VOL. 9, PP. 55-68 (1975)
J. BIORIED. 31ATEII. RES.
Antimicrobial Polymers W. B. ACKART, R. L. CAMP*, W. L. WHEELWRIGHT?, and J. S. BYCKS. Research and Development Department, Chemicals and Plastics, Vnion Carbide Corporation, Bound Brook, Xew Jersey
Summary A number of carboxyl-containing ethylene copolymers have been prepared which exhibit long term antibacterial and antifungal properties. These materials, containing antimicrobial agents bound to the copolymer backbone as carboxglate salts, have been tested for their applicability to hospital products as a means of providing “self-sanitizing” articles. Tests have shown that these materials, although not bactericidal, do inhibit microbial growth. Investigations of the compatibility of these polymers with commodity polymers have been made and water emulsions of the polymers have been tested for applicability as components of product protectant coatings.
INTRODUCTION As part of a biomedical polymers research program, a project was initiated for the purpose of developing antimicrobial polymer systems. The specific objective was to create new polymer compositions consisting of antibacterial and/or antifungal molecules reversibly bonded t o suitable polymer backbones. Through gradual release of the active agent, the polymer should, a t minimum, be self-sanitizing over prolonged periods of time and might, in fact, act as a germicide reservoir to prevent proliferation of microorganisms in contiguous materials. As initially conceived, such a polymer was expected t o have commercial utility as a material of construction for such medical specialty products as catheters, sutures, respirator masks and tubing, and drainage bags. The antimicrobial ability, by itself, could also *Present address: BASF/Wyandotte Corporation, Wyandotte, Michigan. ?Present address : Drew Chemical Company, Boonton, New Jersey. fPresent address: Union Carbide Corporation, South Charleston, West Virginia 25303.
55
91975 by John Wiley & Sons, Inc.
56
ACKART ET AL.
find utility in a broad spectrum of industrial and consumer products where protection of the article itself against microbial attack is the requirement. Although biocidal properties, particularly mildew resistance, are presently available in commercial polymer compositions containing low concentrations of physically incorporated antimicrobial agents (e.g. chlorinated aromatics, organomctallic compounds), i t was anticipated that a chemically bonded system might lead to a variety of industrial, medical, and consumer products having improved resistance to microbial attack.
DISCUSSION Preparation of Antimicrobial Benzalkonium Ionomers I n order to achieve controlled release of an antimicrobial agent from such a polymer system, one must bond the agent to the polymer by some chemically reversible process. This has been achieved through the ability of a number of antimicrobial compounds to be readily converted to cationic forms. Using carboxyl-containing ethylene copolymers such as poly (ethylene-co-acrylic acid) (E/AA) , salts of these copolymers have been prepared in which the cation is based on nitrogen containing organic antimicrobial agents (Reaction 1, p. 57). Using alkylbenzyldimethylammonium (benzalkonium) cations (e.g. IV), as the antimicrobial agents, antimicrobial ionomers of Type I1 can be prepared by two methods. The carboxyl-containing copolymer can be neutralized directly by the benzalkonium hydroxide (111), or the partial sodium salt of the copolymer (V) can be ion exchanged with benzalkonium chlorides. The latter approach is preferred because of the commercial availability of benzalkonium antibacterials only as chlorides. Antimicrobial ionomers have been prepared in this manner from a varieLy of polymer backbones, including poly (ethylene-co-acrylic acid) (E/AA), partially saponified poly(ethy1ene-co-ethyl acrylate) (E/EA), poly(ethy1ene-co-methacrylic acid), and poly(styrene-co-acrylic acid). However, attention has centered on ethylene/acrylic acid copolymers' because of their better balance of mechanical and antimicrobial properties. Three processes have been investigated for the preparation of the benzalkonium ionomers of E/AA by the ion exchange route: an emulsion process, a pellet diffusion process, and a hot melt process.
ANTIiMICROBIAL POLY LIEHH
55
I U
Y n
II
J-
s
a
g.tf o x u-u
I
N
3 U
+8 ?
8 4 o x u--q
m u
I
+x L
n
+
58
ACKART E T AL.
Since sodium salts (ionomcrs) of E/AA arch easily emulsified in water, a convmirnt mcthod of accomplishing the ion rxchangc of thc bcnzalkonium ions into the polymer is by addition of bcnzalkonium halide to an aqueous emulsion of the sodium ionomer of E/AA. l’rrcipitation of thc bcnzalkonium ionomrr results as i t is formed. Ion exchange of the benzalkonium halide by diffusion into pellets of sodium ionomrrs of E/AA can be accomplished by reaction in a dilutr aqurous solution of the benzalkonium halide a t 25-60°C. Although it is possible t o obtain antimicrobially active bcnzalkonium ionomc~sby this method (as well as by a similar diffusion process employ ing dirrct neutralization of E/AA pellets by benzalkonium hydroxidr), thc levels of activity obtainrd arp not as high as with emulsion or mclt processes. Since a melt process represents a potrntially more cconomical rout(. to the benzalkonium ionomers, dirrct preparation of benzalkonium ionomers in t h r mclt was attempted. Sodium ionomers of lI:/AA or partially saponified E/EA copolymers were combined with concentrated aqueous solutions of benzalkonium cholride, water was removed under vacuum near room temperature and the resulting mixtures wrre fluxed on a two roll mill. Benzalkonium ionomers \\ rrc obtained which had activities equal to, or greater than, those obtained by thc othcr two proccsses.
Preparation of Antimicrobial 8-Hydroxquinolinium Ionomers A survey was made of other possiblc antimicrobial agcnts capable of salt formation with carboxylatcs in ordcr to ascertain whether a diffrrent antimicrobial agent would yield activities significantly superior to those of the benzalkonium ionomers. Ionomers of E/AA werc prcparcd by ion rxchangc with hydrochloride salts of the following compounds, all reported t o exhibit antimicrobial activity : bis-(pdimcthylaminophenyl) phenylmet~hane, tris-(p-dimethylaminophenyl) methane, 3,6-diaminoacridin(~,8-hydroxyquinoline, sulfadiazine, sulfathiazole, diaminodiphenylsulfone, 2,6-dichloro-4-nitroanilinr. Of thrsc ionomers, the 8-hydroxyquinolinium ionomer possessed a particularly high antibacterial activity. Preliminary analysis of its activity against Staphylococcus aureus gave values as much as 5fold greatm than the maximum achieved in the benzalkonium systems (Table I).
