U.S. patent application number 11/107412 was filed with the patent office on 2005-11-10 for antimicrobial polymeric surfaces.
Invention is credited to Klibanov, Alexander M., Lewis, Kim, Liao, Chun-Jen, Tiller, Joerg C..
Application Number | 20050249695 11/107412 |
Document ID | / |
Family ID | 27494502 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249695 |
Kind Code |
A1 |
Tiller, Joerg C. ; et
al. |
November 10, 2005 |
Antimicrobial polymeric surfaces
Abstract
Bactericidal compositions are disclosed that comprise a
polymeric compound immobilized on a material. Medical devices are
also disclosed which comprise such a bactericidal composition.
Methods are disclosed for covalently derivatizing the surfaces of
common materials with an antibacterial polycation, e.g.,
poly(vinyl-N-pyridinium bromide); the first step of the methods
involves coating the surface with a nanolayer of silica. Various
commercial synthetic polymers derivatized in this manner are
bactericidal, i.e., they kill on contact up to 99% of deposited
Gram-positive and Gram-negative bacteria, whether deposited through
air or water.
Inventors: |
Tiller, Joerg C.; (Freiburg,
DE) ; Liao, Chun-Jen; (Taipei, TW) ; Lewis,
Kim; (Newton, MA) ; Klibanov, Alexander M.;
(Newton, MA) |
Correspondence
Address: |
PATREA L. PABST
PABST PATENT GROUP LLP
400 COLONY SQUARE
SUITE 1200
ATLANTA
GA
30361
US
|
Family ID: |
27494502 |
Appl. No.: |
11/107412 |
Filed: |
April 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11107412 |
Apr 14, 2005 |
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10123860 |
Apr 16, 2002 |
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60285883 |
Apr 23, 2001 |
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60340078 |
Dec 10, 2001 |
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60368495 |
Mar 29, 2002 |
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Current U.S.
Class: |
424/78.3 ;
424/423 |
Current CPC
Class: |
A01N 43/40 20130101;
A61L 27/34 20130101; A61L 27/50 20130101; A01N 43/40 20130101; A01N
25/10 20130101; A01N 2300/00 20130101; A01N 25/34 20130101; A01N
43/40 20130101; C08L 33/14 20130101; A61L 27/34 20130101 |
Class at
Publication: |
424/078.3 ;
424/423 |
International
Class: |
A61K 031/785 |
Goverment Interests
[0002] This invention was made with support provided by the
National Institutes of Health (Grant No. GM26698) and the National
Science Foundation (Grant No. DMR-9400334); therefore, the
government has certain rights in the invention.
Claims
1. A composition, comprised of a material and a compound
immobilized at said material, wherein said immobilized compound is
a polymer, wherein said polymer is not insoluble in water.
2-35. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 60/285,883, filed Apr. 23,
2001; U.S. Provisional Patent Application Ser. No. 60/340,078,
filed Dec. 10, 2001; and U.S. Provisional Patent Application Ser.
No. 60/368,495, filed Mar. 29, 2002.
BACKGROUND OF THE INVENTION
[0003] Due to the ever-growing demand for healthy living, there is
a keen interest in materials capable of killing harmful
microorganisms. Such materials could be used to coat surfaces of
common objects touched by people in everyday lives, e.g., door
knobs, children toys, computer keyboards, telephones, etc., to
render them antiseptic and thus unable to transmit bacterial
infections. Since ordinary materials are not antimicrobial, their
modification is required. For example, surfaces chemically modified
with poly(ethylene glycol) and certain other synthetic polymers can
repel (although not kill) microorganisms (Bridgett, M. J., et ak,
S. P. (1992) Biomaterials 13, 411-416. Arciola, C. R., et al
Alvergna, P., Cenni, E. & Pizzoferrato, A. (1993) Biomaterials
14, 1161-1164. Park, K. D., Kim, Y. S., Han, D. K., Kim, Y. H.,
Lee, E. H. B., Suh, H. & Choi, K. S. (1998) Biomaterials 19,
851-859.)
[0004] Alternatively, materials can be impregnated with
antimicrobial agents, such as antibiotics, quarternary ammonium
compounds, silver ions, or iodine, that are gradually released into
the surrounding solution over time and kill microorganisms there
(Medlin, J. (1997) Environ. Health Persp. 105, 290-292; Nohr, R. S.
& Macdonald, G. J. (1994) J. Biomater. Sci., Polymer Edn. 5,
607-619 Shearer, A. E. H., et al (2000) Biotechnol. Bioeng. 67,
141-146.). Although these strategies have been verified in aqueous
solutions containing bacteria, they would not be expected to be
effective against airborne bacteria in the absence of a liquid
medium; this is especially true for release-based materials, which
are also liable to become impotent when the leaching antibacterial
agent is exhausted.
[0005] Infection is a frequent complication of many invasive
surgical, therapeutic and diagnostic procedures. For procedures
involving implantable medical devices, avoiding infection can be
particularly problematic because bacteria can develop into
biofilms, which protect the microbes from clearing by the subject's
immune system. As these infections are difficult to treat with
antibiotics, removal of the device is often necessitated, which is
traumatic to the patient and increases the medical cost.
[0006] Any material left embedded in the body provides a surface
for accumulation of infectious microorganisms, particularly
bacteria and occasionally fungi. This is understood to take place
through the formation of biofilms. A biofilm is a type of fouling
that occurs when microorganisms attach to surfaces and secrete a
hydrated polymeric matrix that surrounds them. Microorganisms
existing in a biofilm, termed sessile, grow in a protected
environment that insulates them from attack from antimicrobial
agents. These sessile communities can give rise to nonsessile
individuals, termed planktonic, which rapidly multiply and
disperse. These planktonic organisms are responsible for invasive
and disseminated infections. They are the targets of antimicrobial
therapy. Conventional treatments fail to eradicate the sessile
communities rooted in the biofilm. Biofilms are understood to be a
frequently occurring reservoir for infectious agents. The biology
of biofilms is described in more detail in "Bacterial biofilms: a
common cause of persistent infection," J. Costerson, P. Stewart, E.
Greenberg, Science 284: 1318-1322 (1999), and "The riddle of
biofilm resistance," K. Lewis, Antimicrob. Agents Chemother., 45,
999-1007 (2001), incorporated herein by reference.
