N-halamine biocidal materials with superior antimicrobial efficacies for wound dressings

Article abstract of DOI:10.3390/molecules22101582

This work demonstrated the successful application of N-halamine technology for wound dressings rendered antimicrobial by facile and inexpensive processes. Four N-halamine compounds, which possess different functional groups and chemistry, were synthesized. The N-halamine compounds, which contained oxidative chlorine, the source of antimicrobial activity, were impregnated into or coated onto standard non-antimicrobial wound dressings. N-halamine-employed wound dressings inactivated about 6 to 7 logs of Staphylococcus aureus and Pseudomonas aeruginosa bacteria in brief periods of contact time. Moreover, the N-halamine-modified wound dressings showed superior antimicrobial efficacies when compared to commercially available silver wound dressings. Zone of inhibition tests revealed that there was no significant leaching of the oxidative chlorine from the materials, and inactivation of bacteria occurred by direct contact. Shelf life stability tests showed that the dressings were stable to loss of oxidative chlorine when they were stored for 6 months in dark environmental conditions. They also remained stable under florescent lighting for up to 2 months of storage. They could be stored in opaque packaging to improve their shelf life stabilities. In vitro skin irritation testing was performed using a three-dimensional human reconstructed tissue model (EpiDerm). No potential skin irritation was observed. In vitro cytocompatibility was also evaluated. These results indicate that N-halamine wound dressings potentially can be employed to prevent infections, while at the same time improving the healing process by eliminating undesired bacterial growth.

Full text of DOI:10.3390/molecules22101582

molecules
Article
N-Halamine Biocidal Materials with Superior
Antimicrobial Efficacies for Wound Dressings
Buket Demir 1, Roy M. Broughton 2, Mingyu Qiao 3, Tung-Shi Huang 3 and S. D. Worley 1,
*
1
Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA;
bzd0003@tigermail.auburn.edu
Center for Polymers and Advanced Composites, Department of Mechanical Engineering,
2
Auburn University, Auburn, AL 36849, USA; brougrm@auburn.edu
Department of Poultry Science, Auburn University, Auburn, AL 36849, USA;
3
mzq0001@tigermail.auburn.edu (M.Q.); huangtu@auburn.edu (T.-S.H.)
*
Correspondence: worlesd@auburn.edu; Tel.: +1-334-844-6980
Received: 21 August 2017; Accepted: 20 September 2017; Published: 21 September 2017
Abstract: This work demonstrated the successful application of N-halamine technology for wound
dressings rendered antimicrobial by facile and inexpensive processes. Four N-halamine compounds,
which possess different functional groups and chemistry, were synthesized. The N-halamine
compounds, which contained oxidative chlorine, the source of antimicrobial activity, were
impregnated into or coated onto standard non-antimicrobial wound dressings. N-halamine-employed
wound dressings inactivated about 6 to 7 logs of Staphylococcus aureus and Pseudomonas aeruginosa
bacteria in brief periods of contact time. Moreover, the N-halamine-modified wound dressings
showed superior antimicrobial efficacies when compared to commercially available silver wound
dressings. Zone of inhibition tests revealed that there was no significant leaching of the oxidative
chlorine from the materials, and inactivation of bacteria occurred by direct contact. Shelf life stability
tests showed that the dressings were stable to loss of oxidative chlorine when they were stored for
6 months in dark environmental conditions. They also remained stable under florescent lighting for up
to 2 months of storage. They could be stored in opaque packaging to improve their shelf life stabilities.
In vitro skin irritation testing was performed using a three-dimensional human reconstructed tissue
model (EpiDerm™). No potential skin irritation was observed. In vitro cytocompatibility was also
evaluated. These results indicate that N-halamine wound dressings potentially can be employed to
prevent infections, while at the same time improving the healing process by eliminating undesired
bacterial growth.
Keywords: N-halamine; antimicrobial efficacies; medical wound dressings; antibacterial materials
1. Introduction
Wound care is of great importance, and it can be a burden for chronic wound treatments caused
by diseases such as ulcers, vascular and diabetes, and acute wounds such as burns and post surgical
wounds where the healing process is extended, or for wounds which fail to heal. As a result, infection
control becomes a major problem [ ]. These types of wounds can easily become infected and life
1
threating if not treated properly [2,3]. In addition to loss of function, decreased quality of life and
morbidity, costs associated with chronic wound care represent a significant burden to healthcare
systems worldwide and are expected to continue to rise. Using antibiotics along with dressings is a
common practice in wound treatments. However, there is an increasing concern in an incidence of
rise of antibiotic resistant microorganisms caused by indiscriminate usage of antibiotics, presenting
serious health concerns worldwide [
4–
6]. As a result, antiseptic treatments are becoming increasingly
necessary in controlling bacterial contamination in health care systems [
7]. Antimicrobial materials
Molecules 2017, 22, 1582; doi:10.3390/molecules22101582
www.mdpi.com/journal/molecules

