CA1087977A - Binding of antimicrobial compounds to a hydroxyl containing substrate with cyanuric chloride - Google Patents

Abstract

Methods and compositions are disclosed for chemically bonding an antimicrobial compound to an hydroxyl bearing substrate such as cellulose, starches or leather. Cyanuric chloride (2, 4, 6 tri-chloro 1, 3, 5 triazine) is bonded to the substrate through the substrate hydroxyl and to the antimicrobial through an amine, guanido or quaternary ammonium group. The composition is: R1 or R2 may be chlorine or the same or different amine, guanido or quaternary ammonium-containing antimicrobial. The bonding process is carried out in an aqueous solution having a pH of about 9 - 10.

Description

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BACKGROUND OF THE INVENTION
This invention relates to the chemical binding of antimicrobials onto a substrate bearing a hydroxyl group and particularly to substances such as cellulose, starches and leather.

The binding of antimicrobial substances to substrates such as textiles has been of interest for many years. Prior art efforts in this direction have not been entirely satlsfactory because it has been difficult to attach the antimicrobial to the substrate and still have the antimicrobial retain biostatic or biocidal qualities.
Further problems exist in that the antimicrobials of the ! prior art could generally be readily washed away from the treated substrate with water and/or with detergents.

It has been known to bond enzymes to a substrate through the use of cyanuric chloride, and in such cases the enzymes are immobilized. To our knowledge, however, anti-microbials have not been successfully chemically bonded to a substrate such as cellulose to resist repeated detergent ,~0 washing and still retain inhibitory effort on the growth of microorganisms. Accordingly, it is an object of this ~ invention to provide methods and compositions for the chemical `, bonding o~ antimicrobial compounds to substrates such as cellulose, starches, and leather which cannot be easily removed by washing with water and/or detergents.

The examples of some of the uses of antimicrobials chemically bonded to cellulose would be in the treatment of cloth used in hotels or hospitals for uniforms, bedding and ' the like, to prevent or minimize the presence of bacteria. ~ --- 1 -- .
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The antimicrobials may also be bonded to bandages or bandage pads, to be used direc~ly on superficial wounds, for example.
Besides the use o~ the bonded antimicrobials on clothing in general, still other uses would be for diapers, and other undergarments. Further, the bonded antimicrobials may be used in industrial processes on filter media or on packaging and the like to maintain sterility or to reduce the number of undesirable microorganisms. The non-migrating, bonded antimicrobials are not absorbed into the human body when used on bedding, clothing, bandages or the like. When used on ~ilter media or packaging surface~ active antimicrobials will not contaminate the filtered or packaged substance.
On leather, such as in shoes, the bound antimicrobial can provide built in resistance to fungi, molds and bacteria.
When bound to starch, the antimicrobials can be readily ; mixed into body powders and the like. Thus, bonded non-migrating antimicrobials which could render surfaces sterile have substantial economic value.
Accordingly, it is an object of the invention to provide methods and compositions for chemically bonding ` `
antimicrobials to substrates.
It is a further object of the invention to provide methods and compositions as set ~orth above wherein the chemically bonded antimicrobial resists removal from the substrate by detergent washing.
The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to each of the others, and the composition possessing the features, properties, and the relation of cohstituents, which are examplified in the following detailed disclosure, ... - . .

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and the scope of the invention will be indicated in the ` claims.

Other objects of the invention will in part be - obvious and will in part appear hereinafter.

SUMMARY OF THE INVENTION
- The invention comprises the bonding of antimicrobials onto hydroxyl containing substrates such as cellulose, starches, or leather by reacting the hydroxyl groups of the substrate with cyanuric chloride and then reacting of amine, guanido or quaternary ammonium groups of the antimicrobials with the attached cyanuric chloride. The general reactions are believed to be as follows:

-:

Substrate -O ~ C ~C-Cl ,N
Substrate -O- C ~C-Cl iN ~ ~N ¦l I
, C ~C ~ +HCl i Cl Cl ;20 !~1) Cyanuric Chloride .

NH2-R Amine containing Antimicrobial : .
HCl ~ Substrate -O-C ~C-Cl . Il I ~ . , N~ ~N

HN-R
~30 _ 3 _ ~ ~1 0~5179~7 .:~
` The reaction ta~es place in an aqueous alkaline solution such as a sodium hydroxide solution with a pH of about 9 to 10. The cyanuric chloride is preferably prepared in a ;:~
solvent such as aioxane or acetone. The process is ~:.
particularly useful in bonding antimicrobial compounds having amino groups to cellulosic substrates, such as cotton, rayon, cellulose acetate, etc.

: The invention is ef~ective in chemically bonding antimicrobials to hydroxyl containing substrates and particularly those antimicro~ials having amine, guanido or quaternary ammonium hydroxyl groups. The cyanuric chloride bonds to the substrate and to the antimicrobial to provide .chemical bonds which resist removal by detergent washing while maintaining inhibitory action against one or more microorganisms.

