Effect of UV Curing on Peptide Antimicrobials and Considerations for Incorporation in Polymeric Coatings
Fatima Alim, Jennifer A. Neff, Allvivo Vascular, Inc., Lake Forest, CA
Statement of Purpose
Increases in hospital acquired infection rates and their associated costs have lead to a demand for surface active antimicrobial coatings. Numerous strategies exist to impart antimicrobial activity on the surface of medical devices. Commonly used approaches involve incorporation of an antimicrobial agent directly into the material used to form the device, application directly onto the surface of the device, or encapsulation in a polymeric surface coating. In the latter case, UV curing is a preferred method for bonding polymer coatings to device surfaces due to quick processing speeds and low manufacturing costs. Despite the high use of UV, studies that describe its effects on antimicrobial activity are lacking.
There is a great deal of interest in the use of antimicrobial peptides for medical device coatings because they are not likely to promote the development of antibiotic resistant bacteria. However, when attempting to incorporate an antimicrobial peptide, one is faced with multiple interactions that occur from UV exposure that may reduce or completely eliminate an antimicrobial peptide's activity. These interactions narrow the coating formulation and application approaches that can be effectively utilized and more information is needed to understand how UV and UV initiated crosslinking reactions within a coating layer affect peptide activity.
In this study, coatings comprised of the lantibiotic, nisin, incorporated in a polymer gel composed of Pluronic F127 (F127) were evaluated. Nisin displays excellent antimicrobial activity against gram positive bacteria including Staphylococcus epidermidis .1 This study provides insight into the affect of UV exposure on the activity of two natural variants of nisin, nisin A and nisin Z, taking into account the effect of peptide concentration, UV intensity, and UV duration. This study also examines the affect of UV on the antimicrobial activity of peptide- polymer combination coatings.
High purity nisin (≥95%, Handary) was dissolved in 0.02M degassed HCl at various concentrations. Solutions were then UV treated in UV grade quartz tubes at different intensities and durations using a fusion electrode-less microwave excited UV system. Two UV intensities were chosen for comparison, 277 mW/cm2 (Intensity A) and 554 mW/cm2 (Intensity B). Solutions were diluted to 1 mg/ml and antimicrobial activity assessed by the ZOI assay. Petri dishes were overlay with 25ml agar enriched with trypticase soy broth (TSB). A liquid culture of Staphylococcus Epidermidis (ATCC, P5984) that was prepared by incubating a single colony in TSB was then streaked onto the agar plates. Wells were bore into each quadrant of the plate and 30 µl aliquots of solution added to each well. Plates were incubated at 37oC for 18 hours. ZOI diameters were measured and used for calculating ZOI areas. The ZOI assay provides a quantitative measurement of changes in antimicrobial activity where reduction in ZOI correlates with a reduction in antimicrobial activity.
To determine the effect of UV on nisin A's activity in the presence of F127, two sets of solutions with varying concentrations of F127 (0, 0.5, 1, 5, 10 and 20% w/v) and 2 mg/ml nisin A were prepared. An additional sample type, 20% F127, with a higher, 20 mg/ml, nisin A concentration was also prepared. One set was treated with UV at intensity B for 1 min and the other served as a no UV control. These solutions were subjected to the ZOI assay as described above and the turbidometric assay as follows. A liquid culture of S. Epidermidis was diluted to 107 colony forming units/ml. Aliquots of 190 µl of the bacteria culture were added to each well of a 96 well plate. Sample types were diluted and 10 µl aliquots were subsequently added in triplicates to each well to obtain 5, 10, 20, 40 or 100 µg Nisin/ml Staph. E., where these concentrations are above the minimum inhibitory concentration (MIC), (4.2 µg/ml).1 The 96-well plate was incubated at 37oC for 18 hours and the turbidity of the wells measured spectrophotometrically at 600 nm. Any absorbance obtained indicates growth of S. Epidermidis and hence a loss in nisin A antimicrobial activity.
Nisin A's antimicrobial activity decreased linearly with increasing UV duration (Figure 1). For the doses and times evaluated here, the effect of UV on nisin A activity depended primarily on the total dose of UV delivered. There was a slight trend toward greater retention of activity with the application of higher intensity UV for shorter times vs lower intensities for longer times.