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(biocides) have been used as antiseptic treatments for centuries and are regaining their importance
due to their advantages over antibiotics. In addition, unlike antibiotics, biocides are straight-forward
in use, available without prescription and less expensive than antibiotics [8].
Numerous antimicrobial agents have been developed and explored in an effort to inhibit the
growth of bacteria and to prevent infections in health care applications [
are metal ions, silver nanoparticles, biguanides, and quaternary ammonium compounds, which are
commonly used biocides for wound dressing applications [10 11]. However, technologies utilizing
9]. Among these agents
,
biocidal compounds, such as silver nanoparticles, triclosan and biguanides, that are known to cause
environmental and toxicity problems, have been employed recently [12]. High levels of exposure to
these agents raise health concerns. Research has shown that silver nanoparticles were released from
textiles during washing cycles and bio-accumulated into the environment, which elevated the exposure
of humans to silver metal [13
,14]. In addition to these toxicity issues, it has been well documented
that some biocides such as silver have shown bacterial resistance [15
,16]. Moreover, the antimicrobial
efficacy of silver dressings is generally poor. For example, silver-incorporated materials can require
about 24 h of contact time in order to deactivate pathogenic microorganisms [17]. It has also been
noted that rapid inactivation of bacteria is necessary for an ideal wound dressing in order to prevent
infections and control cross-contamination [2].
Since the discovery of the halogens chlorine and iodine, they have been used for centuries as
disinfectants. The main limitation of using the halogens as disinfectants is their stability. This problem
was solved with development of organic N-halamines. N-halamines consist of a diverse class of
biocides, which are characterized in the form of amines, amides or imides. These functional groups can
form covalent bonds with halogens where the halogen ion (chlorine, bromine or iodine) is stabilized as
oxidative halogen, which is biocidal.
Among other biocides used for antimicrobial applications, N-halamines offer many benefits.
For over three decades extensive work on N-halamine antimicrobial compounds has been progressing
in the Worley laboratories and elsewhere [18–46]. They are generally the most effective antimicrobial
materials due to their rapid inactivation efficacies against a broad spectrum of microorganisms
(Gram-positive and Gram-negative bacteria, yeasts, fungi and viruses) [20,31–35]. Depending on
the chemical structure, they have long-term halogen stability and antimicrobial functionality.
Their most unique characteristic is that once oxidative halogen inactivates the microorganisms and
is exhausted, they can be recharged and therefore can continue the inactivation of pathogens after
being recharged [33,36–38]. They are relatively inexpensive, and they are generally non-leaching
when they are covalently attached onto surfaces [39]. The mechanism of action of stable N-halamines
involves a direct contact of the N-halamine with the bacterial cell during which the oxidative halogen
is transferred to the cell. The molecule does not dissociate into free oxidative chlorine before transfer
to the cell [40]. In addition, it has been reported that the N-halamines do not show any evidence of
bacterial resistance [41].
N-halamines can be employed in a variety of applications. N-halamine molecules can be
incorporated into various substrates [21
,34], modified to produce desirable functional groups
that allow covalent attachment to surfaces by chemical bonds or ionic interactions [27
,
36,42–44],
impregnated into fibrous materials [28], polymerized onto surfaces or used as monomers or polymers
as disinfectants [18,20,43].
Today most wound dressings lack long term antimicrobial function, stability, and rapid
disinfection ability, and may have a risk of resistance to bacteria. There is a need for development
of new, inexpensive, biodegradable, non-leaching, less toxic, environmentally friendly antimicrobial
wound dressings. This could potentially be achieved when N-halamine compounds are incorporated
as biocides into the wound dressings. In an effort to address and overcome the challenges and
limitations, the current research has primarily focused on developing an ideal antimicrobial wound
dressing with use of N-halamine biocides. In this regard four different N-halamine compounds
have been synthesized and incorporated into wound dressing materials. Antimicrobial efficacies,

Molecules 2017, 22, 1582
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stabilities, skin sensitivities and cytotoxicities of the N-halamine-coated wound dressing materials
have been evaluated.
2. Results and Discussions
2.1. Characterization of the Synthesized Compounds
The methods applied in this work were straightforward and have been discussed in the
Materials and Methods section in detail. The 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC)
compound was synthesized by halogenation of its precursor 2,2,5,5-tetramethyl-1,3-imidazolidinone
(TMIO) (Scheme 1); the precursor was prepared as described previously [20]. Synthesis routes
of the precursor products are illustrated in Scheme 2. The 3-glycidyl-5,5-dimethylhydantoin
and 3-triethoxysilylpropyl-5,5-dimethylhydantoin were synthesized by the nucleophilic reaction
of 3-chloropropyltriethoxysilane and epichlorohydrin, respectively, with the potassium salt of
5,5-dimethylhydantoin. HASL copolymer was obtained by free radical polymerization of 2-acrylamido-
2-methyl-1-(5-methylhydantoinyl)propane (HA) and a siloxane functional methacrylate (SL). These
three precursors were rendered antimicrobial by exposure to dilute household bleach after they were
attached to wound dressings.
Scheme 1. 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC) synthesis route by chlorination of
2,2,5,5-tetramethyl-1,3-imidazolidinone (TMIO) precursor.
O
O
H3C
H3C
H3C
H3C
H
KOH
H2O
Cl
O
K
N
N
O
HN
N
25 °C
O
O
Hy-Ep
Scheme 2. Schematic synthesis routes of 2-acrylamido-2-methyl-1-(5-methylhydantoinyl)propane/siloxane
functional methacrylate (HASL) copolymer, BA-1 and Hy-Ep monomer precursors.

Molecules 2017, 22, 1582
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Information concerning the application of the N-halamine compound or its precursor onto
the wound dressings is crucial since the type of N–Cl bond is important for the chlorine stability,
and especially for the toxicity and skin sensitivity data. The structures of the N-halamine precursors
were confirmed by NMR and FTIR characterization. NMR data for the synthesized compounds have
been included in the Materials and Methods Section and Supplementary Materials.
2.2. Preparation and Characterization of the Coatings
Wound dressings were prepared by impregnating N-halamine or its precursor moieties into
or onto the wound dressings by coating processes. Structures of the synthesized N-halamine or
precursor compounds used for the wound dressing application are shown in Figure 1. In order to
obtain optimum chlorine loadings, wound dressings were treated in N-halamine or precursor coating
solutions prepared at different concentrations depending upon the N-halamine compound desired.
Due to the difference in the chemical structure and available N–H sites in each compound, the number
of bound chlorine atoms could differ. Therefore, the coating concentrations of HASL, BA-1, Hy-Ep
and MC were adjusted to obtain approximately the same number of chlorine atoms on each of the
N-halamine-treated wound dressings. After the coating and curing processes, three of the synthesized
N-halamine precursors of BA-1, HASL and Hy-Ep were covalently bound onto the wound dressings
through siloxane or epoxy moieties as represented in the precursor structures, respectively. BA-1,
HASL and Hy-Ep functionalized wound dressings were then rendered antimicrobial by reaction of the
N–H sites with free chlorine during a dilute bleach chlorination process. The MC compound included
the N-chloramine moiety prior to the impregnation process. It was not covalently bound to the fibrous
matrix of the wound dressing; rather, it was bound by electrostatic attraction, but it could not be
mechanically removed from the dressing. After the chlorination process, the presence of oxidative
chlorine bonded to the N-halamine precursors was confirmed by analytical titration of the N-halamine
treated samples. The weight percent of oxidative chlorine amount on the samples of the MC, BA-1-Cl,
Hy-Ep-Cl and HASL-Cl coated wound dressings to be used in the antimicrobial testing procedure
were measured to be 0.22 wt % Cl+, 0.21 wt % Cl+, 0.19 wt % Cl+ and 0.23 wt % Cl+, respectively.
Figure 1. Synthesized N-halamine compound or precursors.
Surface characterization of untreated controls, MC, BA-1, HASL and Hy-Ep-treated and
chlorinated-treated (BA-1-Cl, HASL-Cl, Hy-Ep-Cl) was performed by Fourier Transform Infrared
(FTIR) spectroscopy. FTIR spectra of N-halamine-treated and halogen-activated wound dressings
are shown in Figure 2a. In this spectra data collected from N-halamine wound dressings after the
chlorination process can be compared to the data collected from the untreated native wound dressing
(A). MC, Hy-Ep-Cl, BA-1-Cl and HASL-Cl-treated wound dressing spectra are demonstrated as B, C,