ANTIMICROBIAL POLYMERS
59
TABLE I Activity of Novel Antimicrobial Ionomers Antimicrobial activity Polymer E/AA E/AA E/AA E/AA E/AA E/AA E/AA E/AA Styrene/AA E/Methacrylic Acid E/AA
Biocide Malachite Green Crystal Violet sulfathiazone sulfadiazine 3,6-diaminoacridine 8-hydroxyquinoline diaminodiphenylsulfone 2,6-dichloro-4-nitro-aniline benxalkonium benzalkonium benzalkonium
Bacteria"
Fungib
0 0 0 0.062-0.064 0.42-0.50 45.5-750 0 0 0.24-0.25 0.065-0.068 0.02-155
a Against Staphylococcus, aureus, by zone of inhibition assay, expressed as activity equivalent to mg benzalkonium. h ASTM-11-1924.
After preparation of 8-hydroxyquinolinium ionomers was investigated in greater detail, it was found that ionomers could be obtained having reproducibly high activities by ion exchange of S-hydroxyquinolinium/hydrochloride with E/AA/NaA by either an emulsion or a pellet diffusion process. I n the pellet process, pellets of E/AA copolymer (19.6 wt % acrylic acid, melt index z.50) were immersed in aqueous sodium hydroxide a t 60°C until ~ 6 mole 0 yoneutralization of the acrylic acid groups was attained, after which they were immersed in a n aqueous solution of 8-hydroxyquinolium hydrochloride. The product (containing approximately 60 mol % acrylic acid, 16 mol % sodium acrylate and 24 mol % 8-hydroxyquinolinium acrylate) displayed a much more reproducible antimicrobial activity than could be obtained for benzalkonium ionomers prepared by this process. Benzalkonium ionomers prepared by the diffusion process suffered from significant disintegration of pellet surfaces during the reaction, probably due to a plasticizing effect of the benzalkonium moiety. Since the 8-hydroxyquinolinium ionomers prepared by this method do not suffer disintegration of the pellets, this may account for the better reproducibility obtained in this case. As in the case of benzal-
ACKAKT E T AL.
60
TABLE I1 8-Hydroxyqninolinium Ionomer" Blends in Low-Density Polyethyleneb
Sample
Tensile Modulus (Psi)
Polyethylene 2% Ionomer 50/, Ionomer 3oy0Ionomer 50% Ionomer 8-Hydroxyquinolinium
20,000 21,400 21,700 17,800 13,100 39,200
Strength (psi)
Elongation Yield a t Break Strength (psi) (%I
Antimicrobial Activityc
_.___
1,800 1,950 1,750 1,700 1,800 4,700
600 640 570 465 395 300
1,250 1,210 1,050 900 2,250
none 22.4 21.0 27.9
a The ionomer used in this experiment wm an ionomer containing 24 mol yo 8-hydroxyquinolinium acrylate, 36 mol yosodium acrylate, and 40 mol % acrylic acid. b Bakelite Polyethylene DFDA 4141 Natural. The antimicrobial activity (vs. Staphylococcus aureus) is expressed as the area of the inhibition zone in cm2.
konium ionomers prepared by the pellet diffusion process, the pellets are inhomogeneously reacted with the antimicrobial compound, but homogenization of the product can be accomplished during blending of the ionomer with other polymer systems.
Blends of Antimicrobial Ionomers with Commodity Polymers I n order to obtain the desired mechanical properties for a given end use of the antimicrobial ionomers and take maximum advantage of their antimicrobial properties, ionomers must form compatible blends with commodity polymers. It was desirable, therefore, to determine both the maximum levels of compatibility of these ionomers in a variety of commodity polymers and the minimum levels of incorporation which would provide adequate long-term protection for the polymers into which they are blended. Use of the ionomers as antibacterial and antifungal agents in commodity polymers represents an attractive alternative to arsenic ,lead, and other heavy metal compounds which presently account for most of the antimicrobial additivcs used in these areas. Blends of both the benzalkonium and 8-hydroxyquinolium ionomers were prepared with low- and high-density polyethylenes, polypropylene, poly(vinylchloride), and impact polystyrene, either on a two-roll
ANTIMICROBIAL POLYJIERS
61
mill or in a 1 in. extruder and their mechanical properties measured. The level of compatibility of polymers blends can be estimated from the composition in a series of blends a t which there is a significant drop in elongation a t break. This upper limit of compatibility appears to be around 20 wt % for the ionomers in these commodity polymers, with 10 w t representing a practical use limit. In order to determine the long-term antimicrobial efficacy of these polymer blends and compare their behavior with commercial antimocrobial polymer additives, two series of simulated use tests were devised. Blends of the ionomers and blends of commercial antimicrobial polymer additives with low-density polyethylene were compression molded into 4 in. x 4 in. x 0.020 in. plaques and were then subjected to a distilled water spray for 1000 hr a t 60°C. The antimocrobial activities of these blends were determined a t intervals during the test, and the rcsults are shown in Table 111. The 8TABLE I11 Blends of Antimicrobial Ionomers and Commercial Additives with Polyethylenea Weatherometer Studiesb Activity0 Germicide 100% Benzalkonium ionornerd 10% Benzalkoninm ionomer 0.5% 2 . 0 % &HQ Ionomer 5 . 0 % &HQ Ionomer 100% &HQ Ionomer 0.125% Brand X 1 . 3 % Brand Y 5 . 0 % Brand Y 0 . 2 % Brand Z 1 . 0 % Brand Z
0 hr
750 hr
1000 hr
8.7 8.3 11.2 23.3 16.7 28.5 25.1 13.2 11.0 0.7 3.7
2.8 0.6 0.3 0.1 15.3 26.0 12.5 2.5 2.9 zero zero
:3 . 8 1.5 0.3 8.3 26.9 39.4 13.1 2.3 3.2
zero 0.1
Bakelite Polyethylene DFDA 4141 Natural. Compression molded plaques of each sample were exposed to a continuous distilled water spray for the lengths of time given above. c Zone of inhibition (vs. Staphylococcus aureus) in em2, as the area of the inhibition zone. d Prepared by the pellet diffusion process, 100 mol % benzalkonium acrylate. Prepared by the pellet diffusion process, 30 mol % &hydroxgquinolinir~m acrylate (8 HQ). a
b
62
ACKAllT ET AL.
