Introduction: Some blood-materials devices could suffer thrombosis caused by physiological environment. In order to avoid this complication, one approach is to coat metallic devices with a thin polymer layer. Chitosan is a promising candidate, since it has low cytotoxicity and biodegradability. Moreover, along all its structure, there are amino and hydroxyl groups, which can be easily modified or used for grafting. In this work, a metallic surface was grafted with chitosan and sulfonated chitosan through dopamine and PEG as anchors, then characterized by XPS and contact angle. Thereafter, their effects on platelet adhesion and clotting time were investigated.
Materials and Methods: Chitosan solution at 2% (w/v) was prepared in aqueous solution of acetic acid 3% (v/v)[1]. The chitosan sulfonation was made according the method described by Amiji[2]. Prior to use, stainless steel (SS) discs were successively washed in ultrasonic bath with acetone, water and methanol, for 10 min each, then electropolished[3]. Dopamine solution[4], at pH 8.6, was used to functionalize the metallic surface, which was afterwards activated by PEGb grafting. Chitosan solution was let react for 3h onto the PEGb modified samples, pre-activated with EDAC in MES buffer. Each step of surface modification was characterized by XPS and CA. Whole human blood was used in biological tests. For platelet adhesion, samples were incubated in blood at 37ᵒC and 50 rpm for 15 min and visualized by AFM. Clotting times at 20 min and 80 min were evaluated by colorimetric tests by measuring the free haemoglobin absorbance at 540 nm[5].
Results and Discussion: XPS analysis showed that dopamine fully covered the SS as no metallic compound was detected. PEG and chitosan grafting was supported by the changes in C and O percentages (Table 1), as well as a significant increase in C-O contributions, as expected.
Also, the grafting efficiency was clearly evidenced by the presence of S from the sulfonated moieties on modified chitosan. Besides, these groups led to an increase in the hydrophilic character of chitosan coating. Also, sulfonated chitosan exhibited no platelet adhesion and a significant difference compared to the other coatings regarding the free hemoglobin at 20 min (Fig. 1). Although no significant difference was observed at 80 min, sulfonated chitosan still presented higher free hemoglobin absorbance, meaning a delay in clot formation.
Conclusion: XPS and CA results show that the different surface modification steps were successful, and that chitosans were effectively grafted on modified stainless steel. Sulfonated chitosan show no platelet adhesion and exhibited anticoagulant properties, compared to non-sulfonated chitosan surfaces. Based on these results, this modified surface has potential to be used as blood-contact material.
This work was partially supported by Funcap-Brazil, CNPq-Brazil, MFATD-Canada, NSERC-Canada, CIHR-Canada, CFI-Canada, FRQ-NT-Quebec, and MRI-Quebec.
References:
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