Molecules 2017, 22, 1582
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D, E, respectively. The characteristic carbonyl stretching bands of the hydantoin ring (imide and amide
groups) of the compounds were observed at about 1777 and 1712 cm−1. The band at 1673 cm−1
of the halogen-activated N-halamine dressing (B) was suggestive that the MC compound was
successfully impregnated into the dressing (an additional spectrum of the MC compound is included
in Supplementary Materials Figure S5). Similar to the other N-halamine treated dressings, the bands
due to the hydantoin ring were also present in the FTIR spectra of HASL and HASL-Cl (Figure 2b).
After chlorination, a slight shift to a higher wavenumber of the vibrational stretching bands of the
hydantoin carbonyl groups of HASL-Cl was observed due to transformation of N–H bonds present
in HASL to N–Cl bonds. This data showed that the HASL polymer was bound to the fibrous matrix
and remained bound after the chlorination process. In addition, no new bands appeared after the
halogenation process when compared to the spectrum of the untreated dressing, indicating that the
halogenation process did not affect the structure of the native dressing. Similar results to those seen in
Figure 2b for the HASL dressings were observed for the other Hy-Ep and BA-1 coated dressings for
which the transformation of the N–H bond to an N–Cl bond was also detected.
A
B
100
98
1676
Untreated
HASL
C
D
1673
96
1771
1777
1714
94
92 HASL-Cl
E
90
2978
2889
1777
88
86
84
82
2978
2889
2938
1708
1712
1712
80
3600 3200 2800 1800160014001200
Wavenumbers (cm-1)
3000
2800
1800
1600
Wavenumbers cm-1
(a)
(b)
Figure 2. Fourier Transform Infrared (FTIR) specta of untreated and N-halamine treated wound
dressings. ( ) FTIR spectra of A: untreated, B: MC-treated, C: Hy-Ep-Cl-treated, D: BA-1-Cl-treated
a
and E: HASL-Cl-treated dressings (spectra taken after chlorination of Hy-Ep, BA-1 and HASL-treated
wound dressings); (b) FTIR spectra of untreated, HASL-treated and HASL-Cl-treated dressings.
2.3. Shelf Life Stability
Shelf life (storage) stability of the BA-1-Cl, HASL-Cl, Hy-Ep-Cl and MC-treated wound dressings
are shown in Table 1. Bound chlorine in MC-treated dressings remained for 24 weeks of storage in
a dark environment. BA-1-Cl-treated dressings were stored in dark environmental conditions and
retained most of their initial chlorine loadings for 10 weeks. However, HASL-Cl and Hy-Ep-Cl-treated
dressings were somewhat less stable than MC and BA-1-Cl-treated dressings. BA-1-Cl-treated dressings
lost only 24% of chlorine after 6 months of storage in darkness. It was observed that Hy-Ep-Cl and
HASL-Cl-treated dressings retained 65% and 58% of their chlorine amounts after 16 weeks of storage
in a dark environment. The variation in the chlorine loading data for each of the single N-halamine
treatments shown in Table 1 could be attributed to different fiber and structural uniformity of the
commercial wound dressing samples used. Therefore the bonded chlorine amount could slightly differ
from sample to sample. However, the chlorine bond stability changed between N-halamine treated
samples, MC being the most stable of the four N-halamines. There was no significant change or loss in
bound chlorine when MC-treated samples were stored in darkness. These results revealed that the
MC-treated wound dressing samples had excellent storage stability and did not show any significant
loss of oxidative chlorine loading during storage.

Molecules 2017, 22, 1582
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Halogen stability differs according to N–Cl chemistry. The MC compound showed the
highest stability because of its N-chloramine chemistry (the Cl is bonded to an amine N); whereas,
Hy-Ep-Cl, BA-1-Cl and HASL-Cl include N-chloramide and N-chlorimide moieties for which the N–Cl
dissociation constants are higher. As a result, dressings coated with these N-halamines showed some
less stability over time.
It should also be noted that the N–Cl bond dissociation increased with exposure to UVA photons.
When stored under florescent light, MC samples lost only 32% of the oxidative chlorine over 24 weeks
of storage. Compared to previous UVA light stability data for N-halamine compounds, it can be
concluded that the MC compound is the most stable N-halamine compound yet developed in these
laboratories [20].
Table 1. Storage stability of MC, BA-1-Cl, HASL-Cl, and Hy-Ep-Cl under dark and fluorescent
light conditions.
Time (Weeks)
* MC (Cl+ %)
Dark Light
0.20 0.22
* BA-1 (Cl+ %)
Dark
* HASL (Cl+ %)
Dark
* Hy-Ep (Cl+ %)
Dark
0
1
2
3
4
5
6
7
8
0.21
0.20
0.21
0.22
0.20
0.24
0.18
0.23
0.20
0.18
0.19
0.18
0.17
0.17
0.17
0.18
0.19
ND
ND
ND
ND
ND
ND
0.16
0.19
0.18
0.18
0.15
0.16
0.14
0.13
0.14
0.14
0.14
0.16
0.14
ND
0.13
0.12
0.11
ND
ND
ND
ND
ND
ND
ND
0.07
0.23
0.20
0.18
0.14
0.15
ND
ND
ND
0.17
ND
0.14
0.13
ND
ND
0.15
0.15
0.13
ND
ND
ND
ND
ND
ND
0.06
0.19
0.18
0.20
0.21
0.19
0.20
0.19
0.19
0.20
0.19
ND
ND
ND
ND
ND
0.18
0.21
0.18
0.17
0.19
0.21
0.19
0.21
0.22
0.22
0.20
0.20
0.20
0.20
0.19
0.19
ND
0.18
ND
ND
ND
ND
0.13
0.13
0.13
0.14
0.16
0.16
0.13
0.15
0.15
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
* The average deviation in the measured Cl+ loading was
±
0.03%. Each datum represents an average of three
samples. Chlorine loadings are reported in wt % Cl+. ND represents no determination.
2.4. Antimicrobial Efficacy Testing
In both types of the antimicrobial tests (antimicrobial efficacy and zone of inhibition) two species
of bacteria, Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Pseudomonas aeruginosa
ATCC 27853, were employed. Precursor-treated (Hy-Ep, BA-1, HASL) and untreated native dressings
were used as controls. Those control samples, N-halamine-treated ones (Hy-Ep-Cl, BA-1-Cl, HASL-Cl
and MC) and commercial antimicrobial (silver alginate and polybiagunide (PHMB)) dressings were
evaluated for antimicrobial efficacy. Each test was repeated at least twice on different days, and the
results are shown below in Tables 2–6.
As can be seen, all of the compounds showed efficacy against the two bacteria (S. aureus and
P. aeruginosa) at populations of about 106 to 107 CFU (colony forming units). Untreated native and
un-chlorinated-precursor-treated (control) samples exhibited much lower reductions, even after 60 min
of contact time (Tables 2–5). These reductions were due to adherence of live bacteria to the fibers,

