Cotton gauze bearing non-diffusible quaternary ammonium salts ...

Cotton gauze bearing non-diffusible quaternary ammonium salts and featuring
anti-microbial activity: An example of single-use articles tailored to self-sterilize
Damla Köylü 1 , Kıvanç Bilecen 2 , Erinç Şahin 2 , Gülengül Duman 3 and Alpay Taralp 4
1
Faculty of Arts & Sciences, Boğaziçi University, Department of Chemistry, Istanbul 34342; 2 Faculty of Engineering & Natural Sciences, Biology & Bioengineering Program, Sabancı
University; 3 Bilim Pharmaceuticals Inc., Istanbul 80670; 4 Faculty of Engineering & Natural Sciences, Materials Science & Engineering Program, Sabancı University, Istanbul 34956
Summary
Two approaches were envisaged in which single-use cotton gauze could be chemically tailored to display anti-microbial activity. In the first approach, an intertwined organosilane-organic network bearing anti-microbial
quaternary ammonium salts was afforded by penetrating 3-trimethoxysilylpropyloctadecylammonium chloride, commonly known as AEM 5700, into the cotton matrix and polymerizing the monomers about the fibers. In
a second approach, a related quaternary ammonium salt moiety was synthesized directly onto the solid-phase by employing a three-step synthesis consisting of tosyl group activation, amination with long-chain aliphatic
amines, and quaternization using iodomethane. Iodine-based colorimetric methods indicated that quaternary ammonium groups resided on the cotton whereas spectroscopic IR measurements clearly under detection
thresholds implied the loadings must be low. The modified cottons were assayed for anti-microbial activity following their inoculation with gram negative bacteria by way of a procedure utilizing a GFP-synthesizing
recombinant strain of Escherichia coli to facilitate visual observation. The modified cottons and respective controls were wetted with liquid culture and incubated at 37ºC. Following incubation, control cottons afforded
fluorescence, indicative of bacterial growth. A lack of fluorescence noted in modified cottons could not, however, be automatically equated to anti-microbial activity. To verify that the effect of the quaternary salt was
realized through its anti-microbial action as opposed to a subtle mechanism such as the inhibition of protein expression, a secondary inoculation strategy was devised. Operating under the premise that bacterial growth on
modified cottons stored at 4°C was negligible compared to death in the presence of ammonium salts, the control and modified cottons were applied to liquid Luria-Broth (LB) and LB-Ampicillin media and LB agar
plates. In all cases, growth of bacteria was only noted in the native cottons, indicating that the low loading of ammonium groups was more than sufficient to impede bacterial growth. Plates inoculated with native cotton
not only displayed GFP-fluorescence but also non-specific bacterial growth. In contrast, modified cottons afforded plates that were completely plaque-free under visible as well as UV light. It followed that the proposed
syntheses could transform simple medical products such as gauze into materials that would facilitate topical therapies and long-term storage even under harsh conditions. The synthetic methods, principles and current
state of this investigation are presented.
Introduction
Incorporating diffusible anti-microbial agents such as quaternary ammonium salts within the matrix of medical textiles is potentially problematic. Indeed, adverse effects such as allergic reactions may develop upon
contact and diffusion of the exogenous agents into tissue regions that are exposed and weakened by injury or surgical intervention. These worrisome problems have largely been precluded by developing bacteriocins that
are covalently immobilized to textiles such as cotton, as in the example of the agent AEM 5700. To our knowledge, however, the same approach has never been reported in single-use articles such as medical gauze,
which seems unusual in that the active moiety afforded would be non-irritating to skin and unable enter the bloodstream once assembled into the cotton as a polymer. It followed that testing the efficacy of immobilized
quaternary salts on single-use cottons was a logical progression in the development of specialized medical products, namely, self-sterilizing gauze to treat regions that have experienced open trauma in accident as well as
surgical situations. In this preliminary work, the chemical modification of cotton bearing long-chain ammonium salts was achieved and its performance as an anti-microbial agent was characterized. Two syntheses were
examined, affording moieties bearing similar modes of efficacy in light of their structural similarity. In the first method, AEM 5700 was applied onto the cotton gauze and cured thereon in a single step (Synthesis A). The
approach was particularly attractive since in principle, it featured an efficacy that might be tunable by appropriate choice of alkyl chain. In a second approach that featured design flexibility, native gauze was activated
using tosyl chloride, a reactive and spectrophotometrically monitorable moiety. The cotton-tosyl intermediate was subsequently reacted with hexadecylamine, affording long-chain amino groups linked through a
secondary amine bond. Quaternization of the intermediate using iodomethane afforded the final product (Synthesis B). The anti-microbial action of AEM 5700-treated cotton was tested using a GFP-expressing strain of