hydroxyquinolium ionomer blend survived this treatment better than any of the other blends tested, with its blend a t 5.0% retaining a high comparative activity after 1000 hr. The benzalkonium ionomer blend a t 10% did not survive well, falling below blends of two of the commercial additives tested, 0.125% Brand X and 5.0% Brand Y. The third commercial additive tested, 13rand Z (an organometallic compound of undisclosed composition 'i? hich is recommended as a n additive for vinyl compounds), showed very little retention of activity. The closest competitor to the 8-hydroxyquinolinium ionomer, Brand X, showed good retention of activity a t a low level of addition (0.125%). The composition of this material is not disclosed by the manufacturer, but analysis in these laboratories indicates that i t is probably a dispersion of diisobutylphenoxyethyl dimethylbenzylammonium chloride (Rohm and Haas, Hyamine 1622) in polyethylene. Ionomers were prepared using this compound as the quaternary ammonium halide and were found to have antimicrobial activities comparable to the benzalkonium ionomers. A similar series of polymer blends was also subjected to a more strenuous simulated use test. Here, a series of compression molded samples of blends were sprayed for 1000 hr with a 1% aqueous detergent (Igepal) solution a t 50°C, and the antimicrobial activities of the samples were periodically measured as in the previous test. These results, shown in'Table IV, indicate a substantial loss of activity for all samples.
Nature and Scope of Antimicrobial Activity Samples of antimicrobial material were routinely assayed for antibacterial activity by a '(zone of inhibition" technique in which the test microorganism, Staphylococcus aureus, was seeded in a tryptoneglucose-beef extract agar growth medium. Discs (13 mm in diameter) of the sample films were placed on the surface of the medium and the culture was incubated at 32°C for 20 hr. I n this procedure the antibacterial agent diffuses from the disc through the agar medium and a clear zone of no-growth remains surrounding the disc whenever inhibitory concentrations of the agent are attained. The remainder of the agar surface where bacterial growth develops becomes opaque resulting in a halo or ' zone of inhibition" surrounding the disc. The size of the zone of inhibition can be related to the concentration of available inhibitory agent by rigidly controlling experimental vari-
ANTIMICROBIAL POLYMERS
63
TABLE IV Detergent-Water Spray Test" of Antimicrobial Ionomers and Physical Blends of Antimicrobial Polymer Additives in Polyethyleneb Antibacterial Activity0 Sample 2 . 0 % 8-Hydroxyquinoline ionomer in polyethylene 5 . 0 % 8-Hydroxyquinoline ionomer in polyethylene Pure 8-hydroxyquinoline ionomer 0.125% Brand X in polyethylene 5.0% Brand X in polyethylene 0.48% 8-Hydroxyquinoline in polyethylened 1 .O% Hyamine 1622 in polyethylene Hyamine 1622 inomer
Zero hr
500 hr
1000 hr
22.4
2.1
3.2
21.0
1.8
2.3
27.9 16.7 11.2
1.1 1.2 2.5
4.7 2.3 2.0
16.7
NAe
14.6 10.0
1.5 1.2
1.5 1.7
The samples were sprayed with a 1% Igepal/H20 solution at 20°C. Bakelite Polyethylene DFDA 4141 Natural. c The antimicrobial activity of the samples is expressed as the area (in cmz) of the zone inhibition produced. d This blend of 8-hydroxyquinoline in DFDA 4141 Natural corresponds to the same wt % of fbhydroxyquinoline as that found in a blend of 5.0% of 8-hydroxyquinoline ionomer of D X M 238 in DYNH. NA = not active. b
ables. This technique was made quantitative by the use of a doseresponse curve obtained from known quantities of the pure inhibitory agent contained on discs of a suitable substrate, which were then placed on a seeded agar surface. Concentrations were then plotted against resultant zones of inhibition. The activity of the unknown sample was then read off the curve as being equivalent t o mg/disc of the pure compound. A ('check'' disc containing a known concentration and a n unknown were placed on each petri dish t o correct for biological variations. I n this work the known solutions were applied t o discs of sterile agar cut by a cork borer from a poured plate which were placed on the surface of the seeded agar. To determine the effectiveness of the ionomers and their blends in polyethylene against pathogenic bacteria normally encountered out-
64
ACKART
Er
AL.
side the laboratory, a series of blends of the ionomers and of some commercial antimicrobial polymer additives was tested by a commercial testing laboratory (American Biological Control Laboratories, Tenafly, N.J.) against a number of bacteria isolated from hospital sources. Bacteria obtained in this way are hardier and more virulent than Staphylococcus aureus 209, and thus constituted a more stringent test of activity. The tests, run in the same manner as the zone of inhibition antimicrobial assays performed in these laboratories, consisted of placing a circular disc of the sample on the surface of an agar plate seeded with the bacteria. After incubation, the diameter of the circular area around the sample in which no bacterial growth occurred was measured and reported (mm). As reported in Table V, both the benzalkonium and 8-hydroxyquinolinium ionomers and their blends produced measurable zones of inhibition against all the bacteria tested as did Brand X, in polyethylene, the competitive additive previously shown to possess good long-term activity. A primary consideration regarding antimicrobial compounds is whether they function by killing or by simply inhibiting growth. This distinction is of crucial importance t o applications involving ionomers in medical plastics or other uses where their presence is intended to provide antibacterial protection to the user of the article in which they are compounded. Effective protection then required that the antimicrobial agent must kill pathogenic bacteria within a reasonable time period under the following three types of exposure conditions : 1. An environment in wThich optimal provision for liquid-liquid and liquid-solid interphase transfer of the residual chemical is provided through direct contact between the surface of the treated materials and the bacteria with excess moisture present during the entire exposure period, as would be the case with damp surf aces. 2. An environment in which provision for direct liquid-liquid and liquid-solid interphase transfer is not excluded but transfer occurs only as a function of direct contact between the dry surfaces of treated materials and the dry bacterial cells, with such mobilization of the chemical as may result under ambient conditions of relative humidity (50%) and temperature.
b
a
0 = Inactive material. Bakelite Polyethylene DFDA 4141 Natural.
+ = Active material.