Molecules 2017, 22, 1582
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not to inactivation of bacteria. MC-treated dressings appear to be superior as they inactivated 6 logs
of S. aureus in about a maximum of 15 min and inactivated 5.7 to 7.4 logs of P. aeruginosa in about a
maximum of 30 min of contact time (Table 2). In contrast to the MC results, after 60 min of contact
time, BA-1-Cl-treated samples exhibited a complete inactivation of about 6 logs of S. aureus and
P. aeruginosa (Table 3). The log reduction for the BA-1-Cl-treated samples varied from experiment to
experiment, but it is not unusual to observe such inconsistencies in repeated experiments, especially
when a larger inoculum of bacteria (107 CFU) was used. Hy-Ep-Cl-treated dressings exhibited similar
results to BA-1-Cl coated samples; the samples provided about 6 log reductions against S. aureus
and P. aeruginosa in 60 min of contact time (Table 4). HASL-Cl-treated samples provided complete
kill of 6 logs of S. aureus in 15 min. However, in one of the experiments complete inactivation of
S. aureus was observed after 30 min of contact time. Similar to MC-treated samples, only 30 min of
contact time was required to inactivate 6 logs of P. aeruginosa (Table 5). The variability in the data is as
expected due to the difficulty of performing the experiments. One bacterial CFU trapped in the wound
dressing away from any chlorine can lead to these observations. It can be concluded from these results
that all of the N-halamine compounds could be effective antimicrobial materials in wound dressings.
In contrast, for a commercial silver alginate dressing we obtained less than 1 log of inactivation for
both bacterial species in 30 min of contact using the same experimental sandwich test. PHMB-treated
dressings showed poor inactivation efficacy against Gram-negative P. aeruginosa compared to all four
N-halamine-treated dressings. Only in one experiment were they able to inactivate about 7 logs of
S. aureus in 15 min (Table 6). It was observed that N-halamine dressings showed rapid inactivation
and superior antimicrobial efficacy when compared to silver and biguanide dressings.
Table 2. Biocidal efficacy of MC-treated wound dressings.
Contact
Time (min)
Log Reduction *
Exp 1
Log Reduction *
Exp 2
Log Reduction *
Exp 3
Samples
Inoculum
6.07
7.36
6.05
6.11
6.36
5.70
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
Untreated
dressing
(control)
60
0.18
2.13
0.04
1.26
0.20
0.98
5
6.07
6.07
6.07
6.07
2.06
5.24
7.36
7.36
6.05
6.05
6.05
6.05
0.28
2.30
6.11
6.11
2.03
6.36
6.36
6.36
0.89
2.97
5.70
5.70
MC-treated
dressing
15
30
60
Cl+ = 0.23 wt %
* Data represent three different experiments each being performed on different days with different inoculum
concentrations. Average Cl+ % for all three experiments was measured as 0.23.
Table 3. Biocidal efficacy of BA-1-Cl coated wound dressings.
Contact
Time (min)
Log Reduction *
Exp 1
Log Reduction *
Exp 2
Log Reduction *
Exp 3
Samples
Inoculum
6.07
7.36
6.05
6.11
6.36
5.70
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
Untreated
dressing
(control)
60
0.05
0.62
0.31
1.26
0.006
0.91
5
0.56
3.64
3.94
3.54
0.98
1.46
1.79
4.94
3.45
2.78
3.45
3.08
0.71
6.11
6.11
6.11
0.032
2.53
6.36
6.36
0.029
0.494
2.57
BA-1-Cl-treated
dressing
15
30
60
Cl+ = 0.21 wt %
5.70
* Data represent three different experiments each being performed on different days with different inoculum
concentrations. Average Cl+ % for all three experiments was measured as 0.21.

Molecules 2017, 22, 1582
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Table 4. Biocidal efficacy of Hy-Ep-Cl coated wound dressings.
Contact
Time (min)
Log Reduction *
Exp 1
Log Reduction *
Exp 2
Log Reduction *
Exp 3
Samples
Inoculum
6.07
7.36
6.05
6.11
6.00
6.22
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
Untreated
dressing
(control)
60
0.19
2.33
0.65
3.69
0.17
0.68
5
0.33
1.25
2.45
6.07
0.91
1.16
1.46
4.06
0.10
2.81
2.14
3.08
0.13
0.23
2.39
6.11
0.79
0.70
6.00
6.00
0.02
0.06
0.55
6.22
Hy-Ep-Cl-treated
dressing
15
30
60
Cl+ = 0.20 wt %
* Data represent three different experiments each being performed on different days with different inoculum
concentrations. Average Cl+ % for all three experiments was measured as 0.20.
Table 5. Biocidal efficacy of HASL-Cl coated wound dressings.
Contact
Time (min)
Log Reduction *
Exp 1
Log Reduction *
Exp 2
Log Reduction *
Exp 3
Samples
Inoculum
6.05
6.11
6.00
6.22
6.36
5.70
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
Untreated
dressing
(control)
60
0.02
1.42
0.04
1.26
0.20
0.98
5
2.97
6.05
6.05
6.05
3.99
6.11
6.11
6.11
1.34
3.27
6.00
6.00
0.76
2.89
6.22
6.22
2.03
6.36
6.36
6.36
0.89
2.97
5.70
5.70
HASL-Cl-treated
dressing
15
30
60
Cl+ = 0.23 wt %
* Data represents three different experiments each being performed on different days with different inoculum
concentrations. Average Cl+ % for all three experiments was measured as 0.23.
Table 6. Biocidal efficacy of commercial silver alginate and polybiguanide (PHMB) wound dressings.
Contact
Time (min)
Log Reduction *
Exp 1
Log Reduction *
Exp 2
Samples
Inoculum
7.11
7.09
7.39
7.07
S. aureus
P. aeruginosa
S. aureus
P. aeruginosa
Untreated dressing
(control)
30
0.23
0.62
0.08
0.17
1
5
15
30
0.22
0.14
0.29
0.36
0.13
0.18
0.23
0.31
0.11
0.15
0.50
0.98
0.12
0.31
0.42
1.14
Silver alginate-treated
dressing
1
5
15
30
1.59
3.32
3.31
3.63
1.62
2.05
2.22
3.45
4.66
4.78
7.39
7.39
0.41
1.31
2.97
2.58
PHMB-treated dressing
* Data represents two different experiments being performed on different days with different inoculum populations.
2.5. Zone of Inhibition Test
In this test, as for the sandwich test, two species of bacteria, Gram-positive Staphylococcus aureus
ATCC 6538 and Gram-negative Pseudomonas aeruginosa ATCC 27853, were employed to determine
the zone of inhibition. Un-chlorinated precursor-treated (Hy-Ep, BA-1, HASL) and untreated native
samples were used as controls, and N-halamine-treated ones (Hy-Ep-Cl, BA-1-Cl, HASL-Cl and MC)