SPECIFIC DESCRIPTION OF PREFERRED EMBODIMENTS
_ ... . . . _ .
,:' OF THE INVENTION

. The chemical binding of antimicrobial or antiseptic .1 .compounds onto a hydroxyl containing substrate proceeds in 120 accordance with the above reactions to form the composltion:

. . .

~N ~ ..

:i~ N~C~N

.. R2 . ., wherein X is the substrate which had contained the hydroxyl group, one of Rl or ~i2 may be chlorlne and the other an . amine, guanido or quaternary ammonium containing anti-microbial compound or Rl and R2 may each be the same or , ', ' ' ' - ' ' ' ' ~ 79'77 a dif~erent amine, guanido or quaternary ammonium containing antimicrobial compound which is chemically bonded to the -triazine ring through the nitrogen atom o~ the amine, guanido or quaternary ammonium group.

A number of amino, guanido or quaternary ammonium containing antimicrobials have been chemically bonded to a ~ellulosic substrate, such as cotton, by cyanuric chloride.
These are chlorhexidine acetate, streptomycin, trimethyl octyl decyl ammonium chloride, and mixtures o~ all three of these antimicrobials. Other substances which have been bound to a hydroxyl containing substrate are neomycin sulfate, alkyldimethyl benzyl ammonium chloride and cetyl dimethyl benzyl ammonium chloride. Further, of the antimicrobials tested, the ones showing the strongest inhibition against the growth of E. coli were chlorhexidine acetate, streptomycin sulfate and neomycin sulfate as well as ~uaternary ammonium compounds such as alkyldimethyl benzyl ammonium chloride, trimethyl octyl decyl ammonium chloride and cetyl dimethyl benzyl ammonium chloride.

In bacteriological testing the bonded antimicrobials showed inhibitory action against E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Saccharomyces diastaticus.
, It was also found that cyanuric chloride used alone has some inhibitory effect for specific microorganisms.

The chemically bound antimicrobials showed substantial resistance to being washed from cotton fabric by several well-known laundry detergents. -In the above reactions the cyanuric chloride is bound to the hydroxyl bearing substrate through a carbon atom with hydrochloric acid formed as a reaction by-product.
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The amine, guanido or quaternary ammonium containing antimicrobial compound is then bound to the triazine ring ` of the cyanuric chloride, replacing one or both of the other chlorine atoms through the nitrogen of the antimicrobial, again producing hydrochloric acid as a by-product.
- The cyanuric chloride or 2, 4, 6 tri-chloro-l, 3, 5, triazine is, for-example, prepared as a 2.5% solution in dioxane or acetone as a solvent, and the pH of the solution is adjusted to between 9 and 10 with lN NaOH. In the following examples of the invention the procedures for pre-paring the test samples shown in Tables 1 and 2 were as follows. A number of 1 inch square cotton patches were immersed in lN NaOH for about 15 minutes. The excess ., .
~ solution was poured off a~d the squares were washed with ; excess warm tap water (55C.) to wash out the alkali. Then the washed squares were immersed in 2.5% solution of cyanuric i chloride, prepared as above, again with the p~ of the solution being adjusted to between 9 and 10 with lN NaOH.
The cotton squares were permitted to stand in the cyanuric chloride solution for about 30 minutes. The excess cyanuric chloride solution was then poured off and the squares were washed twice with acetone containing 5% glacial acetic acid to stop any further reaction and to neutralize the alkalinity of the solution. The cotton patches were then washed two more times with acetone in order to remove any excess cyanuric chloride and were well rinsed with warm tap water. Some of the cotton patches were then air dried and analyzed for nitrogen by the Kjeldahl method.
; A number of the cyanuric chloride treated cotton squares were then immersed in an aqueous solution of '.:
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chlorhexidine acetate (having 200 mg of chlorhexidine diacetate per 100 ml of solution) and the pH was adjusted to between 9 and 10 with lN NaOH. The cotton patches were stirred with the solution and permitted to stand overnight at room temperature, permitting reaction between the anti-microbial and the cyanuric chloride bound to the cotton.
Other cotton patches were dipped into a streptomycin sulfate -~
solution containing 200 mg of streptomycin sulfate per 100 ml and still other cotton patches were treated with a 0.5%
aqueous solution of trimethyl octyl decyl ammonium chloride.
The patches were all then removed from their respective solution, rinsed with large amounts of warm tap water at ; about 55C. and air dried. A number of patches were then taken for further nitrogen analysis to determine the amount of antimicrobial compound which was bound to the cyanuric chloride. Using the nitrogen values and weight of the patches, the amounts of bound cyanuric chloride and bound antlmicrobials were then calculated. These values are set forth in Tables below.