Benzalkonium ionomer Benzalkonium ionomer (10% in polyethy1ene)b 8-HQ ionomer &HQ ionomer (5% in polyethylene) 8-HQ ionomer (2% in polyethylene) Brand X (0.125% in polyethylene) Brand Z (0.2%in polyethylene) Brand Z (1% in polyethylene) Brand Y (1.3% in polyethylene) Brand Y (5% in polyethylene)
+
0 0
+ 0 + + 0 + + 0
0
+
-t 0
+ + + + +
+ + + + + + + + 0 +
Enteroccus (unknown Escherichia species) coli Klebsiella
TABLE V Antimicrobial Activity Analysis
+0 +
+ + + + + + +
Proteus
+ + + + + + 0 +Q +
Pseudomonas
+ + + + + + + + 0 +
0 0
+ + + + + + + +
Staphylo- Pseudococcus monas aureus aeruginosa
66
ACKART ET AL.
3. An environment in which no direct liquid-liquid or liquid-solid interphase transfer of the residual chemical occurs, as would be the case with dry bacterial cells positioned on dust particles lying on the surface of material containing the residual chemical. Environmental requirements (2) and (3) at face value alone, are not promising for the antimicrobial ionomers since the mechanism by which these agents work is probably by ion-exchange diffusion of the benzalkonium or 8-hydroxyquinolinium ion out of the polymer surface; this necessitates a liquid (aqueous) transfer medium which would not be present under such conditions. Since the zone of inhibition test does not make a distinction between bacteriocidal and bacteriostatic activity, a bacterial eel1 count test was devised based on the least stringent environmental exposure which was used to test thc 8-hydroxyquinolinium ionomer for activity. I n this procedure, a known number of viable bacterial cells (Staphylococcus aureus 209) as a suspension in distilled water were impregnated on tissue paper discs which were then dried and placed on the surfaces of antimicrobial and control polymer specimens. The discs were moistened and held a t room temperature and 1 0 0 ~relative o humidity. The numbers of surviving cells were determined by plate count after drying and after 1.5, 3 and 6 hr exposure. To establish eficacy of the product, there should be significant kill before 3 hr, preferably within f hr. The results of the test, shown in Table VI, demonstrate that the 8-hydroxyquinolinium ionomer inhibits growth of bacteria and does not kill. These ionomers, therefore, would not be expected to provide significant protection to the user from pathogenic bacteria on the surface of an article fabricated from the ionomem. Elimination of antimicrobial ionomers from consideration in applications such as surface treatments for hospital rooms, protective bed and pillow coverings for hospital beds, etc., leaves two general area where the ionomers might be of use. Medical devices such as urinary catheters and tracheotomy tubes function in environments where their use greatly increases patients’ danger of developing new bacterial infections. The wet environment associated with the use of these devices provides optimum conditions for activation of the antimicrobial ionomers. Their use in such devices would classify them as drugs (therapeutic chemicals) instead of economic poisons and thus place them under the jurisdiction of the Food and Drug Administra-
ANTIMICROBIAL POLYhlEHY
67
TABLE VI Viable Cell Recovery Test for 8-Hydroxyquinolinium Ioiiomer Viable cells per disc, initial:
40, OOO ,000 45,000,000
Viable cells per disc, after drying:
30,000,000 20, OOO ,000
Viffible cells per disc, after exposure: Control Surface
Antimicrobial Surface
7,500,000 11,000,000
2,000,000 9,000,000
3 hr
5,000,000 850,000
2,000,000 3,000,000
6 hr
1 ,200,000 3,500,000
2,500,000 msg.
1% hr
tion. Certification for such use through the ICDA rcquires demonstration of both safety and efficacy in a clinical srnsr which is beyond the scope of the present study. The other major area of use is in products whcre t h r antimicrobial agent is intended t o protect only the article itself from microbial deterioration. Two possibilities in this area are treatment of fabrics and coatings for protection from mildew. The 8-hydroxyquinolium ionomer has been shown in zone of inhibition assay to inhibit the growth of four common fungi (Aspergillus niger, Aspergillus flavus, Penicillium funiculosum, and Chaetomium globosum). Samples (20 mil plaques) of the 8-hydroxyquinolinium ionomer and its blends with a n ethylene-vinyl acetate copolymer were also tested by the British Paint Association Laboratories of Middlesex, England, and were found t o allow little or no growth against four fungi (Aureobasidium pullulans, Cladosporium herbarum, Paecilom yces variotii, and Xtemphylium dendriticum) after 42 days exposure. Emulsions of the 8hydroxyquinolium ionomer at 20% solids in water, prepared by concurrent neutralization of most of the free acrylic acid groups in the ionomer with ammonium hydroxide during the emulsification, were tested for antifungal activity in poly(viny1 acetatc) and acrylic emulsion paint formulations by the British Paint Association. These
68
ACKAltT E T AI,.
formulations, however, showed very limited activity against fungi and discouraged further investigation of their use as fungicides for paints.
Toxicity of the Ionomers Both the benzalkonium and 8-hydroxyquinolinium ionomers have been tested for skin irritation by application to the clipped uncovered intact skin of rabbits, with no adverse effects in either test. Inhalation toxicity tests were also carried out in which substantially saturated vapor is prepared by spreading a 50g sample over 200 cm2 area on a shallow tray placed near the top of a 120-liter glass chamber, which is then sealed for a t least 16 hr while an intermittently operated fan agitates the internal chamber atmosphere. Rats are then introduced in a gasketed drawrr-type cage designed and operated to minimize vapor loss. Neither polymer produced adverse effects in these tests. A 10% blend of the 8-hydroxyquinolinium ionomer’ in polyethylene has also been tested for clearance as a Class VI medical packaging material and has passed all elemrnts of this test. The technical support of K. S. Craig, R. Pogers, A. D. Hammerich, L. I). Mathew, and L. M. Robeson in the performance of segments of this project is acknowledged.
References 1. J. H. Blackwell and I). Heister, “Application of the Tooke and Kethley Method for the Measurement of Residual Antimicrobial Activity of Treated Material,” to be published. 2. L. S. Stuart, Develop. Znd. Mzcrobzol, 1, 65 (1960). 3 . W. R. Tooke and T. W. Kethley, Cdem. Spec. Mf. Assoc. Proc. Ann. Meet., 53, 116 (1966).