[0007] Biofilms develop preferentially on inert surfaces or on
non-living tissue, and occur commonly on medical devices and
devascularized or dead tissues. Biofilms have been identified on
sequestra of dead bone and on bone grafts, from which they can
incite an invasive infection called osteomyelitis that can kill
even more bone. Biofilms have been also identified on living,
hypovascular tissues such as native heart valves, where they are
responsible for the devastating infection called endocarditis where
the microorganism not only can colonize distant locations by
seeding throughout the bloodstream, but also can destroy the heart
valve itself. Infections involving implanted medical devices
generally involve biofilms, where a sessile community provides a
reservoir for an invasive infection. The presence of microorganisms
in a biofilm on a medical device represents contamination of that
foreign body. The elicitation by the biofilm of clinically
perceptible host responses constitutes an infection.
[0008] The development of an infection from an area of
contamination is consistent with the natural history of biofilm
growth and development. Biofilms grow slowly, in one or more
locations, colonized by one or a plurality of microorganisms. The
pattern of biofilm development involves initial attachment of a
microorganism to a solid surface, the formation of microcolonies
attached to the surface, and finally the differentiation of the
microcolonies into exopolysaccharide-encased mature biofilms.
Planktonic cells are released from biofilms in a natural pattern of
programmed detachment, so that the biofilm serves as a nidus for
multiple, recurrent acute invasive infections. Antibiotics
typically treat the infection caused by the planktonic organisms,
but fail to kill those sessile organisms protected in the
biofilm.
[0009] Sessile microorganisms also give rise to localized symptoms,
releasing antigens and stimulating antibody production that
activates the immune system to attack the biofilm and the area
surrounding it. Antibodies and host immune defenses are ineffective
in killing the organisms in the biofilm, even though these
organisms have elicited the antibody and related immune response.
The cytotoxic products of the host's immunologically activated
cells can be directed towards the host's own tissues. This
phenomenon is seen in the mouth, where the host's response to the
dental biofilm can inflame tissues surrounding the teeth and give
rise to periodontitis. This phenomenon can also give rise to local
inflammation around implanted medical devices and bone resorption
with loosening of orthopedic and dental implants.
[0010] While host defenses may hold invasive infections in check by
controlling the proliferation of planktonic organisms, this
favorable equilibrium presupposes an intact immune system. Many
patients in a hospital setting have compromised immune systems,
rendering them more vulnerable to invasive infections once a
biofilm community has become established. Patients requiring
implantable medical devices may likewise have compromised immune
systems, whether on a short-term or long-term basis. A poorly
functioning immune system puts the host at greater risk for initial
formation of a contaminated biofilm around a medical device and for
the invasion of planktonic organisms into the surrounding tissues
and the system. Once the planktonic organisms mount a full-scale
infection, the immunocompromised host will be less likely to
contain and control it, with potentially lethal results.
[0011] Protected from antibiotic treatment and host defenses, the
microorganisms in a biofilm typically cause recurrent infections
and low-grade local symptoms. The biofilm, once established, can
only be eradicated surgically. When a foreign object becomes
contaminated with microorganisms, the only way to eliminate local
and systemic infection may be to remove the contaminated foreign
article. If the material being removed is essential for health, a
similar article may need to be replaced in the same location; the
replacement article will be especially prone to infection because
of the residual microorganisms in the area.
[0012] Since the difficulties associated with eliminating
biofilm-based infections are well-recognized, a number of
technologies have developed to treat surfaces or fluids bathing
surfaces to prevent or impair biofilm formation. Biofilms adversely
affect medical systems and other systems essential to public health
such as water supplies and food production facilities. A number of
technologies have been proposed that treat surfaces with organic or
inorganic materials to interfere with biofilm development. For
example, various methods have been employed to coat the surfaces of
medical devices with antibiotics (See e.g. U.S. Pat. Nos.
4,107,121, 4,442,133, 4,895,566, 4,917,686, 5,013,306, 4,952,419,
5,853,745 and 5,902,283) and other bacteriostatic compounds (See
e.g U.S. Pat. Nos. 4,605,564, 4,886,505, 5,019,096, 5,295,979,
5,328,954, 5,681,575, 5,753,251, 5,770,255, and 5,877,243). Despite
these technologies, contamination of medical devices and invasive
infection therefrom continues to be a problem.
[0013] Infectious organisms are ubiquitous in the medical
environment, despite vigorous efforts to maintain antisepsis. The
presence of these organisms can result in infection of hospitalized
patients and medical personnel. These infections, termed
nosocomial, often involve organisms more virulent and more unusual
than those encountered outside the hospital. In addition,
hospital-acquired infections are more likely to involve organisms
that have developed resistance to a number of antibiotics. Although
cleansing and anti-bacterial regimens are routinely employed,
infectious organisms readily colonize a variety of surfaces in the
medical environment, especially those surfaces exposed to moisture
or immersed in fluid. Even barrier materials, such as gloves,
aprons and shields, can spread infection to the wearer or to others
in the medical environment. Despite sterilization and cleansing, a
variety of metallic and non-metallic materials in the medical
environment can retain dangerous organisms trapped in a biofilm,
thence to be passed on to other hosts.
[0014] Any agent used to impair biofilm formation in the medical
environment must be safe to the user. Certain biocidal agents, in
quantities sufficient to interfere with biofilms, also can damage
host tissues. Antibiotics introduced into local tissue areas can
induce the formation of resistant organisms which can then form
biofilm communities whose planktonic microorganisms would likewise
be resistant to the particular antibiotics. Any anti-biofilm or
antifouling agent must furthermore not interfere with the
salubrious characteristics of a medical device. Certain materials
are selected to have a particular type of operator manipulability,
softness, water-tightness, tensile strength or compressive
durability, characteristics that cannot be altered by an agent
added for anti-microbial effects.
[0015] As a further problem, it is possible that materials added to
the surfaces of implantable devices to inhibit contamination and
biofilm formation may be thrombogenic. Some implantable materials
are of themselves thrombogenic. For example, it has been shown that
contact with metal, glass, plastic or other similar surfaces can
induce blood to clot. Heparin compounds, which are known to have
anticoagulant effects, have therefore been applied to certain
medical devices prior to implantation. However, there are few known
products in the medical arsenal whose antimicrobial effects are
combined with antithrombogenic effects. This combination would be
particularly valuable to treat those medical devices that reside in
the bloodstream, such as heart valves, artificial pumping devices
("artificial hearts" or left ventricular assist devices), vascular
grafting prostheses and vascular stents. In these settings, clot
formation can obstruct the flow of blood through the conduit and
can furthermore break off pieces called emboli that are carried
downstream, potentially blocking circulation to distant tissues or
organs.
[0016] Biofilm formation has important public health implications.