Molecules 2017, 22, 1582
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were used as test samples against the bacteria. In contrast to the sandwich antimicrobial efficacy test,
zone of inhibition is a qualitative test method. A zone of inhibition occurs when a material prevents
the growth of bacteria. The diameter of the zone determines the antimicrobial property when the
biocidal agent is released from the treated surface. Therefore, a clear zone where bacteria growth was
prevented can be only observed when active compound of the biocidal agent leaches out from the
test materials. It can be concluded from zone of inhibition testing (Kirby-Bauer testing technique)
for the three compounds (MC, BA-1-Cl and Hy-Ep-Cl) that there was no significant leaching of the
compounds or oxidative chlorine from the wound dressing samples, as no halo was observed around
the tested disks (Supplementary Materials, Figure S6). However, a slight zone was observed around
the edges of the HASL-Cl-coated disks. This could be attributed to less stability of the aliphatic amide
N–Cl bonds represented in the HASL-Cl compound. Zone of inhibition results revealed that strong
N–Cl bonding occurred and suggested that the killing mechanism of the N-halamine compounds
occurred by direct contact of the N–Cl group with bacteria on the wound dressing. Thus leaching of
the antimicrobial from the wound dressing should not create a regulatory problem.
2.6. Cytotoxicity of N-Halamine-Treated Wound Dressings
The effect of precursor N-halamine-coated and N-halogen-activated wound dressings on cell
survivability on the fibroblast (NIH-3T3) cells was evaluated (Figure 3). Cell viability % was calculated
with respect to untreated control test samples. A 5% sodium lauryl sulfate solution (SLS) was used as
the positive control, which was presumed to have high toxicity levels. Only 1% or less cell viability
was observed indicating the toxicity effects of SLS to cells. Un-chlorinated samples (Hy-Ep, BA-1
and HASL) showed an average of more than 80% of cell viability. However, N-halamine-treated
(MC, HyEp-Cl, BA-1-Cl, HASl-Cl) samples showed 60% or less cell viability. Compared to N-halamine
dressings, commercial silver (Ag) and polybiagunide (PHMB) wound dressings showed only 24% and
29% cell viability, respectively, which corresponds to their toxicity to cells. These results suggested
that N-halamine compounds do not significantly inhibit the cell viability; however, some Cl+ could
dissociate from the N-halogonated samples into the culture medium over an extended period of time
and could interfere with the cell viability. Decreasing the percentage of the Cl+ in dressings and test
medium should minimize the effects. This could be achieved without affecting the antimicrobial
efficacy of the dressings. It has been previously reported that 0.04 wt % Cl+ loading would be sufficient
for antimicrobial efficacy [28,39].
Figure 3. Cell viability analysis (MTT assay) on mouse fibroblast (NIH-3T3) of N-halamine
precursor-coated and N-halamine-coated, and commercial silver alginate (Ag) and polybiguanide
(PHMB) wound dressing test specimens.
2.7. In Vitro Skin Irritation
In vitro skin irritation of the MC-impregnated wound dressings was evaluated using a
reconstructed human epidermal (RHE) model (ISO10993-10). Compared to all of the other

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N-halamine-treated dressings, MC-treated samples showed the highest efficacy and chlorine stability.
Due to the results obtained from previous tests and the limitations in number of samples available for
this test, MC-treated dressings were selected as test samples for skin irritation evaluation. For each
chemical treated epidermis, % viability was expressed as [(ODsample
−
ODblank)/(ODcontrol
−
ODblank)]
×
100. The irritation potential of the MC compound was predicted to be a non-irritant, where 100% of
skin epidermis cells survived (Figure 4). In contrast, the positive control (5% sodium lauryl sulfate
solution) caused destruction of cells resulting in no epidermis cell viability. As expected, native
untreated sample and the negative control (D-PBS buffer solution) showed no irritation potential
where 100% cell viability was observed, similar to MC-treated epidermis. It has been reported as
a guideline that compounds are considered as non-irritant when the cell viability is more than 50%
relative to the negative control. Hence, the results demonstrated that this was the case for N-halamine
monomer MC-treated wound dressings.
Figure 4. Skin irritation of PC (positive control), NC (negative control), MC-treated wound dressing
specimen and untreated wound dressing specimen tested in an in vitro reconstructed human epidermis
prediction model EpiDerm™.
3. Materials and Methods
3.1. Materials and Instrumentation
5,5-dimethylhydantoin, 3-chloropropyltriethoxysilane, 2-acrylamido-2-methyl-4-pentanone,
epichlorohydrin, potassium cyanide, ammonium carbonate and most of the other chemicals were
purchased from Acros Organics (Morris Plains, NJ, USA) and VWR Inc. (Radnor, PA, USA).
All chemicals were used without further purification. Band-Aid® brand non-antimicrobial wound
dressing materials were obtained from Johnson & Johnson Co. (Skillman, NJ, USA). CVS/pharmacyTM
brand antimicrobial sterile silver alginate dressings and antibacterial sterile PHMB gauze pads
(dressings) were purchased from CVS Pharmacy Inc. (Woonsocket, RI, USA). Clorox® brand
(Clorox, Inc., Oakland, CA, USA) household bleach (8.25% of NaOCl) was used for the chlorination
process. Bacterial cultures of S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 were purchased
from American Type Culture Collection (Rockville, MD, USA), and Trypticase soy agar was
obtained from Difco Laboratories (Detroit, MI, USA). Newborn calf serum (NCS) was purchased
from HyClone Laboratories (South Logan, UT, USA). Dulbecco’s modification of eagle’s medium
(DMEM) without L-glutamine (4.5 mg/L glucose) was obtained from Lonza Inc. (Walkerville, MD,
USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from
Alfa Aesar (Ward Hill, MA, USA). Mouse embryonic fibroblast cells BALB/c 3T3 clone A31
(
ATCC® CCL163™) were purchased from the American Type Culture Collection (ATCC; Manassas,
VA, USA). A reconstructed human epidermal model EpiDerm
MatTek Corporation (Ashland, MA, USA).
™ kit (EPI-200-SIT) was purchased from
NMR spectra were obtained using a Bruker Avance 400 MHz spectrometer (Bruker, Inc., Billerica,
MA, USA); 1H-NMR spectra were recorded with 16 scans. FTIR data were obtained with a Model
Spectrum 400 (Perkin Elmer Co., Waltham, MA, USA) and recorded with 32 scans at 4 cm−1 resolution.