Figure 1: Nisin A UV stability as determined by ZOI assay. (n=3, standard deviations less than 0.1)
Nisin Z displayed poor stability to UV relative to nisin A. Even at high concentrations, UV treated nisin Z did not yield a measurable ZOI. After UV exposure at the lowest dose, Nisin Z lost more than 90% activity. Nisin A and nisin Z are nearly identical in structure and differ only in the type of amino acid at position 27 (His in nisin A and Asn in nisin Z). The two variants are also equally distributed among nisin-producing Lactococcus lactis strains and are generally thought to have similar activities and chemical stability with the only difference being that Nisin A displays greater solubility at low pH2, 3. To our knowledge, this is the first report that nisin A is substantially more stable to UV exposure than nisin Z.
The percent reduction in the activity of pure nisin A upon UV exposure was found to depend on the peptide's concentration (Figure 2). Nisin A was more stable to UV when exposed at higher concentrations. A similar trend was obtained with commonly used nisin products such as Nisaplin® that contain very low weight percentages of nisin (2.5% nisin, 22.5% denatured milk solids)(data not shown).
Figure 2: Percent loss in ZOI area for nisin A exposed to UV relative to untreated nisin A.
In the secondary study, the effect of UV exposure on nisin A in the presence of varying concentrations of F127 was evaluated. Using a standard ZOI assay, it was found that mixtures of nisin with F127 produced ZOIs that were comparable to those from nisin only. Samples containing a mixture of F127 and nisin that were treated with UV resulted in smaller ZOIs with increasing F127 concentration. Control samples of UV treated nisin, showed a loss in ZOI area in comparison to untreated nisin, but the difference was not as great as that observed for solutions containing high concentrations of F127, indicating that the loss of activity observed in the mixed samples was due to an interaction with F127. Prior NMR studies indicate some crosslinking between the copolymers occurs with irradiation. Based on the results from the agar diffunsion assay, it was not clear if the nisin was entrapped within F127 structures that impaired its diffusion into the agar or possibly forming chemical crosslinks with F127 upon UV exposure.
Figure 3: Effect of F127 concentration on entrapment of Nisin A. (n=3, standard deviations less than 0.1)
In an effort to better understand the system, a similar experiment was conducted using a turbidometric assay (Table 1). UV treated mixtures of 2 mg/ml nisin A and F127 showed increasing MIC with increasing F127 concentration. At concentrations above 10% F127, the resulting MIC was above 100 µg/ml. In comparison, mixtures containing 20 mg/ml nisin and 20% F127 retained an MIC below 5 µg/ml upon UV exposure. This result is consistent with the prior finding that increasing nisin concentration results in higher stability to UV exposure. It also appears that at the higher nisin concentration of 20 mg/mL, it is possible to incorporate higher concentrations of F127 in conjunction with UV exposure without substantial losses in activity.
Exposure of nisin A to UV results in a loss of activity that depends primarily on the total dose of UV delivered. Although nisin A and nisin Z are nearly identical in structure, nisin A displayed substantially higher stability in the presence of UV compared with nisin Z. Nisin Z looses activity rapidly upon UV exposure and may not be suitable for applications involving UV processing. UV treatment of solutions containing 2 mg/ml nisin A resulted in increasing loss of activity with increasing F127 concentration. However, for gels containing 20 mg/ml nisin, the loss of activity appears to be insignificant, even when using higher concentrations of F127, such that gels retaining potent activity are feasible.
1. Mota-Meira, M., et al., “MICs of mutacin B-Ny266, nisin A, vancomycin, and oxacillin against bacterial pathogens.” Antimicrob Agents Chemother, 44 (1), pp 24-29 (Jan. 2000).
2. Rollema, H. S., et al., “Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering.” Appl Environ Microbiol, 61 (8), 2873-8 (Aug. 2000).
3. de Vos, W. M., et al., “Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.” Appl Environ Microbiol, 59 (1), 213-8. (Jan 1993).