Drinking water systems are known to harbor biofilms, even though
these environments often contain disinfectants. Any system
providing an interface between a surface and a fluid has the
potential for biofilm development. Water cooling towers for air
conditioners are well-known to pose public health risks from
biofilm formation, as episodic outbreaks of infections like
Legionnaires' Disease attest. Turbulent fluid flow over the surface
does not provide protection: biofilms can form in conduits where
flowing water or other fluids pass, with the effects of altering
flow characteristics and passing planktonic organisms downstream.
Industrial fluid processing operations have experienced mechanical
blockages, impedance of heat transfer processes, and
biodeterioration of fluid-based industrial products, all
attributable to biofilms. Biofilms have been identified in flow
conduits like hemodialysis tubing, and in water distribution
conduits. Biofilms have also been identified to cause biofouling in
selected municipal water storage tanks, private wells and drip
irrigation systems, unaffected by treatments with up to 200 ppm
chlorine.
[0017] Biofilms are a constant problem in food processing
environments. Food processing involves fluids, solid material and
their combination. As an example, milk processing facilities
provide fluid conduits and areas of fluid residence on surfaces.
Cleansing milking and milk processing equipment presently utilizes
interactions of mechanical, thermal and chemical processes in an
air-injected clean-in-place methods. Additionally, the milk product
itself is treated with pasteurization. In cheese producing,
biofilms can lead to the production of calcium lactate crystals in
Cheddar cheese. Meat processing and packing facilities are in like
manner susceptible to biofilm formation. Non-metallic and metallic
surfaces can be affected. Biofilms in meat processing facilities
have been detected on rubber "fingers," plastic curtains, conveyor
belt material, evisceration equipment and stainless steel surfaces.
Controlling biofilms and microorganism contamination in food
processing is hampered by the additional need that the agent used
not affect the taste, texture or aesthetics of the product.
[0018] There exists, therefore, a need to be able to render general
surfaces bactericidal. General surface coating/derivatization
procedures have been developed that should be applicable to most
materials regardless of their nature.
SUMMARY OF THE INVENTION
[0019] One aspect of the present invention is directed to
antimicrobial surfaces comprised of covalently attached amphipathic
compounds. In certain embodiments, a surface of the present
invention acts to eliminate airborne biologics on contact. In
certain embodiments, a surface of the present invention acts to
eliminate waterborne biologics on contact. In certain embodiments,
a surface of the present invention acts to prevent the formation of
biofilms.
[0020] In certain embodiments, said covalently attached amphipathic
compound is a polymer comprising ammonium ions. In a preferred
embodiment, said polymer has a molecular weight of at least 10,000
g/mol, more preferably 120,000 g/mol, and most preferably 150,000
g/mol.
[0021] In certain embodiments, the surface of the present invention
is glass. In certain other embodiments, the surface of the present
invention is a plastic. In a particular embodiment, the surface of
the present invention is an amino-bearing glass.
[0022] In certain embodiments, the present invention relates to a
composition, comprised of a material and a compound immobilized at
said material, wherein said immobilized compound is a polymer,
wherein said polymer is not insoluble in water. In certain
embodiments, said immobilized compound is a polycation. In certain
embodiments, said immobilized compound is a water-soluble
lipophilic polycation. In certain embodiments, said immobilized
compound is covalently linked to said solid material. In certain
embodiments, said immobilized compound comprises poly(N-alkyl
vinylpyridine) or poly(N-alkyl ethylene imine).
[0023] In certain embodiments, the compound covalently bonded to
the surface is represented by the formula I: 1
[0024] wherein
[0025] R represents individually for each occurrence hydrogen,
alkyl, alkenyl, alkynyl, acyl, aryl, carboxylate, alkoxycarbonyl,
aryloxycarbonyl, carboxamido, alkylamino, acylamino, alkoxyl,
acyloxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl,
thio, alkylthio, thioalkyl, (alkylthio)alkyl, carbamoyl, urea,
thiourea, sulfonyl, sulfonate, sulfonamido, sulfonylamino, or
sulfonyloxy;
[0026] R' represents independently for each occurrence alkyl, an
alkylidene tether to a surface, or an acyl tether to a surface;
[0027] Z represents independently for each occurrence Cl, Br, or I;
and
[0028] n is an integer less than or equal to about 1500.
[0029] In a preferred embodiment, the invention comprises
Poly(4-vinyl-N-alkylpyridinium bromide) or
poly(methacryloyloxydodecylpyr- idinium bromide) (MDPB) covalently
attached to a glass surface. In another preferred embodiment, the
invention comprises an N-alkylated poly(4-vinyl pyridine)
covalently attached to a glass surface.
[0030] In certain embodiments, the biologics killed on the surfaces
of the instant invention are bacteria. In a particular embodiment,
the bacteria killed are Gram-positive bacteria. In another
particular embodiment, the bacteria killed are Gram-negative
bacteria.
[0031] In certain embodiments, the invention is drawn to a method
for rendering a surface bactericidal. In certain embodiments, the
surface to be rendered bactericidal comprises amino functionality.
In certain embodiments the method for rendering a surface
bactericidal comprises derivatizing a surface possessing amino
functionality to form a covalently bound polymer comprising
quaternary amine groups on said surface.
[0032] In certain embodiments, the surface to be rendered
bactericidal does not have amino functionality, but amino
functionality is added to said surface by applying a layer of
SiO.sub.2 nanoparticles to said surface which form Si--OH groups
upon hydration, and which are then treated with an amination agent
to form a surface comprising amino functionality. In certain
embodiments, the amination agent is
3-aminopropyltriethoxysilane.
[0033] In certain embodiments the invention is drawn to a bacteria
resistant article comprising a covalently bound polymer upon its
surface wherein the covalently bound polymer comprises quaternary
ammonium groups. In certain embodiments the bacteria resistant
article is a household item. In certain embodiments the bacteria
resistant article is a medical device.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 depicts a commercial NH.sub.2-glass slide (left) and
a hexyl-PVP-- modified slide (right) onto which aqueous suspensions
(approximately 10.sup.6 cells/mL of distilled water) of S. aureus
cells were sprayed, air dried for 2 min, and incubated under 0.7%
agar in a bacterial growth medium at 37.degree. C. overnight.
[0035] FIG. 2 depicts the percentage of S. aureus colonies grown on
the infected surfaces of glass slides modified with PVP N-alkylated
with different linear alkyl bromides relative to the number of
colonies grown on a commercial NH.sub.2-glass slide.
[0036] FIG. 3 depicts a compound of the present invention prepared
by Method A.
[0037] FIG. 4 depicts a schematic illustration of the
derivatization of polymer surfaces with
poly(vinyl-N-hexylpyridinium bromide) (hexyl-PVP), comprising the
following four steps: coating with a SiO.sub.2 nanolayer using the
combustion chemical vapor deposition; treating with
3-aminopropyltriethoxysilane; alkylating with 1,4-dibromobutane;
and derivatizing with hexyl-PVP in the presence of
1-bromohexane.