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3.2. Synthesis of N-Halamine Compounds
3.2.1. Synthesis of 3-Glycidyl-5,5-dimethylhydantoin (Hy-Ep)
3-Glycidyl-5,5-dimethylhydantoin was prepared according to
a
procedure outlined
previously [34,35]
.
Briefly, 0.05 mol of 5,5-dimethylhydantoin was converted into its sodium
salt by stirring in an equimolar quantity of NaOH in aqueous solution at ambient temperature
for 10 min. Hy-Ep was synthesized by subsequent addition of epichlorohydrin (0.05 mol) into the
mixture and stirring at ambient temperature for 10 h. After reaction, water was removed by vacuum
evaporation, and the hydantoin epoxide derivative was dissolved in acetone. Byproduct sodium
chloride was removed by filtration. The final product was obtained after removing acetone. The yield
obtained was 50% after purification. For analytical testing the hydantoin epoxide derivative could
1
further be purified by column chromatography for spectroscopic analysis. H-NMR (d-CDCl3):
δ 1.43
(s, 6H), 2.87–2.97 (m, 2H), 3.24 (m, 1H), 3.36–3.59 (m, 2H), 4.16 (s, 1H) (Figure S1 in Supplementary
Materials). However, the purification process was not necessary for coating onto the fibrous materials
(wound dressings) used in this study. After coating and covalent attachment onto the surface,
unreacted precursor materials were washed off by an extensive cleaning and washing procedure;
therefore, the crude product was used for coating onto the wound dressing material.
3.2.2. Synthesis of 3-Triethoxysilylpropyl-5,5-dimethylhydantoin (BA-1)
3-Triethoxysilylpropyl-5,5-dimethylhydantoin (BA-1) was prepared according to the procedure
outlined in [39,45]. Equimolar amounts of 5,5-dimethylhydantoin and KOH were dissolved in
100 mL ethanol. The mixture was heated at reflux until the solution became clear for about 10 min.
After removal of the ethanol and water by evaporation of the solvents under reduced pressure,
◦
the potassium salt of 5,5-dimethylhydantoin was isolated. The collected salt was further dried at 60 C
for 4 days and obtained at 90% yield. Then, the dry potassium salt of 5,5-dimethylhydantoin was
reacted with an equivalent amount of 3-chloropropyltriethoxysilane in 100 mL dimethyl formamide
(DMF) at 95 ◦C for 4 h. The KCl produced in the reaction was removed by filtration, and the DMF
solvent was removed by vacuum distillation. Finally BA-1 was obtained as a viscous oil liquid and
1
identified as the desired precursor monomer. H-NMR (d-CDCl3):
δ 0.41 (2H), 1.02 (9H), 1.23 (6H),
1.53 (2H), 2.71–2.79 (2H), 3.63 (6H), 7.29 (1H) (Figure S2 in Supplementary Materials).
3.2.3. Synthesis of 1-Chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC)
The synthesis of MC compound has been described elsewhere [20]. Briefly, 2,2,5,5-tetramethyl-4-
imidazolidinone (14.2 g, 0.1 mol) was dissolved in 100 mL of 1 N sodium hydroxide solution (0.1 mol).
◦
The mixture was stirred at 10 C, and chlorine gas was bubbled into the solution until the pH
reached 7. The precipitated white solid product was obtained by filtration and was recrystallized
from a hexane/ether mixture. 1H-NMR (d-CDCl3):
Supplementary Materials).
δ 1.34 (6H), 1.46 (6H), 7.85 (1H) (Figure S3 in
3.2.4. Synthesis of 2-Acrylamido-2-methyl-1-(5-methylhydantoinyl)propane (HA) and Hydantoin
Acrylamide Siloxane Copolymer (HASL)
In order to synthesize HASL copolymer, first the precursor HA monomer was prepared according
to a method outlined previously [33]. The Bucherer–Berg reaction was used for the synthesis of the HA
monomer in which the hydantoin ring formed. A similar approach, forming a hydantoin ring from
a ketone moiety, was previously used to synthesize N-halamine monomers [46] and polymers [31].
Briefly, 2-acrylamido-2-methyl-1-(5-methylhydantoinyl) propane (HA) was synthesized by reacting
2-acrylamido-2-methyl-4-pentanone, potassium cyanide, and ammonium carbonate in a 1:2:6 molar
ratio in a water/ethanol (1:1 by volume) solvent mixture in a round flask at room temperature for
5 days. After evaporation of ethanol, the crude product was isolated by adding dilute HCl and
collected as a white powder after filtration. HA precursor was recrystallized in acetonitrile and dried