The inhibitory activity of the cotton squares with antimicrobial substances bound to them were tested against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, ' and Saccharomyces diastaticus. The inhibitory effect as to these microorganisms was determined in the following manner.
Cultures of each organism were grown in appropriate ~; broths and then 1 ml of each culture was diluted to 100 ml with water. Two-tenths of a ml of this suspension was pipetted onto a treated cotton square and allowed to stand at room temperaturé for about 15 minutes. Each square was ~, 30 placed in a petri dish and 20 ml of water added and stirred - 7 ;

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-- ~Lq.)~'7S~77 to re-suspend organisms of inoculating culture. Then 1 ml and 0.1 ml samples of that suspension were placed onto a nutrient agar plate. After 24 hours of incubation at 30C.
the colonies on the plate were counted. An untreated -cotton square or ones treated only with sodium hydroxide or cyanuric chloride were similarly processed as controls.
Those cotton squares which showed less than 10% of the number of colonies obtained with the controls are considered to be inhibitoxy.
In khe following Tables the cotton patches to which antimicrobials are bound through cyanuric chloride were tested for retention of antimicrobial action after , washing. For such testing the following procedure was employed. After 18 hours of immersion in antimicrobial solution the cotton patches were removed, well rinsed with warm water, and placed in a detergent solution of about 1 gm of-detergent per liter of solution. The patches were then stirred in the detergent for about 10 minutes, the detergenk is poured off and the cotton was well washed again with warm tap water at about 55C. Such washing with detergent followed by a warm water rinse was repeated two more times and finally the patches were air dried and tested as described above for their microbial inhibitory activity against the organisms shown in the followin~ Tables.
In the Tables the following abbreviations are used:
C = C~anuric Chloride (2, 4~ 6 trichloro 1, 3, 5 triazine) M.W. = 184.5, N = 22.7%. Prepared as a 2.5%
; solution in either dioxane or acetone.

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_ A - Chlorhexidine Formula in Merc~ Inde~, ~ighth Edition, 1968, p. 23~.
M.W. = 505.5, N = 27.7%. Prepared in a concentration of 200 mg as chlorhexidine acetate per 100 ml of H20 -pH = about 9Ø pH adjusted to 10 before use.

SPM = Streptomycln Formula in Merck Index, Eighth Edition, 1968, p. 984-5 M.W. 736.58, N = 13.3%. Prepared in a concentration of 200 mg as streptomycin sulfate per 100 ml of H2O ~
pH about 5Ø pH adjusted to 9 - 10 hefore use.

AQR =- Arquad 18 50 50% aqueous solution of trimethyl octyl decyl ammonium chloride - ClgH39N tCH3)3Cl - M.W. -- 350.0, N - ~.0gO.
Prepared in a concentration of 2 m1 in 200 ml of H2O.
pH about 5.8. p~ adjusted to 9 - 10 with lN NaOII before use.

DM = A mixed solution (dilute) of CHA (133 my), SPM
(133 mg) and AQR (0.67 ml) in 200 ml of water adjusted to pH 9.0 to 10Ø

CM = A mixed solution (concéntration of CHA (~00 mg) SPM (400 mg), and AQR (2.0 ml) in 200 ml of water adjusted to pH 9.0 to 10Ø

A =*All A retail packaged detergent used ln clothes washing machines -Label states that All contains surfactants, sodium carbonate, sodium silicate, bleach, borax, and brighteners. It is ~: , ; used in concentration of 1 g per liter (pH about 10). It is bio-degradable, conta~ns no phosphorous and is manu~actured by Lever Brothers Co., New York, New York.
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, *Trade Mark i 30 ,`.1 ~ 9 `I A~
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. '-" ' , ~ ~(987~'~'7 T ~*Tide rqtail~ ~ .ergent used in cloth~s washing machilles. Label states that Ti~e contains anionl.c surfactants,.sodil~
caxbonate, sodium silicate, sodi~n sulfate, ~abric whit:ener, and perfume. Used in concentration of 1 g per liter ~pll about 10). It is biodegradable and is manufactured by Proctcr & Gamble, Cinc~.nnati, Ohio~

J =*~oy , _ ___ A retail wàshing liquid used for washing dishes and contains anionic and nQnionic surfactants, ethyl alcohol as a stabilizing agent, and perfume. Used in concentrati.on of 1 g per liter (p~ 7.3). It contains no phosphorous and .is manufactured by Proctor & ~amble, Cincinnati, Ohio.
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TNTC

Too numerous to count.

. Examples:

¦ C-CHA-T Indicates cotton treated with cyanuric chlo.~idc (C), followed by reactioll with chlorhexidine acetate ~CHA), followed by washing with.Tide (T).

C-A Indicates cotton treated with ~yanuric chloride (C), followed by washing with A].l (A).

NaOH Indicates cotton washed with lN NaOH for 15 minutes followed by water wash to remove excess ¦ NaOH.

CHA-J Indicates cotton treated with chlorhexidine acetate (no binding with cyanuric chloride) followed by washing with*Joy.

In the Tables, calculations were made on the . following molecular weights and nitrogen contents of the con~pounds employed:

* TradeÇIark ~ 1 0 --. . .

