Catheter-associated infections (CAIs), most of which are caused by microbial biofilms, are still a major problem in health-care and are associated with significant morbidity, mortality, and medical cost. Currently, the use of nanomaterials or creating nanofeatured topographies on material surfaces seem to be among the most promising ways for reducing initial bacteria attachment, biofilm formation and infections. Many researchers have confirmed that nanofeatured surface topographies are a potent tool for selectively increasing desirable cell functions while simultaneously decreasing competitive cell functions. Also, natural surfaces, such as cicada wing surfaces, appear to be bactericidal to Pseudomonas aeruginosa, which was thought to be due to the surface nanostructure of the wing rather than a surface chemical effect. Motivated by these findings, in this study, our objective was to modify the raw surface of a catheter composed of polydimethylsiloxane or PDMS to possess antibacterial nanostructures, and then to develop a model that can correlate nanosurface roughness and associated surface energy with protein adsorption and bacterial adhesion.
Materials and Methods:
Here, we present a simple and cheap method to prepare a nano-patterned PDMS replica by using highly ordered nanotubular anodized titanium as the template. The surface morphogy and elemental composition of PDMS were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Surface contact angle tests were used to determine the surface wettability and associated surface energy for the various samples. In vitro bacterial studies using Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were conducted to assess the effectiveness of the nano-patterned PDMS (nano-PDMS) at inhibiting bacterial growth. In addition, human fibroblast (ATCC, CCL-110) and endothelial cell (Life Technologies) MTT adhesion assays were conducted to determine the influence of the nanostructure on mammalian cell behavior as a measurement of toxicity. To elucidate the mechanisms of how surface nano-topographies affect cell/ bacteria adhesion, protein interactions with different surfaces were also investigated by using the bicinchoninic acid (BCA) protein assay. As a target protein, casein was used in this study since it has been shown to be a key protein in tryptic soy broth (bacterial media). All experiments were completed in triplicate and repeated at three different times.
Results and Discussion:
As expected, the nano-patterned structures were fabricated successfully on the surface of PDMS. The surface roughness values increased from 3nm for the plain-PDMS to 30nm for the nano-PDMS by AFM roughness measurements. The contact angles for distilled water over plain-PDMS and nano-PDMS surfaces were 99.2° and 66.6°, respectively. These values highlighted that upon nanostructuring of the PDMS surface, it became slightly more hydrophilic. XPS revealed similar surface chemistry for the samples before and after nanomodification. In vitro study indicated that nano-PDMS inhibited the adhesion and growth of both gram-positive and -negative bacteria after 24h and 48h compared with plain-PDMS, respectively. Moreover, data suggested the effectiveness of bacteria inhibition reached above 50%, all without employing antibiotics. It was also found that nano-PDMS increased both fibroblast and endothelial cell adhesion after 4h treatment. BCA protein results indicated that the increase of nanoscale surface roughness caused a significant increase of the amount of adsorbed proteins, presumably due to the increased surface area and change of adsoprtion sites (Fig.1). The maximum adsorbed protein occurred at an incubation time of approximately 1h. The adsorbed mass subsequently decreased during the next couple of hours of incubation due to the Vroman effect. The increased protein adsorption on the nano-PDMS in the first several minutes could in part be responsible for the bactericidal properties. Moreover, increased casein adsorption on nano-PDMS also confirmed that increased nanoscale roughness, surface energy could contribute to enhanced protein adsorption and antibacterial properties.
Figure 1. Variation of the amount of protein adsorbed on PDMS surfaces with (and without) nanostructure as a function of incubation time in tryptic soy broth (TSB) media. N = 3. Abbreviations: n-PDMS (nano-patterned PDMS); p-PDMS (plain-PDMS).
The relationship between the nano-topography, protein adsorption and bacterial activities was investigated in this study. Data shows that the nano-topography on PDMS could increase the amount of protein adsorbed, inhibit both bacterial adhesion and growth significantly while remaining non-toxic to mammalian cells, and thus should strongly be considered for reducing catheter-associated infections.
Acknowledgements: The author would like to thank the Webster Nanomedicine Lab and Department of Chemical Engineering, Northeastern University for funding.
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