[0038] FIG. 5 depicts photographs of a commercial HDPE slide (left)
and the hexyl-PVP-derivatized slide (right) onto which aqueous
suspensions (approximately 10.sup.6 cells/mL of distilled water) of
(a) S. aureus or (b) E. coli cells were sprayed, followed by air
drying for 2 min and incubation under 0.7% agar in a bacterial
growth medium at 37.degree. C. overnight.
[0039] FIG. 6 depicts photographs of a commercial HDPE slide (left)
and the hexyl-PVP-derivatized slide (right) onto which (a) S.
aureus or (b) E. coli cells were allowed to adhere from aqueous
PBS, pH 7.0, suspensions (approximately 10.sup.6 cells/mL of PBS),
washed with PBS, and incubated under 1.5% agar in a bacterial
growth medium at 37.degree. C. overnight.
[0040] FIG. 7 depicts the killing efficiency of
hexyl-PVP-derivatized HDPE toward airborne (.circle-solid.) or
waterborne (.smallcircle.)S. aureus cells deposited on the modified
polymer surface as a function of the surface density of the
pyridinium groups. The negative percentages correspond to a greater
number of bacterial cells adhered to the modified slides than to
the unmodified one.
[0041] FIG. 8 depicts graphically the bactericidal activity of
polyethylene slides covalently coated with hexyl-PVP against
wild-type and various antibiotic-resistant strains of S. aureus.
Either airborne (shaded bars) or waterborne (dashed bars) bacterial
suspensions were deposited onto the slide surface. The standard
deviations from the mean killing efficiency values are represented
by error bars.
[0042] FIG. 9 depicts graphically the bactericidal activity of
polyethylene slides covalently coated with hexyl-PVP against two
parent/mutant pairs of S. aureus strains. In the first pair (A),
the mutant is missing the MDR pump encoded by the norA gene (which
had been knocked out), while its otherwise identical parent has it.
In the second pair (B), the parent lacks the MDR pump encoded by
the qacA gene, whereas the plasmid-bearing strain has it.
DETAILED DESCRIPTION OF THE INVENTION
[0043] A. Overview
[0044] In general, the present invention relates to the prevention
of accumulation of microorganisms on any surface wherein such
accumulation has a deleterious effect on human or animal health. In
particular, the present invention relates to the prevention of
those conditions affecting human or animal health that involve
fouling. Fouling events involve recognition between a biologic and
a surface, adhesion of the biologic to the surface, and the
subsequent activity of the biologic. As understood herein, the
formation of a biofilm is a type of fouling. Biofilms with health
effects commonly contain infectious microorganisms.
[0045] In a health-related environment, fouling can result in
biofilm formation. Biofilm formation is understood to cause local
contamination of an affected area with potential for invasive local
infection and for systemic infection. Microorganisms may damage
tissues in at least three ways: 1) they can enter or contact host
cells and directly cause cell death; 2) they can release endotoxins
or exotoxins that kill cells at a distance, release enzymes that
degrade tissue components, or damage blood vessels and cause
ischemic necrosis; and 3) they can induce host-cellular responses
that, although directed against the invader, may cause additional
tissue damage, including suppuration, scarring and hypersensitivity
reactions. An infection, whether local or systemic, represents the
penetration of microorganisms into a host with the production of
tissue damage or the elicitation of host defense mechanisms or
both, leading to clinically identifiable symptoms. Common local
symptoms can include pain, tenderness, swelling and interference
with function. Common systemic symptoms can include fever, malaise
and hyperdynamic cardiovascular effects. Massive bloodstream
invasion by infectious agents can rapidly become fatal.
[0046] When an infection has its origins in a biofilm surrounding
an object in the body, whether a naturally occurring object or a
foreign one, the infection often cannot be controlled without
removing that object. If the object is naturally occurring, like
devascularized or necrotic tissue, it is removed surgically via a
process called debridement. If the object is a foreign one, such as
a medical device, it is removed entirely. At times a rim of tissue
must be removed along with the contaminated object to ensure
maximal removal of contaminating material. If the material being
removed is essential for health, a similar article may need to be
replaced in the same location; the replacement article will be
especially prone to infection because of the residual
microorganisms in the area.
[0047] The present invention is directed to antimicrobial surfaces
comprised of amphipathic compounds that can covalently attached to
a surface. In certain embodiments, this surface acts to eliminate
airborne biologics on contact, such as bacteria. In a preferred
embodiment, the compound is an amphipathic polycation.
[0048] B. Definitions
[0049] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0050] The term "biologic" as used herein refers to any bacterium,
fungus, virus, protozoan, parasite, or other infective agent
capable of causing disease in humans or non-human animals.
[0051] "Contacting" as used herein refers to any means for
providing the compounds of the invention to a surface to be
protected from biofouling. Contacting can include spraying,
wetting, immersing, dipping, painting, bonding or adhering or
otherwise providing a surface with a compound of the invention.
[0052] A "coating" refers to any temporary, semipermanent or
permanent layer, covering or surface. A coating can be a gas,
vapor, liquid, paste, semi-solid or solid. In addition a coating
can be applied as a liquid and solidify into a hard coating.
Examples of coatings include polishes, surface cleaners, caulks,
adhesives, finishes, paints, waxes polymerizable compositions
(including phenolic resins, silicone polymers, chlorinated rubbers,
coal tar and epoxy combinations, epoxy resin, polyamide resins,
vinyl resins, elastomers, acrylate polymers, fluoropolymers,
polyesters and polyurethanes, latex). Silicone resins, silicone
polymers (e.g. RTV polymers) and silicone heat cured rubbers are
suitable coatings for use in the invention and described for
example in the Encyclopedia of Polymer Science and Engineering
(1989) 15: 204 et seq. Coatings can be ablative or dissolvable, so
that the dissolution rate of the matrix controls the rate at which
AF agents are delivered to the surface. Coatings can also be
non-ablative, and rely on diffusion principles to deliver an AF
agent to the surface. Non-ablative coatings can be porous or
non-porous. A coating containing an AF agent freely dispersed in a
polymer binder is referred to as "monolithic" coating. Elasticity
can be engineered into coatings to accommodate pliability, e.g.
swelling or shrinkage, of the surface to be coated.
