-PEGA and PU-PEGA/HPC membranes, where FB, with a higher molecular weight, showed a higher affinity.

and for PU-PPG/HPC = 53/55), appropriate for cardio-vascular applications (Macocinschi et. al., 2009). The physisorption of SA and FB is further highlighted as screening criteria for biocompatibility and, more specifically, haemocompatibility. Very short characteristics of

**SA** is a protein belonging to the so called "soft" class of proteins, with a molar mass of about 65 kD for BSA and 67 kD for human SA (HSA). This protein represents about 60% of the blood proteins. Normal blood concentration of HSA is 35 – 50 mg/ml. This protein is involved in many physiological phenomena as carrier protein for fatty acids, metals, cholesterol, bile pigment, hormones and drugs. SA is also characterised by antioxidant properties (Bourdon et. al. 1999; Kouoh et. al., 1999; H. Lee et. al., 2000) that is higher in alkaline pH, up to 8 (H. Lee et. al., 2000). SA is preponderantly negatively charged, its isoelectric point being close to 4.8 (Carter & Ho 1994; Noinville & Revault, 2006). Approximately 67% of the secondary SA structure is represented by the α-helix. It was demonstrated that the stability of SA secondary structure strictly depends on pH (Freeman, 2006) that influence the protein conformation. Thus, at pH = 5, SA takes almost spherical, native, unfolded shape that forms a thick layer on the adsorptive surfaces. At pH = 7 (close to physiological pH), due to molecular spreading, SA forms an extended contact area with adsorptive surfaces. This behavior can be influenced by surface charge, surface functionality and functionality distribution, surface morphology or wettability conditions (Wilson et. al., 2005). The role of adsorbed SA on biomaterial biocompatibility is still ambiguously described in the literature. While some authors have demonstrated biocompatibility improvement of material with increased adsorption of SA (Eberhart et.al. 1987; Marconi et. al., 1996; Randrasana et. al., 1994), others demonstrated a better biocompatibility of SA-

**FB** is a high molecular weight (340 kD) complex glycoprotein that has 2 molecular domains, each of them consisting of three polypeptide chains called Aα, Bβ and γ. Molecular updated analysis of FB can be found in recent reports (Cardinali et.al, 2010). FB is an important factor of haemostasis. Through fibrin network formation as first cell scaffold, FB is involved in wound healing and tissue regeneration. Its normal blood concentration varies from 2 to 4 mg/ml. In inflammations or in other pathological statuses - as cardiovascular diseases - FB can reach up to 7 mg/ml, therefore adsorption properties of biomaterials for this protein should be carefully analysed, especially for those targeted

The results obtained in adsorption experiments of SA and FB from both individual and

No significant differences between adsorption behavior of both proteins in their pure and mixed solutions were registered, except a small tendency to decrease adsorbed BSA from mixed solution as compared to individual solution, especially on PU-PEGA and PU-PEGA/HPC membranes, where FB, with a higher molecular weight, showed a higher

SA and FB, important for protein-material interaction are given below.

resistant surfaces (Ostuni et. al., 2001; Wan et. al., 2006).

mixed solutions on PU/HPC membranes are presented in Fig. 5.

for blood contact applications.

affinity.

**Figure 5.** Amount of adsorbed BSA (left) and FB (right) from individual protein solutions and in coadsorptive environment (mixed protein solution) of physiological concentrations, i.e., 3 mg/ml for FB and 45 mg/ml for BSA

Figure 6 shows the ratios of adsorbed BSA and FB from mixed protein solutions and from blood plasma. In both studied conditions and for all membranes, the amount of adsorbed SA is higher than that of adsorbed FB, a normal result considering the lower concentration of FB in solutions. The total amount of the adsorbed SA and FB proteins from blood plasma is lower as compared to that adsorbed from mixed solutions due to the competitive adsorption of some other blood plasma proteins. Moreover, the ratio between adsorbed FB and SA is lower in blood plasma than in mixed solutions.

As conclusion, comparing PU-PEGA and PU-PEGA/HPC membranes one can observe that small amount of polysaccharide rich in functional substituents can bio-stabilize PU

structures and improve their resistance for autoclaving procedures as important step in ready to use biomaterials preparation. From all the data presented in this section, one can say that the more hydrophilic PU-PTHF/HPC membrane could be the most appropriate for biomedical applications.

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of the Improved Polyurethane Membranes for Medical Applications 213

antioxidant compounds to materials can improve their resistance against tissue degradation

**Figure 7.** Schematic representation of the oxidative/antioxidative balance with enzymatic and

Antioxidant defensive systems are present in both cells and extracellular environment. SA molecules are the most important antioxidants in blood. Due to their high concentration and polyvalent possibilities to fit with oxygen free radicals, SA molecules are considered to be the main plasmatic components of defence that assure neutralisation of more than 70% of

Assigning to SA molecules the main role in protective effect, we analysed the interaction of PU/HPC membranes with blood plasma, following the plasma antioxidant status. To define the importance of SA adsorption on material surface, the membranes were incubated at 37 oC in blood plasma and TAS was measured periodically. The results are shown in Fig. 8.

Two PU samples (PU-PEGA and the more hydrophobic PU-PPG/HPC) had significant tendency to quickly decrease TAS activity in the first 48 hours. Due to the complexity of TAS, it is difficult to speculate on the mechanism by which the decreasing phenomenon arises and certainly more examinations are needed. However, one can suppose that PU-PEGA alter the TAS activity as a result of plasma pH modification that leads to sustained free radical generation in the presence of the material. The mechanisms by which TAS activity is lowered after PU-PPG/HPC incubation could not be related directly to SA antioxidant activity, but to some other oxidant pathways that need further investigations.

nonenzymatic tissue pathways

ROS (Bourdon & Blache, 2001).

(Oral et. al., 2006; Stachelek et. al., 2006; Wattamwar et. al., 2010).