'7~'77 Antimicrobial Molecular Weight ~ Nitro~en C-Cyanuric Chloride 184.5 22.7 CHA-Chlorhexidine 505.6 22.7 SPM-Streptomycin 581.6 16.86 AQR-Arquad 18-50 350 4.0 Cotton patches were prepared as above and the . results of testing of the cyanuric-antimicrobial treated cotton for inhibition of microbiological growth are set forth in Tables l(a), (b), (c) and (d).
TABLE l(a?
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~acteriological Results - Inhibition Against E. coli Controls Dilution - 1 ml0.1 ml . - .
NaOH TNTC1335 C-J 55 . 7 C--T l'NTC 218 Unbound Antimicrobials . _ _ . _ _ :: 20 CHA-H20 CHA-T g o Bound Antimicrobials .. __ . ... :

C-SPM-J 3 o C-SPM-A O O
:j C-SPM-T 2 0 C-AQR-A 3 l 7 3 0 ;

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TABLE l(b) Bacteriological Results - Inhibition Against B. subtilis Controls Dilution 1 ml 0.1 ml NaOH 3 C-J O O
C-A g o Unbound Antimicrobials CHA-J O O
CHA-T . o o Bound Antimicrobials . .
, C-CHA-H20 7 .- C-CHA-J O O
:;
C-CHA-A 7 o C-CHA-T O . O
C-SPM-H20 .6 0 C-SPM-J O O
. C-SPM-~ o o .~;

C-AQR-J 3 o : . C-AQR-A 2 0 C-AQR-T 4 o , ' . , ., .

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C-A TNTC TNTC

Unbound Antimicrobials Bound Antimicrobials C-CHA-T >300 68 C-SPM-T TNTC TNTC

C-AQR-J >300 210 C-AQR-A >300 41 ', ~'.

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. , , ; . . ., ., ,. . - , , 7~77 TABLE 1 ~d) Bacteriological Results - Inhibition Against S. diastaticus Controls Dilution 1 ml 0.1 ml NaOH 42 5 C-J >300 31 Unbound Antimicrobials CHA-T >300 27 Bound Antimicrobials i -.......... _ ._ i~ . C-CHA-A 2 0 : C-CHA-T 16 . 2 C-SPM-H20 . 17 1 -C-AQR-J
. C-AQR-A O O
C-AQR-T O O

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t7~7 TABLE l(e ?
Mi~ rams of Nitrogen per Gram of Cotton Expressed as I C~anuric Chloride and Antimicrobials - Mg N per g . .
; Mg N per Cotton (Cor- Mg N per gMg Anti- , g of rected forCotton (Cor-microbial SampleCotton NaOH Blank) rected for C) per g cotto~
.. ~
NaOH 0.25 C-H20 9.09 8.84 39.0 C-J 8.45 8.20 3601 C-A 8.35 8.10 35.6 C-T 7.42 7.17 31.6 C-AQR-J 8.87 8.62 0.40 10.0 C-AQR-A 10.33 10.08 1.98 49O6 C-AQR-T 9.92 9.77 1.60 40.0 C-AQR-H2O 9.32 9.07 0.23 5.8 C-SPM-J 11.65 11.40 3.20 19.0 C-SPM-A 10.06 9.81 1.71 10.1 ,., C-SPM-T I5.36 15.11 7.94 47.1 C-SPM-H2O 11.57 11.32 2.48 14.7 C-CHA-J 16.65 16.40 8.20 29.6 C-CHA-A 18.05 17.80 9.70 35.0 C-CHA-T 14.73 14.48 7.31 26.4 C-CHA-H20 14.80 14.55 5.71 20.6 CHA-J. 0.85 0.60 2016 CHA-T 0.91 0.66 2.38 CHA-H20 0.97 0.72 2O60 , -:
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~` 1~)~3'7~ 7 The bac-teriological results for inhibitory activity against the four ~icroorganisms in Tables l(a), (b), (c) and (d) m~y be summarized generally as follows. The con~rols in which cyanuric chloride alone was bound to cotton (followed by washing wi~h water and detergents) showed little orno inhibition of growth against E. coli, Pseudomonas aeruginosa and S. diastaticus, but showed substantial effectiveness against B. subtilis growth in the test results set forth in Tables l(a), (b), (c) and (d). As will be noted, B. subtilis growth was inhibited on all treated and untreated cotton patches. It is believed that either the culture used was weak or the number of organisms used was too small.
The experiment was repeated using a heavier inoculum of B. subtilis and showed less inhibition of R. subtilis by cyanuric chloride alone, as shown in Table 2(a) below. i Two of the antimicrobials, namely chlorhexidine (CHA) and streptomycin (SPM), after binding to cotton were ineffective in preventing the growth of Pseudomonas aeruginosa, ` particularly after the cotton was washed with detergents.
In one instance chlorhexidine showed some inhibition against Pseudomonas aexuginosa after only a water wash. Trimethyl octyl decyl ammonium chloride (AQR) showed the best relative results against this microbe after the cotton patches were washed with water and detergents. A repetition of the experiments as to Pseudomonas aeruginosa, as shown in Table 2(b), generally confirms these results.
In Table l(a) cotton treated with chlorhexidine alone followed by water washing and Tide detergent is shown to have inhibitory activity against E. coli. Further, the ` 30 chlorhexidine could not be completely washed out of the cotton.