[0053] A "component" is a part of an apparatus that is structurally
integrated with that apparatus. A component may be applied to a
surface of an apparatus, contained within the substance of the
apparatus, retained in the interior of the apparatus, or any other
arrangement whereby that part is an integral element of the
structure of the apparatus. As an example, the silicone covering
surrounding the mechanical part of a pacemaker is a component of
the pacemaker. A component may be the lumen of an apparatus where
the lumen performs some function essential to the overall function
of the apparatus. The lumen of a tissue expander port is a
component of the tissue expander. A component can refer to a
reservoir or a discrete area within the apparatus specifically
adapted for the delivery of a fluid to a surface of the apparatus.
A reservoir within an implantable drug delivery device is a
component of that device.
[0054] A "delivery system" refers to any system or apparatus or
component whereby the disclosed antifouling compounds can be
delivered to a surface upon which biofilm formation is to be
inhibited. Representative delivery systems can include
encapsulation of the agent, incorporation of the agent in the
substance of an article of manufacture, or inserting the agent into
the matrices or pores of a suitable object, so that the agent is
able to reach the targeted surface in sufficient amount to inhibit
biofilm. A delivery system can comprise a coating. A delivery
system can comprise a mechanical object adapted for the delivery of
the antifouling compound to a surface. Other mechanisms comprising
delivery systems will be apparent to those of skill in the relevant
arts.
[0055] "Dressing" refer to any bandage or covering applied to a
lesion or otherwise used to prevent or treat infection. Examples
include wound dressings for chronic wounds (such as pressure sores,
venous stasis ulcers and burns) or acute wounds and dressings over
percutaneous devices such as IVs or subclavian lines intended to
decrease the risk of line sepsis due to microbial invasion. For
example, the compositions of the invention could be applied at the
percutaneous puncture site, or could be incorporated in the
adherent dressing material applied directly over the entry
site.
[0056] The phrase "effective amount" refers to an amount of the
disclosed antifouling compounds that significantly reduces the
number of organisms that attach to a defined surface
(cells/mm.sup.2) relative to the number that attach to an untreated
surface. Particularly preferred are amounts that reduce the number
of organisms that attach to the surface by a factor of at least 2.
Even more preferred are amounts that reduce the surface attachment
of organisms by a factor of 4, more preferably by a factor of 6. An
effective amount of the disclosed antifouling compound is said to
inhibit the formation of biofilms, and to inhibit the growth of
organisms on a defined surface. The term "inhibit," as applied to
the effect of an antifouling compound on a surface includes any
action that significantly reduces the number of organisms that
attach thereto.
[0057] The term "health-related environment" is understood to
include all those environments where activities are carried out
that are implicated in the restoration or maintenance of human
health. A health-related environment can be a medical environment,
where activities are carried out directly or indirectly intended to
restore human health. An operating room, a doctor's office, a
hospital room, and a factory making medical equipment are all
examples of medical environments. Other health-related environments
can include industrial or residential sites where activities
pertaining to human health are carried out. Such activities include
food processing, water purification, and sanitation.
[0058] An "implant" is any object intended for placement in a human
body that is not a living tissue. Implants include naturally
derived objects that have been processed so that their living
tissues have been devitalized. As an example, bone grafts can be
processed so that their living cells are removed, but so that their
shape is retained to serve as a template for ingrowth of bone from
a host. As another example, naturally occurring coral can be
processed to yield hydroxyapatite preparations that can be applied
to the body for certain orthopedic and dental therapies. An implant
can also be an article comprising artificial components. The term
"implant" can be applied to the entire spectrum of medical devices
intended for placement in a human body.
[0059] The terms "infectious microorganisms" or "infectious agents"
as used herein refers to disease causing or contributing bacteria
(including Gram-negative and Gram-positive organisms, such as
Staphylococci sps. (e.g. Staphylococcus aureus, Staphylococcus
epidermis), Enterococcus sp. (E. faecalis), Pseudomonas sp. (P.
aeruginosa), Escherichia sp. (E. coli), Proteus sp. (P.
mirabilis)), fungi (including Candida albicans), viruses and
protists.
[0060] "Medical device" refers to a non-naturally occurring object
that is inserted or implanted in a subject or applied to a surface
of a subject. Medical devices can be made of a variety of
biocompatible materials, including: metals, ceramics, polymers,
gels and fluids not normally found within the human body. Examples
of polymers useful in fabricating medical devices include such
polymers as silicones, rubbers, latex, plastics, polyanhydrides,
polyesters, polyorthoesters, polyamides, polyacrylonitrile,
polyurethanes, polyethylene, polytetrafluoroethylene,
polyethylenetetraphthalate and polyphazenes. Medical devices can
also be fabricated using certain naturally-occurring materials or
treated naturally-occurring materials. As an example, a heart valve
can be fabricated by combining a treated porcine heart valve with
an affixation apparatus using artificial materials. Medical devices
can include any combination of artificial materials, combinations
selected because of the particular characteristics of the
components. For example, a hip implant can include a combination of
a metallic shaft to bear the weight, a ceramic artificial joint and
a polymeric glue to affix the structure to the surrounding bone. An
implantable device is one intended to be completely imbedded in the
body without any structure left outside the body (e.g. heart
valve). An insertable device is one that is partially imbedded in
the body but has a part intended to be external (e.g. a catheter or
a drain). Medical devices can be intended for short-term or
long-term residence where they are positioned. A hip implant is
intended for several decades of use, for example. By contrast, a
tissue expander may only be needed for a few months, and is removed
thereafter. Insertable devices tend to remain in place for shorter
times than implantable devices, in part because they come into more
contact with microorganisms that can colonize them.
[0061] The term "soluble" refers to the ability to be loosened or
dissolved.
[0062] The term "surface" or "surfaces" can mean any surface of any
material, including glass, plastics, metals, polymers, and like. It
can include surfaces constructed out of more than one material,
including coated surfaces.
[0063] Biofilm formation with health implications can involve those
surfaces in all health-related environments, including surfaces
found in medical environments and those surfaces in industrial or
residential environments that are involved in those functions
essential to well-being like nutrition, sanitation and the
prevention of disease.
