(623bj) Effect of UV Curing On Peptide Antimicrobials and Considerations for Incorporation In Polymeric Coatings

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.

Methods

 

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 37^oC 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 37^oC 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.

Results

 

 

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 pH^{2, 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.

Conclusions

 

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.

References

 

 

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).