Received January 22, 1974 Revised June 4, 1974
J. BIORIED. 31ATEII. RES.
Antimicrobial Polymers W. B. ACKART, R. L. CAMP*, W. L. WHEELWRIGHT?, and J. S. BYCKS. Research and Development Department, Chemicals and Plastics, Vnion Carbide Corporation, Bound Brook, Xew Jersey
Summary A number of carboxyl-containing ethylene copolymers have been prepared which exhibit long term antibacterial and antifungal properties. These materials, containing antimicrobial agents bound to the copolymer backbone as carboxglate salts, have been tested for their applicability to hospital products as a means of providing “self-sanitizing” articles. Tests have shown that these materials, although not bactericidal, do inhibit microbial growth. Investigations of the compatibility of these polymers with commodity polymers have been made and water emulsions of the polymers have been tested for applicability as components of product protectant coatings.
INTRODUCTION As part of a biomedical polymers research program, a project was initiated for the purpose of developing antimicrobial polymer systems. The specific objective was to create new polymer compositions consisting of antibacterial and/or antifungal molecules reversibly bonded t o suitable polymer backbones. Through gradual release of the active agent, the polymer should, a t minimum, be self-sanitizing over prolonged periods of time and might, in fact, act as a germicide reservoir to prevent proliferation of microorganisms in contiguous materials. As initially conceived, such a polymer was expected t o have commercial utility as a material of construction for such medical specialty products as catheters, sutures, respirator masks and tubing, and drainage bags. The antimicrobial ability, by itself, could also *Present address: BASF/Wyandotte Corporation, Wyandotte, Michigan. ?Present address : Drew Chemical Company, Boonton, New Jersey. fPresent address: Union Carbide Corporation, South Charleston, West Virginia 25303.
55
91975 by John Wiley & Sons, Inc.
56
ACKART ET AL.
find utility in a broad spectrum of industrial and consumer products where protection of the article itself against microbial attack is the requirement. Although biocidal properties, particularly mildew resistance, are presently available in commercial polymer compositions containing low concentrations of physically incorporated antimicrobial agents (e.g. chlorinated aromatics, organomctallic compounds), i t was anticipated that a chemically bonded system might lead to a variety of industrial, medical, and consumer products having improved resistance to microbial attack.
DISCUSSION Preparation of Antimicrobial Benzalkonium Ionomers I n order to achieve controlled release of an antimicrobial agent from such a polymer system, one must bond the agent to the polymer by some chemically reversible process. This has been achieved through the ability of a number of antimicrobial compounds to be readily converted to cationic forms. Using carboxyl-containing ethylene copolymers such as poly (ethylene-co-acrylic acid) (E/AA) , salts of these copolymers have been prepared in which the cation is based on nitrogen containing organic antimicrobial agents (Reaction 1, p. 57). Using alkylbenzyldimethylammonium (benzalkonium) cations (e.g. IV), as the antimicrobial agents, antimicrobial ionomers of Type I1 can be prepared by two methods. The carboxyl-containing copolymer can be neutralized directly by the benzalkonium hydroxide (111), or the partial sodium salt of the copolymer (V) can be ion exchanged with benzalkonium chlorides. The latter approach is preferred because of the commercial availability of benzalkonium antibacterials only as chlorides. Antimicrobial ionomers have been prepared in this manner from a varieLy of polymer backbones, including poly (ethylene-co-acrylic acid) (E/AA), partially saponified poly(ethy1ene-co-ethyl acrylate) (E/EA), poly(ethy1ene-co-methacrylic acid), and poly(styrene-co-acrylic acid). However, attention has centered on ethylene/acrylic acid copolymers' because of their better balance of mechanical and antimicrobial properties. Three processes have been investigated for the preparation of the benzalkonium ionomers of E/AA by the ion exchange route: an emulsion process, a pellet diffusion process, and a hot melt process.
ANTIiMICROBIAL POLY LIEHH
55
I U
Y n
II
J-
s
a
g.tf o x u-u
I
N
3 U
+8 ?
8 4 o x u--q
m u
I
+x L
n
+
58
ACKART E T AL.
Since sodium salts (ionomcrs) of E/AA arch easily emulsified in water, a convmirnt mcthod of accomplishing the ion rxchangc of thc bcnzalkonium ions into the polymer is by addition of bcnzalkonium halide to an aqueous emulsion of the sodium ionomer of E/AA. l’rrcipitation of thc bcnzalkonium ionomrr results as i t is formed. Ion exchange of the benzalkonium halide by diffusion into pellets of sodium ionomrrs of E/AA can be accomplished by reaction in a dilutr aqurous solution of the benzalkonium halide a t 25-60°C. Although it is possible t o obtain antimicrobially active bcnzalkonium ionomc~sby this method (as well as by a similar diffusion process employ ing dirrct neutralization of E/AA pellets by benzalkonium hydroxidr), thc levels of activity obtainrd arp not as high as with emulsion or mclt processes. Since a melt process represents a potrntially more cconomical rout(. to the benzalkonium ionomers, dirrct preparation of benzalkonium ionomers in t h r mclt was attempted. Sodium ionomers of lI:/AA or partially saponified E/EA copolymers were combined with concentrated aqueous solutions of benzalkonium cholride, water was removed under vacuum near room temperature and the resulting mixtures wrre fluxed on a two roll mill. Benzalkonium ionomers \\ rrc obtained which had activities equal to, or greater than, those obtained by thc othcr two proccsses.
Preparation of Antimicrobial 8-Hydroxquinolinium Ionomers A survey was made of other possiblc antimicrobial agcnts capable of salt formation with carboxylatcs in ordcr to ascertain whether a diffrrent antimicrobial agent would yield activities significantly superior to those of the benzalkonium ionomers. Ionomers of E/AA werc prcparcd by ion rxchangc with hydrochloride salts of the following compounds, all reported t o exhibit antimicrobial activity : bis-(pdimcthylaminophenyl) phenylmet~hane, tris-(p-dimethylaminophenyl) methane, 3,6-diaminoacridin(~,8-hydroxyquinoline, sulfadiazine, sulfathiazole, diaminodiphenylsulfone, 2,6-dichloro-4-nitroanilinr. Of thrsc ionomers, the 8-hydroxyquinolinium ionomer possessed a particularly high antibacterial activity. Preliminary analysis of its activity against Staphylococcus aureus gave values as much as 5fold greatm than the maximum achieved in the benzalkonium systems (Table I).
