Investigation on the Design and Development of Novel Antithrombotic and Anti-adhesion Coatings for Cardiovascular Applications

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Mei, Yan (Centre for Blood Research, 2350 Health Sciences Mall, University of British Columbia)
Yu, Kai (Centre for Blood Research, 2350 Health Sciences Mall, University of British Columbia)
Lo, Joey (Department of Urological Sciences, University of British Columbia)
Lange, Dirk (Department of Urological Sciences, University of British Columbia)
Kizhakkedathu, Jayachandran N (Centre for Blood Research, 2350 Health Sciences Mall, University of British Columbia)

Introduction

Cardiovascular diseases account for an estimated 17 million deaths annually around the world. Current treatment options for vascular diseases include balloon angioplasty, stent replacement, graft bypass surgery, and the use of pharmacological agents. Therefore, the demand for improved cardiovascular devices is constantly growing. When a device is implanted into the body, a cascade of events is initiated, including protein adsorption, platelet adhesion and activation, and cellular attachment to the implant surface. These events initiate coagulation cascade that may lead to the thrombus formation on the device surface. In addition, microorganisms may grow on the device surface and form biofilms as implant surface could provide an initial attachment site for bacteria. Hence, developing antithrombotic and anti-adhesion coatings to control the interfacial interactions is of particular importance to sustain their functionality and life-time of cardiovascular devices within the body, thereby reducing the mortality rate and medical costs. Functional coatings (20-400 nm thickness) based on polymer brushes have gained tremendous attention recently due to their excellent biocompatibility and cell-adhesion resistance. However, the composition of polymer brush exhibit the optimal antithrombotic and anti-adhesion/bacterial performance is still unknown. The current screening studies will help us in identifying the optimal coating and a general strategy to reduce the thrombus formation and bacterial adhesion on cardiovascular devices.

Materials and Methods

Polypropylene film was used as a model platform to construct antithrombotic and anti-adhesion coating. We fabricated 36 different polymer brush coatings (6 different N-substituted acrylamide monomers and 6 different thicknesses) (Figure 1) using surface-initiated atom transfer radical polymerization. Polymer brush coatings were characterized by determining the absolute molecular weight of the polymer in solution along with surface grafted polymer, surface composition by attenuated total reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) and by static water contact angle measurements. The protein interaction on various coatings was determined by measuring the absorption of fluorescently labeled bovine serum albumin (BSA) and fibrinogen. The platelet adhesion on the surface in platelet rich plasma (PRP) was determined using scanning electron microscopy. Fluorescently labeled anti-C3b antibody was used to determine the complement activation on the surface as determined by fluorescent microscopy analysis. The adhesion of Pseudomonas aeruginosa on brush coatings was assessed by measuring the number of adhered bacteria by luminescence measurements.

Results

Well-defined functional polyacrylamide brushes with different thicknesses were prepared on polypropylene films by aqueous surface-initiated atom transfer radical polymerization. Protein adsorption on brush coating showed a correlation between protein adsorption and film thickness: all polymer brushes exhibit highest protein resistance at medium grafting thickness (Figure 2B). The poly(N,N-dimethylacrylamide) (PDMA) brushes showed better protein resistance compared to other polymer brushes, and the optimized coating based on PDMA brush reduced 95% of BSA adsorption and 80% of fibrinogen adsorption compared to the original surface (Figure 3A). The PDMA coating with highest grafting thickness prevented platelet adhesion; the number of adhered platelets was 282 platelets per 1 mm2 compared to 20625 platelets per 1 mm2 on control surface (Figure 3C). Complement activation results showed that the degree of activation mainly depends on the brush composition, primarily determined by the functionalities present on the polymer chains (Figure 2C). Hydroxyl group carrying polymer brush coating generated more complement activation than other surfaces. Bacteria adhesion results showed that the PDMA coating at medium grafting thickness exhibited superior anti-adhesion performance against Pseudomonas aeruginosa, preventing 96% of initial bacterial adhesion compared to the control surface (Figure 3D).

Discussion and Conclusion

Our findings show that the polymer chemistry, functionality of the polymers and the thickness of the coating influence coating’s performance in preventing protein adsorption, platelet adhesion, complement activation and bacterial adhesion. Based on the current results, PDMA coating with optimized thickness presents the best non-fouling character. Our ongoing work includes further screening of the polymer coating and development of the coating towards the antithrombotic and antimicrobial coating for the prevention both thrombus generation as well as infection on cardiovascular devices. We anticipate this novel coating will significantly improve the safety and performance of cardiovascular devices.

Figure 1: Representation of the different polymer brush coating on polypropylene (PP) film. Six different N-substituted acrylamide monomers were polymerized from the surface of PP by surface-initiated atom transfer radical polymerization (Si-ATRP) on PP film.

Figure 2: Surface properties and antifouling characteristics of polymer brushes on PP film. Influence of polymer structure on (A) water contact angle (B) adsorption of fluorescently labeled BSA (1mg/ml) (C) complement activation of the coating. All the samples had similar grafting thickness.

Figure 3: Influence of grafting thickness (increased from PDMA-1 to PDMA-6) on PDMA brushes (A) adsorption of BSA (1mg/ml), (B) adsorption of fibrinogen (0.25mg/ml), (C) platelet adhesion from platelet rich plasma, (D) Pseudomonas aeruginosa adhesion after 4h incubation.

Acknowledgements

We acknowledge fundings from Canadaian Institutes of Health Research and the NSERC CREATE program for regenerative medicine.

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