Molecules 2017, 22, 1582
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at 45 ◦C for 24 h prior to the HASL copolymer reaction. The molecular weight of HA was measured
by mass spectrometry to be 239.128 g/mol, in accord with the calculated molecular weight for HA of
239.127 g/mol [32]. HA has a melting point of 178 ◦C.
The hydantoin acrylamide siloxane copolymer (HASL) was synthesized by free radical
polymerization. 10 mmol of HA and of 10 mmol of 3-(trimethoxysilyl)propyl methacrylate (SL)
were dissolved in 15 mL methanol, and 0.05 g AIBN (2,2-Azobis-2-methylpropionitrile) was added into
the mixture. After nitrogen was bubbled into the mixture for 15 min to remove any dissolved oxygen,
the mixture was heated to 65 ◦C, and reaction was continued for 2 h. After evaporation of the solvent,
1
the copolymer was obtained as white solid. H-NMR (DMSO-d6):
δ 0.6 (2H), 0.91 (3H), 1.25 (8H), 1.61
(1H), 1.88 (2H), 2.12 (2H), 2.17 (2H), 2.51 (2H), 3.48 (9H), 4.02–4.13 (2H), 7.59 (1H), 7.83 (1H), 10.59 (1H)
(Figure S4 in Supplementary Materials). The HA mole fraction in the copolymer was calculated by
comparing the signal area of total methyl group protons (Figure S4, 3.6 ppm) attached to the silicon
atom in SL (-Si-(OCH3)3) to the imide proton signal area (Figure S4, 10.6 ppm) of the HA moiety. The
reactivity ratio of mHA/(mHA + mSL) was calculated as 0.40. Thus the reactivity of SL was found to
be slightly higher than that of HA when the feed ratio of monomers was 1:1. The reactivity of the
monomers was observed to be similar to that reported previously [32]. In previous studies the intrinsic
viscosity of the HASL copolymer (1:1 feed ratio) was reported to be 0.55 dL/g (in 2-methoxyethanol at
25 ◦C) [32].
3.3. Preparation of N-Halamine-Modified Wound Dressings
Synthesized BA-1 was dissolved in EtOH/H2O (1/1 w/w) at a concentration of 7.5 wt %. Wound
dressings in the size of 10 cm
uniformly padded through a laboratory wringer (Birch Brothers Southern, Waxhaw, NC, USA). BA-1
coated dressings were then cured at 95 C for 40 min and at 145 C for 20 min. After curing they were
soaked in 300 mL 0.25 (w/v) % AATCC detergent aqueous solution for 15 min, washed with copious
amount of tap water and finally rinsed several times with DI water. Coated dressings were allowed to
dry completely at 45 ◦C for 3–4 h before chlorination.
×
10 cm were soaked in the coating solution for 15 min and then
◦
◦
Wound dressings were coated with 5 wt % of Hy-Ep solutions prepared in acetone/H2O (1/1 w/w).
As in the previous method, samples were uniformly padded through a laboratory wringer after soaking
◦
◦
in Hy-Ep solutions for 15 min. Hy-Ep coated pads were then cured at 95 C for 1 h and then at 145 C
for 20 min. Cured samples were washed and rinsed in detergent solution in the same manner as above
in order to remove the unreacted compounds.
1-Chloro-2,2,5,5-tetramethyl-4-imidazolidi-none (MC) was used as an antimicrobial coating for
the wound dressings. MC (at 1 wt %) was dissolved in ethanol solution at room temperature, and then
the dressings were soaked in the coating solution for 15 min. The samples were padded through a
laboratory wringer (Birch Brothers Southern, Waxhaw, NC, USA). MC-coated dressings were dried at
room temperature for 24 h. Alternatively, they could also be dried at 45 ◦C for 2 h. In contrast to the
other coating processes described here for the other N-halamine compounds, MC compound used
for the coating solutions was chlorinated during the synthesis of the MC; therefore, after the coating
process, no extra chlorination was necessary.
Wound dressings were coated in 3 wt % HASL copolymer solutions, which were prepar◦ed
in EtOH/water (3/1 w/w). HASL copolymer-coated wound dressings were then cured at 145 C
for 1 h. After curing, the HASL-coated samples were washed and dried prior to chlorination as
described above.
3.4. Chlorination Procedure
Precursor N-halamine-treated wound dressings were rendered antimicrobial by a chlorination
process. Samples were chlorinated in dilute household bleach (10 v/v % of 8.25% commercial sodium
hypochlorite solution) at pH 7 at room temperature for 30 min. Chlorinated samples were then washed
thoroughly with distilled water and dried at 45 ◦C for 2 h to remove free chlorine from the surface

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of the wound dressings. The oxidative chlorine loadings on the dressings were determined by an
iodometric/thiosulfate titration procedure. The weight percentage of the bound oxidative chlorine
was calculated according to following formula
35.45 × N × V
Cl+% =
× 100,
(1)
2 × W
where Cl+ % is the weight percent of oxidative chlorine on the samples, N and V are the normality
(equiv/L) and volume (L) of the titrant (Na2S2O3), respectively, and W is the weight of the sample (g)
used for the titration.
3.5. Shelf Life Stability Testing
The storage or shelf life stability of the oxidative chlorine bound onto the wound dressings by
the chlorination procedure was evaluated. Wound dressings were stored in sealed opaque packaging
in a cabinet (dark environment) at room temperature. The stability of the chlorine content over
time was measured for up to 24 weeks. The stabilities of the N-halamine-coated dressings were
determined by measuring the amount of remaining chlorine on the samples by using the standard
iodometric/thiosulfate titration procedure as discussed above.
3.6. Antimicrobial Efficacy Testing
Two types of tests were conducted in order to determine the biocidal efficacies of the
N-halamine-coated wound dressings. Staphylococcus aureus (S. aureus, ATCC 6538) was used as a
Gram-positive bacterium and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) was used as a
Gram-negative bacterium in order to challenge the un-chlorinated and chlorinated-coated wound
dressings and commercial silver alginate and PHMB dressings.
In the first method, a “sandwich test” was used [39]. In this procedure, both S. aureus and
P. aeruginosa were suspended in 100
population (colony forming units, CFU). Then, an aliquot of 25
the center of a swatch, at size of 2.54 cm 2.54 cm, and a second identical swatch was placed on
µM phosphate buffer (pH 7) to produce a suspension of known
µL of this suspension was placed in
×
top. Both swatches were covered by a sterile weight to ensure a good contact with the bacteria. After
predetermined contact times, samples were quenched by 5.0 mL of sterile 0.02 N sodium thiosulfate
solutions to neutralize the oxidative chlorine and thus terminate the disinfection action. Samples were
vortexed for 2 min, and then serial dilutions were prepared using pH 7, 100
µ
M phosphate buffer
◦
solutions which were plated on trypticase soy agar plates. After the plates were incubated at 37 C for
24 h, viable bacterial colonies were counted for the biocidal efficacy analysis. All experiments were
performed at least twice (on different days) using different bacterial inocula.
3.7. Zone of Inhibition Antibacterial Test
Zone of inhibition of the N-halamine-coated wound dressings were evaluated by the Kirby-Bauer
testing technique. A modified version of the American Association of Textile Chemist and Colorist
(AATCC 147) method was applied, and the samples were tested against S. aureus and P. aeruginosa.
In this method S. aureus and P. aeruginosa were suspended in 100
µ
M phosphate buffer (pH 7) to
produce a suspension of known population (approximately 107 CFU). Then, an aliquot of 100
µL of this
suspension was plated on trypticase soy agar plates. N-halamine-coated wound dressings were cut
into 0.79 cm2 disks and placed onto the agar plates and gently pressed to ensure full contact. Control
disks were prepared in the same manner and placed onto agar plates. The plates were incubated at
◦
37 C for 24 h. After the plates were incubated, the formed zones of inhibition (if any) around the disks
were measured. Two replicates of the samples were examined.