ANTIMICROBIAL POLYMERS
59
TABLE I Activity of Novel Antimicrobial Ionomers Antimicrobial activity Polymer E/AA E/AA E/AA E/AA E/AA E/AA E/AA E/AA Styrene/AA E/Methacrylic Acid E/AA
Biocide Malachite Green Crystal Violet sulfathiazone sulfadiazine 3,6-diaminoacridine 8-hydroxyquinoline diaminodiphenylsulfone 2,6-dichloro-4-nitro-aniline benxalkonium benzalkonium benzalkonium
Bacteria"
Fungib
0 0 0 0.062-0.064 0.42-0.50 45.5-750 0 0 0.24-0.25 0.065-0.068 0.02-155
a Against Staphylococcus, aureus, by zone of inhibition assay, expressed as activity equivalent to mg benzalkonium. h ASTM-11-1924.
After preparation of 8-hydroxyquinolinium ionomers was investigated in greater detail, it was found that ionomers could be obtained having reproducibly high activities by ion exchange of S-hydroxyquinolinium/hydrochloride with E/AA/NaA by either an emulsion or a pellet diffusion process. I n the pellet process, pellets of E/AA copolymer (19.6 wt % acrylic acid, melt index z.50) were immersed in aqueous sodium hydroxide a t 60°C until ~ 6 mole 0 yoneutralization of the acrylic acid groups was attained, after which they were immersed in a n aqueous solution of 8-hydroxyquinolium hydrochloride. The product (containing approximately 60 mol % acrylic acid, 16 mol % sodium acrylate and 24 mol % 8-hydroxyquinolinium acrylate) displayed a much more reproducible antimicrobial activity than could be obtained for benzalkonium ionomers prepared by this process. Benzalkonium ionomers prepared by the diffusion process suffered from significant disintegration of pellet surfaces during the reaction, probably due to a plasticizing effect of the benzalkonium moiety. Since the 8-hydroxyquinolinium ionomers prepared by this method do not suffer disintegration of the pellets, this may account for the better reproducibility obtained in this case. As in the case of benzal-
ACKAKT E T AL.
60
TABLE I1 8-Hydroxyqninolinium Ionomer" Blends in Low-Density Polyethyleneb
Sample
Tensile Modulus (Psi)
Polyethylene 2% Ionomer 50/, Ionomer 3oy0Ionomer 50% Ionomer 8-Hydroxyquinolinium
20,000 21,400 21,700 17,800 13,100 39,200
Strength (psi)
Elongation Yield a t Break Strength (psi) (%I
Antimicrobial Activityc
_.___
1,800 1,950 1,750 1,700 1,800 4,700
600 640 570 465 395 300
1,250 1,210 1,050 900 2,250
none 22.4 21.0 27.9
a The ionomer used in this experiment wm an ionomer containing 24 mol yo 8-hydroxyquinolinium acrylate, 36 mol yosodium acrylate, and 40 mol % acrylic acid. b Bakelite Polyethylene DFDA 4141 Natural. The antimicrobial activity (vs. Staphylococcus aureus) is expressed as the area of the inhibition zone in cm2.
konium ionomers prepared by the pellet diffusion process, the pellets are inhomogeneously reacted with the antimicrobial compound, but homogenization of the product can be accomplished during blending of the ionomer with other polymer systems.
Blends of Antimicrobial Ionomers with Commodity Polymers I n order to obtain the desired mechanical properties for a given end use of the antimicrobial ionomers and take maximum advantage of their antimicrobial properties, ionomers must form compatible blends with commodity polymers. It was desirable, therefore, to determine both the maximum levels of compatibility of these ionomers in a variety of commodity polymers and the minimum levels of incorporation which would provide adequate long-term protection for the polymers into which they are blended. Use of the ionomers as antibacterial and antifungal agents in commodity polymers represents an attractive alternative to arsenic ,lead, and other heavy metal compounds which presently account for most of the antimicrobial additivcs used in these areas. Blends of both the benzalkonium and 8-hydroxyquinolium ionomers were prepared with low- and high-density polyethylenes, polypropylene, poly(vinylchloride), and impact polystyrene, either on a two-roll
ANTIMICROBIAL POLYJIERS
61
mill or in a 1 in. extruder and their mechanical properties measured. The level of compatibility of polymers blends can be estimated from the composition in a series of blends a t which there is a significant drop in elongation a t break. This upper limit of compatibility appears to be around 20 wt % for the ionomers in these commodity polymers, with 10 w t representing a practical use limit. In order to determine the long-term antimicrobial efficacy of these polymer blends and compare their behavior with commercial antimocrobial polymer additives, two series of simulated use tests were devised. Blends of the ionomers and blends of commercial antimicrobial polymer additives with low-density polyethylene were compression molded into 4 in. x 4 in. x 0.020 in. plaques and were then subjected to a distilled water spray for 1000 hr a t 60°C. The antimocrobial activities of these blends were determined a t intervals during the test, and the rcsults are shown in Table 111. The 8TABLE I11 Blends of Antimicrobial Ionomers and Commercial Additives with Polyethylenea Weatherometer Studiesb Activity0 Germicide 100% Benzalkonium ionornerd 10% Benzalkoninm ionomer 0.5% 2 . 0 % &HQ Ionomer 5 . 0 % &HQ Ionomer 100% &HQ Ionomer 0.125% Brand X 1 . 3 % Brand Y 5 . 0 % Brand Y 0 . 2 % Brand Z 1 . 0 % Brand Z
0 hr
750 hr
1000 hr
8.7 8.3 11.2 23.3 16.7 28.5 25.1 13.2 11.0 0.7 3.7
2.8 0.6 0.3 0.1 15.3 26.0 12.5 2.5 2.9 zero zero
:3 . 8 1.5 0.3 8.3 26.9 39.4 13.1 2.3 3.2
zero 0.1
Bakelite Polyethylene DFDA 4141 Natural. Compression molded plaques of each sample were exposed to a continuous distilled water spray for the lengths of time given above. c Zone of inhibition (vs. Staphylococcus aureus) in em2, as the area of the inhibition zone. d Prepared by the pellet diffusion process, 100 mol % benzalkonium acrylate. Prepared by the pellet diffusion process, 30 mol % &hydroxgquinolinir~m acrylate (8 HQ). a
b
62
ACKAllT ET AL.