; - 16 -,, ' ~-- i ' Table l(e) shows the extent of antimicrobial bond-ing by-analysis for nitrogen by the Kjeldahl method. As shown in Table l~e), some of the chlorhexidine was directly bound to the cotton (lines 25-27). The inhibiting effect of chlorhexidine against E. coli was thus somewhat affected by detergent washing. Trimethyl octyl decyl ammonium chloride ; also shows a reduction of inhibitory effect on E. coli after detergent washing. Streptomycin showed excellent inhibition of E. coli growth in all tests even after extensive washing ; 10 with water and detergents. Generally, streptomycin and chlorhexidine showed about 70 to 90~ inhibition of S. diastaticus growth. Trimethyl octyl dccyl ammonium ~ chloride was completely inhibitory in all the experiments ,~ against S. diastaticus.

, Thus, these above antimicrobials have been shown ~i, to be chemically bound to cotton via reaction with cyanuric : ~1 , chloride and are useful to inhibit the growth of four different classes of microorganisms even after extensive washing of the treated cottons with water and three commonly used commercially available detergents.
,'1 . .
,,tj, , The tests were repeated for further study of ,~
inhibitory activity against B. subtilis and Pseudomonas aeruginosa and the results are shown in Tables 2(a) and (b).

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TABLE 2(a) Bacteriological Results - Inhibition Against B. subtilis Controls Dilution 1 ml 0.1 ml Bound Antimicrobials 3 C-SPM-A 121 18 :.

C-AQR-H20 o C-AQR-T . 72 0 C-DM-J 72 12 ~ .
' C-DM-A 0 0 '''"
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7~77 TABLE 2(b) Bacteriological Resul~s - Inhibition Against Pseudomonas aeruginose Controls Dilution 1 ml 0.1 ml Bound Antimicrobials C-CHA-J >300 87 C-CHA-T >300 41 C-SPM-HzO 158 C-SPM-J >300 508 C-SPM-A >300 94 . . .
C-SPM-T >300 207 C-AQR-J . >300 84 C-AQR-A >300 179 C-AQR-T >300 182 C-CM-J >300 91 C-CM-T >300 62 ., C-DM-H20 >300 187 C-DM-J >300 224 C-DM-A >300 157 C-DM-T >300 178 ., ' : -: , ~,, ' ' .

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7~77 .
The results shown in Tables 2(a) and (b) furtherillustrate the inhlbition of microorganism growth of Bacillus subtilis and Pseudomonas aeruginosa by chlorhexidine (CHA), streptomycin (SPM), trimethyl octyl decyl ammonium chloride (AQR), and dilute mixtures !DM) and concentrated mixtures (CM) of all three of these antimicrobials.

In the experiments shown in Table 2(a), a heavier inoculum of B. subtilis was used and inhibition of B. subtilis by chlorhexidine and trimethyl octyl decyl ammonium chloride is clearly shown. Detergent washing as well as the concentra-tion of the organism slightly affected the inhibitory ~;
properties of these chemically bound antimicrobials.
.
A dilute mixture of all three antimicrobials (DM) shows good inhibition of growth of B. subtilis even after extensive washing with water and detergentsO It has been found that use of a more concentrated solution (DM) of anti-~i microbial compounds shows even greater inhibition against B. subtilis when bound to cotton regardless of the type of washing which is used.
.~, . . .
~ 20 Chlorhexidine and trimethyl octyl decyl ammonium .; . . , chloride show good inhibition toward Pseudomonas aeruginosa when the cotton was washed only with water. There was less . .
inhibitory effect when the cotton was washed with detergents,confirming the results shown in Table l(c). The dilute mixture (DM) and streptomycln showed the least effectiveness against Pseudomonas aeruginosa. The best resulks were obtained by a concentrated mixture (CM) of all three anti-microbials in the inhibition of Pseudomonas aeruginosa growth.
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Tables 2(a) and (b) show that good inhibition of ~rowth-of B. subtilis and Pseudomonas aeruginosa can be obtained when these organisms are exposed to cotton trea~ed With chlorhexidine or trLmethyl octyl decyl ammonium chloride or mixtures of these two antimicrobials in the proper concentrations. Cyanuric chloride alone had some inhibitory effect against B. subtilis, but little effect against Pseudomonas aeruginosa.
As shown in Table 2(c), the nitrogen values again show substantial antimicrobial bonding to the cotton.

