Introduction: Prebiotic chemistry is the study of the chemistry associated with the origin of life [1],[2]. HCN derived polymers have been studied for many years in this context as possible sources for the precursors of proteins and nucleic acids [3],[4]. However, the application of these polymers in the field of materials science and in particular in biomaterials science has not been reported. Here, we have investigated the polymerization of the HCN trimer aminomalononitrile as a simple generic method providing adhesive and versatile coatings. Our results suggest that the resulting rich surface chemistry is highly suitable for biomedical applications.
Experimental Methods: Aminomalononitrile p-toluenesulfonate (AMN) was used as a precursor material. PBS buffer solutions containing AMN were adjusted to pH 8.5 using sodium hydroxide to initiate polymerization. Copolymerization reactions were carried out by adding compounds carrying suitable functional groups, such as aldehydes and amines, during coating formation while secondary immobilization reactions, including metallization, were carried out after coating deposition. Surface analytical data were obtained using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and contact angle measurements. Cell culture experiments were carried out using L929 mouse fibroblasts. Biofilm assays were carried out using S. Epidermidis and P. Aeruginosa strains.
Results and Discussion: Aminomalononitrile polymerizes spontaneously to give a brown protein-like polymer. We found that this polymerization reaction, when carried out in simple aqueous buffer solutions, can be used to produce adherent coatings on a wide range of organic and inorganic substrate materials (Figure 1).
Figure 1. Contact angles determined on a range of materials before and after AMN coating deposition.
We demonstrated that the coating thickness can be contolled by the polymerization time, while the coating morphology can be controlled via the buffer concentration as well as co-solvents. We also demonstrated that the robust, non-cytotoxic AMN coatings provide cell attachment equivalent to tissue culture polystyrene (TCPS), thereby providing access to a range of biomedical applications (Figure 2-3).
Figure 2. L929 cell attachment after 24 h on Corning Ultra-low attachment surface (ULA) before and after AMN coating deposition in comparison to TCPS.
Figure 3. Spatial control over cell attachment was achieved by micro-array printing of AMN solutions on an ULA substrate surface.
In addition, the nitrogen-rich coating chemistry allowed the covalent immobilization of compounds carrying suitable complementary functional groups, such as aldehydes and amines, both during coating formation and via secondary immobilization reactions. Furthermore, AMN coatings were successfully used in metallization reactions. This was exploited for the formation of antimicrobial coatings via the release of silver ions, leading to the prevention of biofilm formation for both Gram-positive and Gram-negative strains.
Conclusion: We have demonstrated that prebiotic chemistry inspired coatings derived from AMN can be deposited on a wide range of substrate materials. These coatings combine excellent biocompatibility with a rich surface chemistry, allowing the immobilization of bioactive signals and the complexation of metal ions. Combined with an extraordinarily simple coating process, it is expected that these coatings will be translated into a number of biomedical applications in vitro and in vivo.
Aylin Koegler (CSIRO) is acknowledged for her help with cell culture assays.; Yue Qu and Trevor Lithgow (both Monash University) are acknowledged for their help with biofilm assys.
References:
[1] Miller S.L., Science 117 (1953) 528.
[2] Bada, J.L., Chem. Soc. Rev. 42 (2013) 2186.
[3] Matthews C.N. et al., Nature, 215 (1967) 1230.
[4] Matthews C.N. et al., Faraday Discuss. 133 (2006) 393.