[0064] A surface of an article adapted for use in a medical
environment can be capable of sterilization using autoclaving,
biocide exposure, irradiation or gassing techniques like ethylene
oxide exposure. Surfaces found in medical environments include the
inner and outer aspects of various instruments and devices, whether
disposable or intended for repeated uses. Examples include the
entire spectrum of articles adapted for medical use, including
scalpels, needles, scissors and other devices used in invasive
surgical, therapeutic or diagnostic procedures; implantable medical
devices, including artificial blood vessels, catheters and other
devices for the removal or delivery of fluids to patients,
artificial hearts, artificial kidneys, orthopedic pins, plates and
implants; catheters and other tubes (including urological and
biliary tubes, endotracheal tubes, peripherably insertable central
venous catheters, dialysis catheters, long term tunneled central
venous catheters peripheral venous catheters, short term central
venous catheters, arterial catheters, pulmonary catheters,
Swan-Ganz catheters, urinary catheters, peritoneal catheters),
urinary devices (including long term urinary devices, tissue
bonding urinary devices, artificial urinary sphincters, urinary
dilators), shunts (including ventricular or arterio-venous shunts);
prostheses (including breast implants, penile prostheses, vascular
grafting prostheses, heart valves, artificial joints, artificial
larynxes, otological implants), vascular catheter ports, wound
drain tubes, hydrocephalus shunts, pacemakers and implantable
defibrillators, and the like. Other examples will be readily
apparent to practitioners in these arts.
[0065] Surfaces found in the medical environment include also the
inner and outer aspects of pieces of medical equipment, medical
gear worn or carried by personnel in the health care setting. Such
surfaces can include counter tops and fixtures in areas used for
medical procedures or for preparing medical apparatus, tubes and
canisters used in respiratory treatments, including the
administration of oxygen, of solubilized drugs in nebulizers and of
anesthetic agents. Also included are those surfaces intended as
biological barriers to infectious organisms in medical settings,
such as gloves, aprons and faceshields. Commonly used materials for
biological barriers may be latex-based or non-latex based. Vinyl is
commonly used as a material for non-latex surgical gloves. Other
such surfaces can include handles and cables for medical or dental
equipment not intended to be sterile. Additionally, such surfaces
can include those non-sterile external surfaces of tubes and other
apparatus found in areas where blood or body fluids or other
hazardous biomaterials are commonly encountered.
[0066] Surfaces in contact with liquids are particularly prone to
biofilm formation. As an example, those reservoirs and tubes used
for delivering humidified oxygen to patients can bear biofilms
inhabited by infectious agents. Dental unit waterlines similarly
can bear biofilms on their surfaces, providing a reservoir for
continuing contamination of the system of flowing and aerosolized
water used in dentistry.
[0067] Sprays, aerosols and nebulizers are highly effective in
disseminating biofilm fragments to a potential host or to another
environmental site. It is understood to be especially important to
health to prevent biofilm formation on those surfaces from whence
biofilm fragments can be carried away by sprays, aerosols or
nebulizers contacting the surface.
[0068] Other surfaces related to health include the inner and outer
aspects of those articles involved in water purification, water
storage and water delivery, and those articles involved in food
processing. Surfaces related to health can also include the inner
and outer aspects of those household articles involved in providing
for nutrition, sanitation or disease prevention. Examples can
include food processing equipment for home use, materials for
infant care, tampons and toilet bowls.
[0069] The term `Gram-positive bacteria` is an art recognized term
for bacteria characterized by having as part of their cell wall
structure peptidoglycan as well as polysaccharides and/or teichoic
acids and are characterized by their blue-violet color reaction in
the Gram-staining procedure. Representative Gram-positive bacteria
include: Actinomyces spp., Bacillus anthracis, Bifidobacterium
spp., Clostridium botulinum, Clostridium perfringens, Clostridium
spp., Clostridium tetani, Corynebacterium diphtheriae,
Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus
faecium, Erysipelothrix rhusiopathiae, Eubacterium spp.,
Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp.,
Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium
chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium,
Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium
marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis,
Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium
ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus
spp., Proprionibacterium spp., Staphylococcus aureus,
Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus
cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus,
Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus
saccharolyticus, Staphylococcus saprophyticus, Staphylococcus
schleiferi, Staphylococcus similans, Staphylococcus warneri,
Staphylococcus xylosus, Streptococcus agalactiae (group B
streptococcus), Streptococcus anginosus, Streptococcus bovis,
Streptococcus canis, Streptococcus equi, Streptococcus milleri,
Streptococcus mitior, Streptococcus mutans, Streptococcus
pneumoniae, Streptococcus pyogenes (group A streptococcus),
Streptococcus salivarius, Streptococcus sanguis.
[0070] The term "Gram-negative bacteria" is an art recognized term
for bacteria characterized by the presence of a double membrane
surrounding each bacterial cell. Representative Gram-negative
bacteria include Acinetobacter calcoaceticus, Actinobacillus
actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes
xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella
bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella
catarrhalis, Brucella spp., Campylobacter spp., Chalmydia
pneumoniae, Chlamydia psittaci, Chlamydia trachomatis,
Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens,
Enterobacter aerogenes, Escherichia coli, Flavobacterium
meningosepticum, Fusobacterium spp., Haemophilus influenzae,
Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella
spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii,
Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria
meningitidis, Pasteurella multocida, Plesiomonas shigelloides,
Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas
aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia
rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi,
Serratia marcescens, Shigella spp., Treponema carateum, Treponema
pallidum, Treponema pallidum endemicum, Treponema pertenue,
Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia
enterocolitica, Yersinia pestis.
[0071] An amphipathic molecule or compound is an art recognized
term where one end of the molecule or compound is polar and another
end is non-polar.
[0072] The term "polar" is art-recognized. A polar compound
contains substances with asymmetric charge distribution. In
general, a non-polar substance will dissolve non-polar molecules,
and a polar substance will dissolve polar molecules, e.g. water, a
polar substance, dissolves other polar substances. An amphipathic
compound has a portion which is soluble in aqueous solvents, and a
portion which is insoluble aqueous solvents.
[0073] The term "ligand" refers to a compound that binds at the
receptor site.
[0074] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0075] The term "electron-withdrawing group" is recognized in the
art, and denotes the tendency of a substituent to attract valence
electrons from neighboring atoms, i.e., the substituent is
electronegative with respect to neighboring atoms. A quantification
of the level of electron-withdrawing capability is given by the
Hammett sigma (.sigma.) constant. This well known constant is
described in many references, for instance, J. March, Advanced
Organic Chemistry, McGraw Hill Book Company, New York, (1977
edition) pp. 251-259. The Hammett constant values are generally
negative for electron donating groups (.sigma.[P]=-0.66 for
NH.sub.2) and positive for electron withdrawing groups
(.sigma.[P]=0.78 for a nitro group), .sigma.[P] indicating para
substitution. Exemplary electron-withdrawing groups include nitro,
acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the
like. Exemplary electron-donating groups include amino, methoxy,
and the like.
[0076] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 3-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0077] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Preferred alkyl
groups are lower alkyls. In preferred embodiments, a substituent
designated herein as alkyl is a lower alkyl.