TABLE 2(c) .
Milligrams of Nitrogen per Gram of Cotton Expressed as Cyanuric Chloride and Antimicrobials ~ Mg N per g ¦ Mg N per Cotton (Cor- Mg N per g Mg Anti-i g ofrected for Cotton (Cor- microbial per Sample CottonN H Blank) rected for C) ~ Cotton NaOH 0.25 -~
C-H2O 8.45 8.20 36.1 C-AQR-J 9.28 9.03 0.83 20.7 C-AQR-T 9.68 9.43 1.23 30.8 C~SPM-T 11.77 11.52 3.32 19.7 C-CHA-J 16.26 16.01 7.81 28.2 C-CHA-T 14.87 14.62 6.42 23.2 C-CM-J 15.13 14.98 6.78 38.6 C-CM-A 14.08 13.83 5.63 32.1 C-CM-T 16.40 16.15 7.95 45.3 C-CM-H2O 14.7 13.92 5.72 32.6 C-DM-J 12.77 12.52 4.32 24.6 ; C-DM-A 13.33 13.08 4.88 27.8 C-DM-T 13.61 13.36 5.16 29.4 C-DM-H2O 12.20 11.95 3.75 21.4 .~ .
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~` ~ ` ~087977 ; In further work done, the antimicrobials were bound'onto insoluble starch by cyanuric chloride. The antimicrobials we~e applied in unbound and bound form on a starch powder which a~ter exposure to the cyanuric chloride and the antimicrobials, was well washed with water, followed by an acetone rinse to remove excess reagent and to then permlt air drying. It is indicated that chlorhexidine acetate (CHA) and streptomycin (SPM) are adsorbed onto the starch and are not completely removed by water washing since they exhibit good antimicrobial activity without being chemically bouncl with cyanuric chloride. Arquad 18-50 (AQR) also exhibited some adsorption onto the starch, but is shown to be effective against E. coli only when bound via cyanuric chlorid'e. ' The antimicrobials, streptomycin, chlorhexidine acetate and*Arquad 18-50 have also been bound onto leather .. . . .
(cowhide) via cyanuric chloride. In the case of chlorhexidine acetate and Arquad 18-50, these antimicrobials appear to be as effective unbound as when bound by cyanurlc chloride, since there is substantial adsorption o~ these antimicrobials on~to the leather. In the case of streptomycin, however, there appears to be little adsorption of the SPM onto the leather and when bound to leather by cyanuric chloride, it is indicated that streptomycin' has substantial antimicrobial activity against B. subti~is and S. diastaticus.
In later work, to examine the necessity for the use of acetone or dioxane solvents for cyanuric chloridè, an aqueous solution at a pH of about 9-10 was used and the concentration of cyanuric chloride was reduced from 2.5%
`30 -to 1~. In two different sets of experiments the time of reaction of the antimicrobials with the cyanuric chloride * Trade Mark was reduced from 18 hours to one hour, as shown in Tables 3~a), (b), (c) and ~d). From these Tables the results lead to the conclusion that the cyanuric chloride can be ~ound to a substrate (in this case, cotton squares, as for the Tables l(a), (b), (c) and (d) above) in an aqueous alkaline solution and that the concentration of cyanuric chloride may be substantially reduced when i-t is to be used as a -blnding agent only, in most cases.

The reduction of reaction time of the antimicrobials from 18 hours to one hour indicates that a substantial amount of the antimicrobial is bound to the cyanuric chloride within the first hour of the reaction and that additional reaction time does not add much more antimicrobial material.
Accordingly, it should be concluded that reaction time of the antimicrobials need be no more than an hour, except in those cases where an absolute maximum of antimicrobial bound onto the substrate is required. The reaction time is indicated by the number suffix 1 or 18 in the Tables.

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TABLE 3(a) Bacteriological Results - Inhibition Against E. coli . .
Controls Dilution 1 ml 0.1 ml NaOH TNTC TNTC
C TNTC TNTC

Unbound Antimicrobials ~'l l AQR-l TNTC 250 i~ AQR-18 TNTC 500 CHA-l 500 64 SPM-l 107 21 :

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Controls Dilution 1 ml 0.1 ml Bound Antimicrobials :
C-AQR-l 18 4 C-CHA-l 12 C-SPM-l 14 2 C-SPM-18 . 57 8 ~ ~:

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TABLE 3(b) Bacteriological Results - Inhibition Against B. subtilis Controls Dilution :
, 1 ml 0.1 ml .1 - . . .
NaOH . 148 16 ~ :

Unbound Antimicrobials AQR-l 0 0 CHA-l 1 0 CHA-18 0 0 : :
SPM-l 65 5 Bound Antimicrobials C-AQR-l 0 0 C-CHA-l 0 0 :ii C-SPM-l 31 3 .~ C-SPM-18 7 o - 24 - .

.

