(741c) Understanding the Role of Nanoscale Topography of Polymer Surfaces on Protein Adsorption and Bacterial Adhesion for Reducing Catheter-Associated Infections

Introduction:

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).

Conclusions:

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.