E. coli and the results are reported herein.
M 3 O Si
Synthethesis A
Synthesis B
N + (CH 2 ) 12
N +
OH
1. 10% MeOH/H 2 O, 1h 1. Cotton gauze is incubated (10%NaOH, 20
o
2.70C, 2h
o min) and placed in 1wt% 3-trimethoxysilylpropyldimethyloctadecyl
ammonium
3. H 2 O wash; dry 70C, 2h
chloride/10% methanolic water solution (1h)
OH
OH
2. Cotton gauze in oven dried (70ºC, 2h)
OH O 3. Cotton is rinsed and dried (70ºC, 2h)
Si N + (CH
O 2 ) 12 Note - The same procedure was repeated
OH OH with aminopropyltirmethoxysilane in place
of 3-trimethoxysilylpropyldimethyloctadecyl
OH
ammonium chloride to afford a colorimetric
control of the iodine test (vide infra)
OH
OH
A. Direct Immobilization
OH
OH OH
OH
HO
N +
N + O Si OH
OH
O
Si O Si
O O
MeO OMe
B. Immobilized via self-crosslinking
HO
Si O
Si O Si
O O OH
Si
HO
Figure 1 outlines the synthetic method and reaction conditions used to afford anti-microbial
cotton bearing quaternary ammonium moieties starting with the reagent 3-trimethoxysilylpropyldimethyloctadecylammonium
chloride. Direct bonding (A) and intertwined (B) modes of
binding to cotton are portrayed.
Iodine-based Colorometric Analysis of Synthetic Route A
HO
N +
N
OH
OH
H
OH
O
Cl S
(CH 2) 13
O
MeI/DMF
Figure 2 outlines the synthetic method and reaction conditions used to afford anti-microbial cotton
bearing quaternary ammonium groups starting with tosylation, proceeding through a hexadecylamination
step, and terminating with a quaternization step. The activation of primary hydroxyl groups of
cotton are portrayed:
1. Activation of cotton using tosyl chloride
2. Hexadecylamination of activated cotton with concomitant loss of tosylate anion
3. Quaternization of nitrogen centers using methyl iodide
OH
O
O S
O
OH
+ N
CH 3
CH 3
(CH 2) 13
ATR-IR Analysis of Synthetic Route A
H 2N
-
- O
(CH 2) 13
O
S
O
Quat Silane Apte Silane Native
Figure 3 outlines the color yields of a test used to verify the presence of quaternary amino
groups in the cotton:
1. The cotton was dipped in an methanolic solution of iodine (2%, 1min)
2. The cotton was washed thrice in methanol and once in water.
Result - The appearence of a fixed orange-red color on the quaternized moiety (AEM 5700) and a
freely diffusing violet color (not shown) indicates a positive test. The native and aminopropylsilylated
cottons tested negative, as shown by a fixed yellow color and lack of diffusable color.
Figure 4 shows the following:
1. IR spectra of native and modified cottons are identical. The peak at 1630cm -1 is the bending mode of water.
2. Since a sensitive ATR accessory was employed in the analysis, the degree of loading was likely low.

Anti-microbial Efficacy, Results and Discussion
(A) (B) (C) (D) (E) (F) (G)
Figure 6 shows that GPF was expressed on native gauze but not AEM 5700-treated gauze:
The photographs taken under UV light (in B&W and color) show that treating gauze with
trimethoxysilylpropyldimethyloctadecyl ammonium chloride at least curbs the expression of GPF in
gram (-) E coli bearing the GFP gene. The question remaining to be answered is whether or not E coli
was actually killed by the action of AEM 5700. Inoculation method: Cotton was autoclaved for 30 min.
Ampicillin resistant bacterial strain was inoculated with ampicillin (0.1mg /mL) and incubated at 37ºC
with 300 rpm shaking until A = 0.5, then diluted in LB+Amp. Native and modified cottons were put in
liquid media and were incubated either at 4ºC (results not shown) or 37°C (results, vide supra)]
[Photographs: Images were captured using a UVITEC-JAI camera and processed using UVITEC-
UVIPhotoMW Version 10.02 software]
under UV light
(A) (B) (C) (D)
under visible light
A B C D
Figure 7 shows the post-incubation appearance of Luria-Broth Agar plates streaked with native and modified cotton gauze under aseptic
conditions. Plates were incubated at 37°C for 24 hours and visualized under visible and UV light to monitor bacterial growth. On the LB agar plate
that was streaked with native cotton gauze, growth of GFP producing and non-producing bacteria can be followed (A, C). In contrast, no growth was
observed in the case of modified cotton (B, D).
Figure 8 shows under UV light the post-incubation appearance of LB and LBA (100µg/ml
ampicillin) media following inoculation with native and modified cottons
LB and LBA (100µg/ml ampicillin) media (5ml) were inoculated with native and modified cotton. The
tubes were incubated at 37°C with 300 rpm shaking for 24 hours. Bacterial growth in LB (F) and LBA
(G) media that were inoculated with native cotton gauze contrasted notably against sterile LB medium
containing a piece of cotton gauze (A). LB (B, D) and LBA (C, E) media inoculated with modified cotton
gauze showed no bacterial growth.
Conclusions
The quaternary amine strategy shows merit in the matter of producing improved
single-use cottons and related self-sterilizing products of medical significance.
References
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Background Material - The Bacterial Wall and Quaternary Ammonium Salts
Figures 9 and 10 illustrate the difference
in wall structure of gram (+) and (-)
bacteria. Gram positive bacteria have a
thick peptidoglycan layer outside their
plasma membrane. In contrast, gram
negative bacteria have a thin
peptidoglycan layer and an outer (lipid
bilayer) lipid membrane (OM) containing
lipopolysaccharide (LPS). Quaternary
ammonium salts bearing long alkyl chains
have the property of disrupting the delicate
cell membrane and therefore do not need
to be absorbed in solution to show their
bactericidal effect.
Figure 9: Gram (+) bacterial wall
Figure 10: Gram (-) bacterial wall