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:
~ABLE 3(c) Bacteriological Results - Inhibition Against Pseudomonas aerugino sa . ' :
; Controls Dilution 1 ml 0.1 ml NaOH TNTC TNTC
I C TNTC TNTC
.~ Unbound Antimicrobials ; - AQR-l TNTC 127 - AQR-18 320 45 -^
CHA-l TNTC 380 ,; SPM-l - . TNTC 320 Bound Antimicrobials ~ . .
. C-AQR-l TNTC 167 ` C-AQR-18 174 26 ;~
!l C-CHA-l TNTC 420 `1 C-CHA-18 TNTC 84 .
I C-SPM-l TNTC 624 ,; C-SPM-18 TNTC 87 , ~i .', , . ~ .
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'7~77 , TABLE 3(d) .
Bacteri-ological Results - Inhibition Against S. diastaticus Controls Dilution 1 ml 0.1 ml NaOH TNTC 106 Unbound Antimicrobials AQR-l 0 0 ~
AQR-18 2 0 ;:
CHA-l 54 6 SPM-l 810 95 Bound Antimicrobials ~.
, C-AQR-l 0 0 C-AQR-18 . 3 1 ~ .

C-CHA-18 28 . 3 C-SP~-18 82 9 .~ .`. .:

.

:

9~7~7 TABLE 3(e) Milligrams of Nitrogen per Gram of Cotton Expressed as , Cyanuric Chloride and Antimicrobials . Corrected for Mg Anti-Mg N per NaOH Blank Mg microbial per , Sample g of Cotton N per g Cotton g Cotton NaOH 0.44 AQR-l 0.66 0.22 5.5 CHA-l 1.41 0.97 3.5 SPM-l 0.54 ~ 0.10 0.53 AQR-18 0.87 0. 43 10.0 CHA-18 1.66 1.22 4.4 SPM-18 0.86 0.42 2.5 ~ C 2.24 1.80 8.0 - .
.~l C-AQR-l 2.42 1.98 4.5 C-CHA-l 3.45 3.02 4.4 i, C-SPM-l 2.73 2. 29 2.9 .~ C-AQR-18 2.46 2.02 5.4 .l C-CHA-18 3.72 3.28 5.3 `~ C-SPM-18 2.99 2.55 4.4 "~ .

Notes: C = Cyanuric Chloride - 1~ aqueous solution pH 9 - 10.
: AQR-l = Exposure to AQR of one hour.
;~ AQR-18 = Exposure to AQR of eighteen hours.

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The results shown in Tables 3(a), (b), (c) and (~) indicate generally that the inhibition ~f E. coli is enhanced by the binding of the antimicrobials through -cyanuric chloride. In the case of B. subtilis, the unbound antimicrobials, as well as the bound antimicrobials, inhibited yrowth of that organism, but it should be noted that in all of the experiments shown in Tables 3(a), (b), (c) and (d), a water wash only was used. Pseudomonas aeruginosa was somewhat inhibited by the bound and unbound . I .
10 . AQR and it was measurably affected by an 18 hour exposure of antimicrobial rather than the one hour treatment. Accordingly, for difficult to inhibit microorganisms such as Pseudomonas aeru~inosa, it is recommended that a maximum of antimicrobial ::~ be employed by the use of a more concentrated solution of ~j ; cyanuric chloride as the binding agent and by a more extensive exposure of antimicrobial to the cyanuric chloride J treated substrate.
.. . . .
S. diastaticus was inhibited by both bound and unbound AQR and CHA, while bound SPM somewhat inhibited the growth of S. diastaticus. Again, the extended exposure of the antimicrobial over 18 hours has a marked effect on the inhibition of S. diastaticus.
From the Tables 3(a), (b), (c) and (d), it is also indicated that the cyanuric chloride alone when used in a 1% aqueous solution was not as antimicrobial as that used for the Tables l, wherein a 2.5% solution in dioxane .. . .
was used.
Table 3(e) shows the milligrams of antimicrobial ` per gram of cotton deposited or bound on the substrate as determined by the Kjeldahl method. As shown in Table 3(e), .
., . ' `

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8~77 there was only abou-t 8 mg of cyanuric chloride per græ.m of cotton bound to the substrate as compared to from 31.~ ~o 39 mg of cyanuric chloride per gram of cotton as shown in ~able l~e). Accordingly, the binding of cyantlric chloride ~o the - cellulosic substrate appears to be proportional to the concentration of the cyanuric chloride solution used. As the concentration o cyanuric chloride is increased, ~he antimicrobial effect of the cyanuric chloride comes apparent.

In early work done the results showed that cyanuric chloride when used in a concentration of 2.5~ substantially inhibited growth of E coli When chlorhexidine acetate O ~ , streptomycin and Arquad 18-50 were bound to the cotton substrate via cyanuric chloride, the results showed variable efficiency depending, for example, on the test organlsrn used. In general, the chlorhexidine acetatej whether washed with water or commercial detergents showed complete inhibi-tion of E. coli and the streptomycin nearly comp]ete inhibition. The*Arquad 18-50, however, only showed complete inhibition when washed with All and very poor inhibition when washed with Joy or*Tide.
.
Furthermore, it was also found during work leading up to the present invention that not all antimicrobial compounds retained their activity on being bound to a substrate material. ~sing the procedures described herein-~efore to determine the inhibitory properties of various agents, it can be seen from the data presented in Table 4 that, using E. coli as test organism, neomycin effects practically total inhibition of growth when bound to the cotton substrate but, in contrast, cyanuric chloride (CYC), pxoflanine (POF), tetracycline (TCC) and bacitracin * Trade Mark ~ 29 -, . , ' . .