hydroxyquinolium ionomer blend survived this treatment better than any of the other blends tested, with its blend a t 5.0% retaining a high comparative activity after 1000 hr. The benzalkonium ionomer blend a t 10% did not survive well, falling below blends of two of the commercial additives tested, 0.125% Brand X and 5.0% Brand Y. The third commercial additive tested, 13rand Z (an organometallic compound of undisclosed composition 'i? hich is recommended as a n additive for vinyl compounds), showed very little retention of activity. The closest competitor to the 8-hydroxyquinolinium ionomer, Brand X, showed good retention of activity a t a low level of addition (0.125%). The composition of this material is not disclosed by the manufacturer, but analysis in these laboratories indicates that i t is probably a dispersion of diisobutylphenoxyethyl dimethylbenzylammonium chloride (Rohm and Haas, Hyamine 1622) in polyethylene. Ionomers were prepared using this compound as the quaternary ammonium halide and were found to have antimicrobial activities comparable to the benzalkonium ionomers. A similar series of polymer blends was also subjected to a more strenuous simulated use test. Here, a series of compression molded samples of blends were sprayed for 1000 hr with a 1% aqueous detergent (Igepal) solution a t 50°C, and the antimicrobial activities of the samples were periodically measured as in the previous test. These results, shown in'Table IV, indicate a substantial loss of activity for all samples.
Nature and Scope of Antimicrobial Activity Samples of antimicrobial material were routinely assayed for antibacterial activity by a '(zone of inhibition" technique in which the test microorganism, Staphylococcus aureus, was seeded in a tryptoneglucose-beef extract agar growth medium. Discs (13 mm in diameter) of the sample films were placed on the surface of the medium and the culture was incubated at 32°C for 20 hr. I n this procedure the antibacterial agent diffuses from the disc through the agar medium and a clear zone of no-growth remains surrounding the disc whenever inhibitory concentrations of the agent are attained. The remainder of the agar surface where bacterial growth develops becomes opaque resulting in a halo or ' zone of inhibition" surrounding the disc. The size of the zone of inhibition can be related to the concentration of available inhibitory agent by rigidly controlling experimental vari-
ANTIMICROBIAL POLYMERS
63
TABLE IV Detergent-Water Spray Test" of Antimicrobial Ionomers and Physical Blends of Antimicrobial Polymer Additives in Polyethyleneb Antibacterial Activity0 Sample 2 . 0 % 8-Hydroxyquinoline ionomer in polyethylene 5 . 0 % 8-Hydroxyquinoline ionomer in polyethylene Pure 8-hydroxyquinoline ionomer 0.125% Brand X in polyethylene 5.0% Brand X in polyethylene 0.48% 8-Hydroxyquinoline in polyethylened 1 .O% Hyamine 1622 in polyethylene Hyamine 1622 inomer
Zero hr
500 hr
1000 hr
22.4
2.1
3.2
21.0
1.8
2.3
27.9 16.7 11.2
1.1 1.2 2.5
4.7 2.3 2.0
16.7
NAe
14.6 10.0
1.5 1.2
1.5 1.7
The samples were sprayed with a 1% Igepal/H20 solution at 20°C. Bakelite Polyethylene DFDA 4141 Natural. c The antimicrobial activity of the samples is expressed as the area (in cmz) of the zone inhibition produced. d This blend of 8-hydroxyquinoline in DFDA 4141 Natural corresponds to the same wt % of fbhydroxyquinoline as that found in a blend of 5.0% of 8-hydroxyquinoline ionomer of D X M 238 in DYNH. NA = not active. b
ables. This technique was made quantitative by the use of a doseresponse curve obtained from known quantities of the pure inhibitory agent contained on discs of a suitable substrate, which were then placed on a seeded agar surface. Concentrations were then plotted against resultant zones of inhibition. The activity of the unknown sample was then read off the curve as being equivalent t o mg/disc of the pure compound. A ('check'' disc containing a known concentration and a n unknown were placed on each petri dish t o correct for biological variations. I n this work the known solutions were applied t o discs of sterile agar cut by a cork borer from a poured plate which were placed on the surface of the seeded agar. To determine the effectiveness of the ionomers and their blends in polyethylene against pathogenic bacteria normally encountered out-
64
ACKART
Er
AL.
side the laboratory, a series of blends of the ionomers and of some commercial antimicrobial polymer additives was tested by a commercial testing laboratory (American Biological Control Laboratories, Tenafly, N.J.) against a number of bacteria isolated from hospital sources. Bacteria obtained in this way are hardier and more virulent than Staphylococcus aureus 209, and thus constituted a more stringent test of activity. The tests, run in the same manner as the zone of inhibition antimicrobial assays performed in these laboratories, consisted of placing a circular disc of the sample on the surface of an agar plate seeded with the bacteria. After incubation, the diameter of the circular area around the sample in which no bacterial growth occurred was measured and reported (mm). As reported in Table V, both the benzalkonium and 8-hydroxyquinolinium ionomers and their blends produced measurable zones of inhibition against all the bacteria tested as did Brand X, in polyethylene, the competitive additive previously shown to possess good long-term activity. A primary consideration regarding antimicrobial compounds is whether they function by killing or by simply inhibiting growth. This distinction is of crucial importance t o applications involving ionomers in medical plastics or other uses where their presence is intended to provide antibacterial protection to the user of the article in which they are compounded. Effective protection then required that the antimicrobial agent must kill pathogenic bacteria within a reasonable time period under the following three types of exposure conditions : 1. An environment in wThich optimal provision for liquid-liquid and liquid-solid interphase transfer of the residual chemical is provided through direct contact between the surface of the treated materials and the bacteria with excess moisture present during the entire exposure period, as would be the case with damp surf aces. 2. An environment in which provision for direct liquid-liquid and liquid-solid interphase transfer is not excluded but transfer occurs only as a function of direct contact between the dry surfaces of treated materials and the dry bacterial cells, with such mobilization of the chemical as may result under ambient conditions of relative humidity (50%) and temperature.
b
a
0 = Inactive material. Bakelite Polyethylene DFDA 4141 Natural.
+ = Active material.