[0078] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0079] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0080] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more cyclic rings in
which two or more carbons are common to two adjoining rings (the
rings are "fused rings") wherein at least one of the rings is
aromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
[0081] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0082] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0083] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
[0084] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0085] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0086] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula: 2
[0087] wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a group permitted by the rules of valence.
[0088] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 3
[0089] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above.
[0090] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 4
[0091] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0092] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.8, wherein m and R.sub.8 are defined
above. Representative alkylthio groups include methylthio, ethyl
thio, and the like.
[0093] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula: 5
[0094] wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.1 1 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.8 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0095] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen radical attached
thereto. Representative alkoxyl groups include methoxy, ethoxy,
propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an alkoxyl,
such as can be represented by one of --O-alkyl, --O-alkenyl,
--O-alkynyl, --O--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8
are described above.
[0096] The term "sulfonate" is art recognized and includes a moiety
that can be represented by the general formula: 6
[0097] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0098] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0099] The abbreviations Me, Et, Ph, Tf, Ts, Ms represent methyl,
ethyl, phenyl, trifluromethanesulfonyl, nonafluorobutanesulfonyl,
p-toluenesulfonyl and methanesulfonyl, respectively. A more
comprehensive list of the abbreviations utilized by organic
chemists of ordinary skill in the art appears in the first issue of
each volume of the Journal of Organic Chemistry; this list is
typically presented in a table entitled Standard List of
Abbreviations. The abbreviations contained in said list, and all
abbreviations utilized by organic chemists of ordinary skill in the
art are hereby incorporated by reference.
[0100] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 7
[0101] in which R.sub.41 is as defined above.
[0102] The term "sulfonylamino" is art recognized and includes a
moiety that can be represented 8
[0103] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by 9
[0104] The term "sulfonyl", as used herein, refers to a moiety that
can be represented by the general formula: 10
[0105] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,
or heteroaryl.
[0106] The term "sulfoxido" as used herein, refers to a moiety that
can be represented by the general formula: 11
[0107] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0108] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0109] As used herein, the definition of each expression, e.g.
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0110] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0111] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0112] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
[0113] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g., functioning as
analgesics), wherein one or more simple variations of substituents
are made which do not adversely affect the efficacy of the compound
in binding to opioid receptors. In general, the compounds of the
present invention may be prepared by the methods illustrated in the
general reaction schemes as, for example, described below, or by
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions,
it is also possible to make use of variants which are in themselves
known, but are not mentioned here.
[0114] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover.
[0115] C. Compounds
[0116] One aspect of the present invention is directed to
antimicrobial surfaces comprised of covalently attached amphipathic
compounds. In certain embodiments, a surface of the present
invention acts to eliminate airborne biologics on contact. In
certain embodiments, a surface of the present invention acts to
eliminate waterborne biologics on contact. In certain embodiments,
a surface of the present invention acts to prevent the formation of
biofilms.
[0117] In certain embodiments, said covalently attached amphipathic
compound is a polymer comprising ammonium ions. In a preferred
embodiment, said polymer has a molecular weight of at least 10,000
g/mol, more preferably 120,000 g/mol, and most preferably 150,000
g/mol.
[0118] In certain embodiments, the surface of the present invention
is glass. In certain other embodiments, the surface of the present
invention is a plastic. In a particular embodiment, the surface of
the present invention is an amino-bearing glass.
[0119] In certain embodiments, the compound covalently bonded to
the surface is represented by the formula I: 12
[0120] wherein
[0121] R represents individually for each occurrence hydrogen,
alkyl, alkenyl, alkynyl, acyl, aryl, carboxylate, alkoxycarbonyl,
aryloxycarbonyl, carboxamido, alkylamino, acylamino, alkoxyl,
acyloxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl,
thio, alkylthio, thioalkyl, (alkylthio)alkyl, carbamoyl, urea,
thiourea, sulfonyl, sulfonate, sulfonamido, sulfonylamino, or
sulfonyloxy;
[0122] R' represents independently for each occurrence alkyl, an
alkylidene tether to a surface, or an acyl tether to a surface;
[0123] Z represents independently for each occurrence Cl, Br, or I;
and
[0124] n is an integer less than or equal to about 1500.
[0125] In a preferred embodiment, the invention comprises
Poly(4-vinyl-N-alkylpyridinium bromide) or
poly(methacryloyloxydodecylpyr- idinium bromide) (MDPB) covalently
attached to a glass surface. In another preferred embodiment, the
invention comprises an N-alkylated poly(4-vinyl pyridine)
covalently attached to a glass surface.
[0126] In certain embodiments, the biologics killed on the surfaces
of the instant invention are bacteria. In a particular embodiment,
the bacteria killed are Gram-positive bacteria. In another
particular embodiment, the bacteria killed are Gram-negative
bacteria.
[0127] Modification of Surface Containing Reactive Amino Groups
[0128] In one embodiment, the surface is modified by graft
copolymerization. In this case (Method A), the surface is prepared
by acylating a NH.sub.2-glass slide with an acylating agent to
introduce double bonds. In particular embodiment, the acylating
agent is acryloyl chloride. This is followed by copolymerization
with a copolymerizing agent, for example, 4-vinylpyridine. A
immobilized polymer, such as polyvinylpyridine, PVP, was found to
afford approximately the same number of viable S. aureus cells
after spraying the bacterial suspension onto its surface as a plain
NH.sub.2-glass slide.
[0129] The final step in creating the surface is to introduce
positive charges into the polymer chains attached to glass. For
example, the PVP pyridine rings may be N-alkylated by seven linear
alkyl bromides (with chain lengths varying from propyl to
hexadecyl). This procedure is exemplified in Example 1. This method
tends to produce mostly linearly attached, straight chain polymers
to the surface.
[0130] The resultant slide surfaces were examined with respect to
their ability to kill on contact S. aureus cells sprayed on them.
As seen in FIG. 2, propylated, butylated, hexylated, and octylated
immobilized PVP chains were effective in markedly reducing the
number of viable bacterial cells, with the most effective,
hexyl-PVP affording a 94.+-.4% reduction (right portion of FIG. 1).
In contrast, the immobilized PVP N-alkylated by decyl through
hexadecyl bromides, as well as the non-alkylated chains, were
totally ineffective (FIG. 2).
[0131] This behavior pattern may correlate with the visual
appearance of the alkylated PVP-slides. While non-alkylated,
decyl-, dodecyl-, and hexadecyl-PVP-modified slides were all cloudy
(absorbance at 400 nm, A.sub.400, of some 0.1), an
octyl-PVP-modified slide was much less so (A.sub.400=0.03), and
propyl-, butyl-, and hexyl-PVP-modified slides were clear
(A.sub.400<0.002). The cloudiness reflects polymer aggregates
formed on the surface, with such aggregates apparently being unable
to interact with bacterial cells. The non-alkylated immobilized PVP
chains, as well as those modified with long alkyl moieties, may
stick to each other due to hydrophobic interactions; in contrast,
in immobilized PVP chains modified with short alkyl groups, such
interactions are not strong enough to overcome the electrostatic
repulsion of the positively charged polymers.