:~ .
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797~7 -:

(BCT) proved ineffective from a practical viewpoint compared with the value for the C-NaOH control. Since the type, number and location of reactive moieties in sueh agents is eritical to the aetivity the compound exhibits, it must be eonsidered highly surprising that when such moieties are involved in the bonding of the moleeule to the substrate, and are therefore unavailable, activity is still found to exist. These data teac~ then, that not all antimicrobial agents on being so bound, successfully retain a desirable level of activity.

Baeteriological Results - Inhibition against E. coli ; Controls Dilution 1 ml 0~1 ml C-~aOH TNTC 7,300 Bo_nd Antimicroblals ~, TCC TNTC 1,399 BCT TNTC 2,226 :
Attempts to chemically bond antimicrobials with eyanuric fluoride indicate that the amount of cyanuric fluoride which would bond to the cotton was substantially `30 less than the cyanuric chloride bound under the same experimental conditions. Accordingly, the amount of antimicrobial that could be bound to a cellulosic substrate by cyanuric fluoride was also substantially less, i.e.
, _ 30 _ '~

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~ 791~7 !~ in the order of about 20~ of that which could be bonded via cyanuric chloride.
lt has also been foudn that a fresh solution ; o cyanuric chloride is more effective than aged solutions.
~.
Accoxdingly, it is recommended that the cyanuric chloride be used within 24 hours after being prepared to assure maximum bonding to the substrate.
The method and composition of the invention -' thus provide antimicrobial surfaces for a variety of uses wherein persistence, but non-migrating antimicrobial action, is necessary or desirable. The chemical bonding of ,l the antimicrobial to the substrate provides resistance to removal by washing and accordingly, is particularly suitable for use on clothing, bedding, bandages, filters, packaging, shoes, powders, and a number of other applications.
~ Chemical bonding of the antimicrobial to the substate also 3l prevents ingestion or other undesirable removal of the antimicrobial from the substrate.
It will thus be seen that the objects set forth above, among those made apparent from the preceding .~ , description, are efficiently attained and, since certain changes may be made in carrying out the above process and in ` the composition set forth without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.
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Claims (5)

The embodiments of the present invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of bonding an amine, guanido or quaternary ammonium containing antimicrobial compound selected from the group consisting of chlorohexidine acetate, streptomycin sulfate, neomycin sulfate, alkyl dimethyl benzyl ammonium chloride, trimethyl octyl decyl ammonium chloride, cetyl dimethyl benzyl ammonium chloride and mixtures thereof to a hydroxyl containing substrate selected from the group consisting of cellulose, starches and leather comprising the steps of:
A. reacting the substrate with cyanuric chloride in a solution to chemically bond the cyanuric chloride to the substrate; and B. thereafter reacting an effective amount of said antimicrobial in solution with the cyanuric chloride on the substrate to chemically bond said antimicrobial to the cyanuric chloride, said cyanuric chloride being present in an amount effective to bond said antimicrobial to the substrate through the cyanuric chloride.

2. The method according to claim 1, wherein the bonding of the antimicrobial compound is carried out in an aqueous alkaline solution having a pH of from 9 to 10.

3. The method according to claim 2, wherein the solution is an aqueous solution of sodium hydroxide.

4. An antimicrobial composition comprising a hydroxyle containing substrate selected from the group consisting of cellulose, starches and leather, to which is bound cyanuric chloride and one or more amine, guanido or quaternary ammon-ium containing antimicrobial compound selected from the group consisting of chlorhexidine acetate, streptomycin sulfate, neomycin sulfate, trimethyl octyl decyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, cetyl dimethyl benzyl ammonium chloride and mixtures thereof, said antimicro-bial compound being chemically bonded to the hydroxyl contain-ing substrate through the cyanuric chloride.

5. A persistent antimicrobial composition comprising a cellulosic substrate having chemically bound thereto a cyanuric chloride radical and one or more amine, guanido or quaternary ammonium containing antimicrobial radical chemically bound to said cyanuric chloride radical to provide a composition of the formula:

wherein X is the substrate, R1 or R2 may be chlorine and one or each of R1 and R2 is an antimicrobial radical either the same or different, selected from the group consisting of chlorhexidine acetate, streptomycin sulfate, neomycin sulfate, alkyl dimethyl benzyl ammonium chloride, trimethyl octyl decyl ammonium chloride and cetyl dimethyl benzyl ammonium chloride.