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3.8. Cytotoxicity of N-Halamine-Treated Wound Dressings
In vitro cytocompatibilities of N-halamine-treated dressings as well as commercial silver alginate
and PHMB dressings were evaluated. The influence of the antimicrobial compounds on cell growth
and viability was examined after 24 h incubation in the presence of antimicrobial N-halamine-treated
and antimicrobial silver and PHMB commercial dressings by a direct contact test. For this test NIH 3T3
◦
mouse fibroblast cells were cultured in DMEM media with 10% NCS and 1% Pen/Strep at 37 C and 5%
CO2. All samples were cut into disks (0.32 cm2) to fit into 96-well tissue culture plates. Prior to testing,
the disk samples were sterilized by UV exposure for 15 min. Untreated wound dressing samples
were used as negative controls. Unchlorinated precursor-treated and chlorinated N-halamine-treated
materials were used as test samples. Individual specimens of the test samples were placed on the
bottoms of the 96-well plates. Then, 104 of NIH-3T3 mouse fibroblast cells were seeded onto each
specimen. After 24 h of contact time for cell attachment, MTT assays were performed to quantify
the cell viability using a micro-plate reader. Optical density values of the viable cell medium were
recorded at 540 nm. Cell culture plates (TCPS) and untreated samples were used as controls. The cell
viability was determined as the percentage compared to the untreated sample controls. The test was
repeated twice with a total of 12 replicates recorded for each treatment. The results were presented as
means ± standard errors.
3.9. Skin Irritation Testing
In vitro skin irritation of the MC-impregnated wound dressings was tested using the EpiDerm
™
reconstructed human epidermal (RHE) model (ISO10993-10). A reconstructed human epidermal model
EpiDerm™ kit (EPI-200-SIT) was purchased from MatTek Corporation (Ashland, MA, USA), and the
Epiderm
™ skin tissue inserts were transferred into 6-well plates and were conditioned overnight at
◦
37 C, 1% CO2, 95% RH (relative humidity), following the instructions of the manufacturer. Untreated
and MC-impregnated dressings were cut into 0.32 cm2 disks, and two layers of each sample were
placed on top of the EpiDerm
Saline) was added onto the test specimens to moisten the skin tissue surface as described in the
protocol for solid samples. 30 L of D-PBS were used as a negative control, and 5% SLS (sodium lauryl
sulfate) solution was used as a positive control. Three EpiDerm skin tissues were used for each test
material (untreated and MC-treated samples), as well as for the positive control and a negative control.
After 60 min exposure, the test samples were discarded, and the EpiDerm skin tissues were rinsed
15 times with D-PBS and washed with copious amount of saline solution. After 42 h of post-incubation,
the EpiDerm™ skin tissue inserts were transferred into a 24-well plate prefilled with 300 L of MTT
™ skin tissue. Then, 25 µL of D-PBS (Dulbecco’s Phosphate Buffer
µ
™
™
µ
[3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium] (1 mg/mL) and incubated for 3 h. Then MTT
medium was discarded, and the tissue inserts were extracted with 2 mL of isopropyl alcohol. Then
200 µL of the MTT extracts were transferred into 96-well plates, and the optical density (OD) of the
extract was measured at a wavelength of 540 nm using a microplate reader. For each chemical-treated
epidermis, the % viability was expressed as [(ODsample − ODblank)/(ODcontrol − ODblank)] × 100.
4. Conclusions
This study has revealed that N-halamine chemistry offers significant potential for producing an
antimicrobial wound dressing. Hy-Ep and BA-1 monomer precursors, HASL polymer precursor and
MC compound were successfully coated onto or impregnated into (in the case of MC) wound dressings,
and these N-halamine-treated dressings were rendered antimicrobial by a simple chlorination process
with a dilute sodium hypochlorite solution (MC was chlorinated during its synthesis). MC-treated
wound dressings were stable to loss of oxidative chlorine when they were stored in darkness and
also under florescent light for 6 months. Other N-halamine treated dressings showed less stability
over time with BA-1-Cl being the second most stable treatment among the N-halamine compounds.
Their shelf life stabilities would be improved upon storage in opaque packaging. Zone of inhibition

Molecules 2017, 22, 1582
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tests performed to determine any leaching of the antimicrobials from the N-halamine-employed
wound dressing materials demonstrated that there was no leaching of the oxidative chlorine from
three of the samples (Hy-Ep-Cl, BA-1-Cl and MC) and only minimally from HASL-Cl. Antimicrobial
efficacies of the materials were determined by a sandwich test in which viable bacteria were quantified.
The N-halamine treated wound dressings exhibited a complete inactivation of the bacteria within
15 to 60 min against S. aureus (Gram-positive bacteria) and P. aeruginosa (Gram-negative bacteria).
In addition, the N-halamine-treated dressings showed rapid inactivation rates when compared to
commercially available silver alginate dressings. In vitro cytotoxicity tests designed to show potential
toxicity of the N-halamine compounds indicated that they did not inhibit cell viability. In fact,
the potential toxicity of the N-halamine compounds was observed to be insignificant and less toxic
when compared to commercially available Ag and PHMB dressings. MC-treated wound dressings
showed no skin irritation, as indicated by 100% skin cell viability. All factors considered, compound
MC would seem to be the optimum N-halamine compound for employment in a wound dressing.
It is inexpensive, available commercially, easily applied to wound dressings, stable when stored in
opaque packaging, effective in the inactivation of pathogenic bacteria in brief contact times, and it
shows minimal skin sensitivity in wound dressing materials.
Supplementary Materials: Supplementary materials are available online. That contains spectral data confirming
the structures of the synthesized materials and zone of inhibition data for the wound dressings.
Acknowledgments: This research was supported by a LAUNCH grant from Auburn University.
Author Contributions: B.D. assisted in the design and performance of all of the experimental work; R.M.B. and
S.D.W. directed the work; M.Q. and T.-S.H. performed the antimicrobial, cytotoxicity and skin irritation studies;
B.D. and S.D.W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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