Characterization of Anti-infective Coatings for Biomedical and Environmental Applications
Researchers at NDSU have developed a suite of novel antifouling coatings based on contact active or non-leaching technologies. The primary reason for developing these types of coating systems was to prevent the unwanted accumulation of sea life on surfaces and structures immersed in the marine environment, with a particular emphasis on ship hulls. A logical extension of this technology would be the development of anti-infective coatings for a variety of biomedical and environmental applications, including antimicrobial protection for medically implanted devices (i.e., urinary catheters, tracheal tubes, wound dressing, stents etc.) and protection of military personnel and infrastructure (i.e., uniforms, vehicles, tents, etc.) from biological warfare agents. A suite of high-throughput assays are currently being developed at NDSU to rapidly characterize coatings for these types of applications.
The high-throughput characterization of antimicrobial coatings for biomedical applications is achieved with the same multi-well plate technology utilized to screen antifouling coatings for ship hull applications. However, instead of employing marine bacteria and algae, microbial organisms associated with infection of implanted medical devices are used in the screening assays. As with the marine coating characterizations, biofilm based assays are predominantly utilized to assess the antimicrobial properties of the coatings due to their relevance and contribution to chronic and persistent infections.
A variety of microorganisms implicated in medical device related infections are used to screen antimicrobial coating libraries. This includes the use of Gram-negative (i.e., P. aeruginosa, E. coli) and Gram-positive (S. epidermidis, S. aureus) bacteria and yeast (C. albicans) to evaluate the broad spectrum antimicrobial activity of coating candidates.
Biofilm Growth and Retention Assays
The same multi-well plate assays used to screen antifouling coatings with marine bacteria is employed to screen antimicrobial coatings. This includes the rapid determination of leachate toxicity and the use of the biomass indicator dye, crystal violet, to quickly quantify biofilm growth and retention on the coating surfaces. In certain situations, the crystal violet dye can interact and bind with the coating material itself (i.e., without a microbial biofilm). In these instances, a variety of alternative spectrophotometric based biofilm quantification assays can be used to circumvent this issue, including additional absorbance based dyes (i.e., dimethyl methylene blue, alcian blue) and fluorescence based lectin staining (i.e., wheat germ agglutinin, concanavalin A) techniques.
An antimicrobial coatings library, consisting of 30 unique coating compositions, was prepared from silanol terminated polydimethylsiloxane (PDMS) of three different molecular weights. The library was comprised of five different quaternary ammonium salts (QASs) with different alkyl chains attached with nitrogen (top left diagram). A low and high concentration of QASs was generated for each PDMS molecular weight. Visual inspection of the crystal violet stained array plates (top right picture) clearly shows an antimicrobial effect for several coating candidates. Broad spectrum activity was observed for the coatings containing the C18 QAS, as both the Gram-negative (E. coli) and Gram-positive (S. epidermidis) bacterium showed a substantial reduction in biofilm growth and retention (Bottom charts).
Microbial Viability Assays
The viability of microbial cells, on antimicrobial coatings prepared in multi-well plates, is determined in addition to total retained biomass. Two quantification assays are utilized in this regard. The first assay is based on the reduction of a tetrazolium salt compound (XTT) by actively respiring microbial cells. This process produces an orange colored compound that can be measured by absorbance at 490 nm. The second assay is based on a bioluminescence measurement of ATP extracted from viable microbial cells on the coating surfaces.
Advanced Screening Capabilities
Traditional antimicrobial characterization techniques have been implemented at NDSU for the advanced testing and evaluation of promising antimicrobial coating candidates identified with the high-throughput screening workflow.
Agar Contact/Diffusion Assay
Antimicrobial coatings are prepared on glass or metal discs and placed in contact with nutrient agar plates seeded with a lawn of the appropriate microorganism. The nutrient agar contains a respiratory indicator dye (TTC), which is converted from a colorless to red compound by viable microbial cells, to visualize microbial growth on the coating surfaces. Coatings that exhibit no growth on the coating surfaces (i.e., absence of red color) and lack a zone of inhibition are determined to be contact active.
Flow Cell Assay
Antimicrobial coatings are prepared on glass or metal discs and placed in the channels of a dual chamber flow cell. A physiological saline suspension of the appropriate microorganism is delivered to the coating discs for 1 hr (using a peristaltic pump) to facilitate microbial attachment. A nutrient growth medium is then supplied at a continuous flow rate for several days to weeks to promote colonization and biofilm growth. The microbial attachment and colonization periods are monitored with a fluorescence microscope via the visualization of green fluorescent protein (GFP) produced by the viable microbial cells.
Coating compositions that show promising antimicrobial activity will also be evaluated for their biocompatibility or potential to cause adverse reactions when implanted in the body. In this regard, the biocompatibility of coatings and materials used in medically implanted devices is equally or more important than its antimicrobial properties. We are currently adapting standard laboratory assays (for assessing biocompatibility of materials) to our high-throughput screening methodology in multi-well plates.
Protein Adsorption Assay
Laboratory based protein adsorption assays are widely utilized by material scientists to obtain a first approximation of the biocompatibility properties of polymers and materials used in implanted medical devices. At NDSU, an immunoassay based detection kit is used to quantify the amount of bovine serum albumin (BSA) adsorption on the surface of coatings prepared in multi-well plates. BSA is incubated for 4 hours on the coating surfaces and quantified with a HRP (horseradish peroxidase) conjugate of the BSA detection antibody. The modified method has been shown to be reproducible on a variety of different surfaces in which a general trend is consistently observed from one trial to the next (above figure).
Mammalian Cell Culture Assay
Researchers at NDSU are currently developing a high-throughput mammalian cell culture assay for rapidly evaluating the in vitro ctyotoxicity of antimicrobial coating materials. L929 mouse fibroblasts (ATCC, CCL-1) are seeded directly onto the experimental coatings prepared in multi-well plates and are allowed to attach for 1 hour. After initial attachment, coatings are rinsed with PBS to remove non-adherent cells and replenished with fresh cell culture medium. Fibroblast proliferation on each surface is then determined after 7 days with a tetrazolium salt (MTT/PMS) colorimetric assay. Comparisons of fibroblast proliferation are made between the experimental antimicrobial coatings and non-toxic (silicone elastomer) and toxic controls (Triton X-100) to determine the degree of biocompatibility.
Red Blood Cell Hemolysis Assay
NDSU researchers are also developing a mammalian red blood cell (RBC) hemolysis assay for investigating biocompatibility of antimicrobial coatings. This assay has been traditionally used to evaluate the cytotoxicity profile of new antimicrobial compounds and biomedical materials. The degree of RBC hemolysis is determined by the amount of free hemoglobin released after direct contact of RBC’s with the material being evaluated. Specifically, a saline suspension of fresh RBC’s, collected from healthy white rabbits, is delivered to coatings deposited in multi-well plates and incubated for 1-4 hours at 37°C. Each RBC suspension is then removed and the optical density of the supernatant is measured at 545 nm with a multi-well plate reader. As with the mammalian cell culture assay, comparisons of RBC hemolysis are made between the experimental antimicrobial coatings and non-toxic (silicone elastomer) and toxic controls (Triton X-100) to determine the degree of biocompatibility.