(525f) Engineering Antibacterial Nanosurfaces for Field Hospitals

Antibiotic resistance (ABR), although not a
new phenomenon, is rapidly gaining attention within the scientific community as
an escalating threat to the global health care infrastructure. The increasing
frequency of occurrence of resistant bacterial strains, specifically multidrug
resistant (MDR), extensively drug resistant (XDR), and totally drug resistant
(TDR) phenotypes, would seemingly necessitate demand for a new generation of
small-molecule drugs for clinical implementation. Unfortunately; the compound
effect of administrative barriers, technical complications, and selective
economic pressures has significantly reduced the rate of discovery for new
antibiotic species. In addition to this, systemic overuse and misuse of
currently available therapeutics is generally accepted as the principle driving
force behind the induction of resistance. This is a crucial factor that has yet
to be effectively mitigated through international regulation, and would only be
perpetuated with the development of new classes of antibiotics. While
attempting to develop new engineering solutions that surpass the specified
shortcomings, novel approaches towards the management of bacterial growth and
colonization in clinical settings are highly desirable.

One such approach is the development
of nanoscale surface functionalization for materials which are commonly seen in
a clinical setting, ranging from general architectural elements to
instrumentation. Nanoscale functionalization, which includes both physical and chemical
components interacting with biological systems, in this work has been motivated
by macrophage biomimetics. When macrophages phagocytize bacteria for systemic
clearance, the bacteria are entrained within endosomal compartments which
subsequently mature with a reduction in pH, concentration of enzymatic species,
and generation of reactive oxygen species (ROS). Although these factors work
synergistically to induce cytotoxicity within the complex environment of the
endosome, ROS generation along nanoparticulate surfaces (in addition to other
nanoscale phenomena present within colloidal systems) has prove sufficient in
demonstrating antibacterial properties in vitro and in
vivo. Although metallic nanoparticulates, specifically silver, are
frequently implemented within antibacterial applications, this work opted to
utilize selenium nanoparticles for achieved therapeutic effect. Selenium
was selected on the grounds that its nanoparticulate formulations are capable
of ROS generation within cellular targets, while still serving as an overall
trace elemental nutrient in the human diet.

Selenium nanoparticle
functionalization was achieved through heterogeneous nucleation along a diverse
range of solid supports provided by academic and federal collaborators. These materials
were coated in small-batch processes, in which the solid supports were immersed
in a reaction solution for 60 seconds, followed several successive washing
stages with deionized water. Nanoparticulate coatings were characterized with
atomic force microscopy (AFM), scanning electron microscopy (SEM), energy
dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS),
and contact angle measurement. Physical characterization revealed that
functionalization yielded heterogeneous surface coverage of
nanoparticles, with average interspacings within the sub-micron range. Chemical
characterization verified that the observed nanoparticulates were composed of
elemental selenium. Following physical and chemical characterization,
functionalized surfaces were assessed for antibacterial properties by means of
colony forming unit (CFU) assay. Treated materials were immersed in cultures of
gram-positive (Staphylococcus aureus) or gram-negative (Escherichia
coli) bacterial strains, and the concentration of viable, adsorbed bacteria
were determined at set intervals following initial
exposure.  Statistically significant reduction in S. aureus bacterial
colonization was observed for the two current test materials, carbon-microfiber
sheets and basalt-epoxy blocks. For carbon-microfiber sheets, nanoparticulate
treated surfaces resulted in 32 % reduction and 63 % reduction in CFU at the
material’s targeted operational temperatures of 50 degrees Celsius and 40
degrees Celsius, respectively. For basalt-epoxy blocks, nanoparticulate treated
surfaces resulted in a 77 % reduction and 82 % reduction in CFU at the
material’s targeted operational temperatures of 60 degrees Celsius
and 50 degrees Celsius, respectively. A general trend of reduced bacterial
colonization has been observed for  E. coli in
preliminary studies.

The future directions of this project
are focused on elucidating the mechanism of antibacterial activity, assessing
mammalian cell biocompatibility, determining efficacy under biological fouling,
and investigating the general mechanical properties of nanoparticle
functionalized composites. The mechanism of antibacterial activity is of
significance, as this may pose serious implications on the potential for
subsequent bacterial resistance. While the understanding of resistance
developed under the selective pressures of antibiotics is historically well
established, current studies in the literature have demonstrated select cases
for the development of resistant phenotypes in bacterial populations exposed to
silver nanoparticle treatments. It is of great interest to determine if this
pattern is similarly observed with the implementation of selenium
nanoparticulate coatings. As ROS are believed to serve as a principle factor in
prokaryotic cytotoxicity, which is inherently non-specific for select
biomolecular targets within pathogens, there is additional interest in
determining whether selenium nanoparticles are either cytotoxic or mutagenic to
various eukaryotic cell lines. In addition to this, if ROS generation is
crucial for achieving antibacterial efficacy, than direct exposure of
colonizing bacterial cells to the nanoparticulate coating is essential. Steric
constraints on this exposure may result from the formation of a fouling layer,
arising either through the directed-assembly of bacterial biofilms or the
random deposition of organic/inorganic debris, and thus there is also interest
in determining the antibacterial properties of selenium nanoparticulate
coatings under biofouling conditions. Finally, the mechanical properties of
nanoparticulate coated composites will also be assessed under
variant environmental conditions, such as extreme heat and humidity,
that may be experienced in field hospitals operating in diverse locations
throughout the globe.

Figure
One: Appearance and antibacterial property of basalt-epoxy blocks with selenium
nanoparticle coating. (A.) An SEM image of a basalt-epoxy surface following
selenium nanoparticulate functionalization. Observe that the nanoparticles that
have nucleated along the surface are polydisperse, both in their sizing and
morphology. The interspacings between nanoparticulates are smaller than the
dimensions of common bacteria, such as S. aureus and E. coli.
Nanoparticulates have even formed within crevices along the treated surface,
demonstrating that coverage extends to all areas that are accessible to the
reactant solution during particle nucleation. The inlaid scale bar is 2
micrometers in length. (B.) The antibacterial efficacy of selenium
nanoparticulate coatings of basalt-epoxy blocks at two targeted operational
temperatures set by collaborators. Functionalization with selenium
nanoparticles yielded a statistically significant reduction in bacterial colonization
at both temperatures, as demonstrated by CFU assay. Treated surfaces
experienced a 77 % and 88 % reduction in CFU at the targeted temperatures of 60
degrees Celsius and 50 degrees Celsius, respectively. N = 3.