Benzalkonium ionomer Benzalkonium ionomer (10% in polyethy1ene)b 8-HQ ionomer &HQ ionomer (5% in polyethylene) 8-HQ ionomer (2% in polyethylene) Brand X (0.125% in polyethylene) Brand Z (0.2%in polyethylene) Brand Z (1% in polyethylene) Brand Y (1.3% in polyethylene) Brand Y (5% in polyethylene)
+
0 0
+ 0 + + 0 + + 0
0
+
-t 0
+ + + + +
+ + + + + + + + 0 +
Enteroccus (unknown Escherichia species) coli Klebsiella
TABLE V Antimicrobial Activity Analysis
+0 +
+ + + + + + +
Proteus
+ + + + + + 0 +Q +
Pseudomonas
+ + + + + + + + 0 +
0 0
+ + + + + + + +
Staphylo- Pseudococcus monas aureus aeruginosa
66
ACKART ET AL.
3. An environment in which no direct liquid-liquid or liquid-solid interphase transfer of the residual chemical occurs, as would be the case with dry bacterial cells positioned on dust particles lying on the surface of material containing the residual chemical. Environmental requirements (2) and (3) at face value alone, are not promising for the antimicrobial ionomers since the mechanism by which these agents work is probably by ion-exchange diffusion of the benzalkonium or 8-hydroxyquinolinium ion out of the polymer surface; this necessitates a liquid (aqueous) transfer medium which would not be present under such conditions. Since the zone of inhibition test does not make a distinction between bacteriocidal and bacteriostatic activity, a bacterial eel1 count test was devised based on the least stringent environmental exposure which was used to test thc 8-hydroxyquinolinium ionomer for activity. I n this procedure, a known number of viable bacterial cells (Staphylococcus aureus 209) as a suspension in distilled water were impregnated on tissue paper discs which were then dried and placed on the surfaces of antimicrobial and control polymer specimens. The discs were moistened and held a t room temperature and 1 0 0 ~relative o humidity. The numbers of surviving cells were determined by plate count after drying and after 1.5, 3 and 6 hr exposure. To establish eficacy of the product, there should be significant kill before 3 hr, preferably within f hr. The results of the test, shown in Table VI, demonstrate that the 8-hydroxyquinolinium ionomer inhibits growth of bacteria and does not kill. These ionomers, therefore, would not be expected to provide significant protection to the user from pathogenic bacteria on the surface of an article fabricated from the ionomem. Elimination of antimicrobial ionomers from consideration in applications such as surface treatments for hospital rooms, protective bed and pillow coverings for hospital beds, etc., leaves two general area where the ionomers might be of use. Medical devices such as urinary catheters and tracheotomy tubes function in environments where their use greatly increases patients’ danger of developing new bacterial infections. The wet environment associated with the use of these devices provides optimum conditions for activation of the antimicrobial ionomers. Their use in such devices would classify them as drugs (therapeutic chemicals) instead of economic poisons and thus place them under the jurisdiction of the Food and Drug Administra-
ANTIMICROBIAL POLYhlEHY
67
TABLE VI Viable Cell Recovery Test for 8-Hydroxyquinolinium Ioiiomer Viable cells per disc, initial:
40, OOO ,000 45,000,000
Viable cells per disc, after drying:
30,000,000 20, OOO ,000
Viffible cells per disc, after exposure: Control Surface
Antimicrobial Surface
7,500,000 11,000,000
2,000,000 9,000,000
3 hr
5,000,000 850,000
2,000,000 3,000,000
6 hr
1 ,200,000 3,500,000
2,500,000 msg.
1% hr
tion. Certification for such use through the ICDA rcquires demonstration of both safety and efficacy in a clinical srnsr which is beyond the scope of the present study. The other major area of use is in products whcre t h r antimicrobial agent is intended t o protect only the article itself from microbial deterioration. Two possibilities in this area are treatment of fabrics and coatings for protection from mildew. The 8-hydroxyquinolium ionomer has been shown in zone of inhibition assay to inhibit the growth of four common fungi (Aspergillus niger, Aspergillus flavus, Penicillium funiculosum, and Chaetomium globosum). Samples (20 mil plaques) of the 8-hydroxyquinolinium ionomer and its blends with a n ethylene-vinyl acetate copolymer were also tested by the British Paint Association Laboratories of Middlesex, England, and were found t o allow little or no growth against four fungi (Aureobasidium pullulans, Cladosporium herbarum, Paecilom yces variotii, and Xtemphylium dendriticum) after 42 days exposure. Emulsions of the 8hydroxyquinolium ionomer at 20% solids in water, prepared by concurrent neutralization of most of the free acrylic acid groups in the ionomer with ammonium hydroxide during the emulsification, were tested for antifungal activity in poly(viny1 acetatc) and acrylic emulsion paint formulations by the British Paint Association. These
68
ACKAltT E T AI,.
formulations, however, showed very limited activity against fungi and discouraged further investigation of their use as fungicides for paints.
Toxicity of the Ionomers Both the benzalkonium and 8-hydroxyquinolinium ionomers have been tested for skin irritation by application to the clipped uncovered intact skin of rabbits, with no adverse effects in either test. Inhalation toxicity tests were also carried out in which substantially saturated vapor is prepared by spreading a 50g sample over 200 cm2 area on a shallow tray placed near the top of a 120-liter glass chamber, which is then sealed for a t least 16 hr while an intermittently operated fan agitates the internal chamber atmosphere. Rats are then introduced in a gasketed drawrr-type cage designed and operated to minimize vapor loss. Neither polymer produced adverse effects in these tests. A 10% blend of the 8-hydroxyquinolinium ionomer’ in polyethylene has also been tested for clearance as a Class VI medical packaging material and has passed all elemrnts of this test. The technical support of K. S. Craig, R. Pogers, A. D. Hammerich, L. I). Mathew, and L. M. Robeson in the performance of segments of this project is acknowledged.
References 1. J. H. Blackwell and I). Heister, “Application of the Tooke and Kethley Method for the Measurement of Residual Antimicrobial Activity of Treated Material,” to be published. 2. L. S. Stuart, Develop. Znd. Mzcrobzol, 1, 65 (1960). 3 . W. R. Tooke and T. W. Kethley, Cdem. Spec. Mf. Assoc. Proc. Ann. Meet., 53, 116 (1966).
Received January 22, 1974 Revised June 4, 1974