[0132] Using hexyl-PVP (henceforth referred to as that prepared by
method A), the bactericidal effect of this surface toward another
Gram-positive bacterium, S. epidermidis, as well as two
Gram-negative bacteria, E. coli and P. aeruginosa is investigated.
The first two formed colonies of the same size as S. aureus (FIG.
1, left portion) when sprayed on NH.sub.2-glass slides, whereas the
colonies of P. aeruginosa were larger but still distinguishable. As
seen in Table 1 (method A), the number of colonies all three
bacteria formed after spraying onto hexyl-PVP-slides dropped more
than 100-fold compared to the plain NH.sub.2-glass.
1TABLE 1 The ability of hexyl-PVP attached to glass slides by two
different methods to kill various airborne bacteria on contact.
Percentage of bacteria killed Bacterium Gram Method A Method B S.
aureus (+) 94 .+-. 4 94 .+-. 3 S. epidermidis (+) >99 >99 P.
aeruginosa (-) >99.8 >99.8 E. coli (-) >99 >99
[0133] In another embodiment, the surface is prepared by alkylation
of the surface using halogenated alkyl groups. These reactive alkyl
groups are then used for attachment of the polymer. The polymer
chains were then further alkylated to increase the positive charge.
This method yields mostly branched and looped polymers covalently
bonded to the surface.
[0134] For example, a NH.sub.2-glass slide was alkylated with
1,4-dibromobutane to introduce reactive bromobutyl groups which
were subsequently used for the attachment of PVP. To increase the
positive charge of the attached PVP chains, the chains were further
N-alkylated with hexyl bromide (found optimal in method A, see FIG.
2). After S. aureus cells were sprayed, air dried, and cultured,
the resultant hexyl-PVP-slides (henceforth referred to as those
prepared by method B) looked essentially the same as shown in the
right portion of FIG. 1. Compared to a NH.sub.2-glass slide,
94.+-.3% of the deposited S. aureus cells were killed (the last
column, 1st line, of Table 1).
[0135] The molecular weight of the immobilized polymer was found to
be important for the antibacterial properties of the surface. For
example a hexyl-PVP-slide prepared using method B with shorter PVP
chains, 60,000 instead of 160,000 g/mol, killed only 62.+-.8% of
the deposited S. aureus cells.
[0136] Inspection of the remainder of the data in Table I reveals
that method B afforded the slide surface which was as deadly toward
S. epidermidis, P. aeruginosa, and E. coli as that obtained via
method A.
[0137] Previously described materials that kill bacteria by
releasing bactericidal agents are eventually depleted of the active
substance due to its release into the surrounding solution. To test
whether the antibacterial polymers immobilized using our
methodology can leach from the glass surface, hexyl-PVP-slides
prepared via methods A and B were placed into polystyrene petri
dishes, and aqueous suspensions of S. aureus cells were sprayed on
them as well as on the non-antibacterial polystyrene. After air
drying and incubation under growth agar, it was observed that only
a few (some 3 per cm.sup.2) colonies were growing on both types of
the hexyl-PVP-glass slides, while a far larger number of colonies
(60.+-.10 per cm.sup.2) was growing on the surrounding petri
dishes, even in the immediate proximity of the slides. The lack of
inhibition zones around the hexyl-PVP slides, typical of the
release of bactericidal agents (23), indicates that the immobilized
bactericidal polymer hexyl-PVP does not leach from the slides,
i.e., that the bacteria are indeed killed on contact with the
slide's surface.
[0138] To test how unique the ability to kill airborne bacteria is
among dry surfaces, an S. aureus cell suspension was sprayed on
various common materials, including metals, synthetic and natural
polymers, and ceramics. The number of colonies remaining viable in
all instances was compared to that on NH.sub.2-glass. As seen in
Table 2, none of the materials examined significantly lowered the
amount of bacterial cells remaining viable after spraying.
2TABLE 2 The number of S. aureus cells remaining viable after their
aqueous suspensions have been sprayed onto various materials.
Relative numbers of Material viable bacterial cells % plain glass
83 .+-. 10 polystyrene 105 .+-. 15 polypropylene 97 .+-. 9 aluminum
72 .+-. 7 steel 95 .+-. 15 paper 77 .+-. 8 wood (birch) 102 .+-. 10
porcelain 85 .+-. 10 PVP slide 115 .+-. 16 HPVP slide (method A) 6
.+-. 4 HPVP slide (method B) 6 .+-. 3
[0139] Modification of Surface that does not Contain Active Amino
Groups
[0140] Since it would be desirable to make numerous diverse objects
bactericidal, a surface derivatization approach potentially
applicable to any material was developed. Ordinary commercial
synthetic polymers, namely polyolefins, a polyamide, and a
polyester, which by themselves exhibit no antibacterial activity,
were used to test the approach. A slide made of high-density
polyethylene (HDPE) was selected as the initial target. This
polymer, as many other materials, lacks reactive groups suitable
for a facile chemical modification. Therefore, we decided to coat
it with an ultrathin silica layer by a combustion chemical vapor
deposition technique. Schinkinger, B., Petzold, R., Tiller, H.-J.
& Grundmeier, G. Chemical structure and morphology of ultrathin
combustion CVD layers on zinc coated steel. Appl. Surf. Sci. 179,
79-87 (2001). To this end, we employed (step #1 in FIG. 4) a
pen-like torch containing a compressed mixture of 0.6%
tetramethylsilane with 7:3 propane-butane (Pyrosil.RTM.). When this
mixture burns in the air, tetramethylsilane is oxidized to form
2-5-nm SiO.sub.2 nanoparticles which cover a surface to which the
flame is applied. Tiller, H. J., Goebel, R., Magnus, B., Garschke,
A. & Musil, R. A new concept of metal-resin adhesion using an
intermediate layer of silicon oxide (SiO.sub.x)-carbon. Thin Solid
Films 169, 159-168 (1989). The resulting dense, 100-nm thick
SiO.sub.2 layer chemically resembling glass (polysiloxane), can
thereafter be readily chemically modified in a uniform fashion
regardless of the nature of the bulk material. Schinkinger, B.,
Petzold, R., Tiller, H.-J. & Grundmeier, G. Chemical structure
and morphology of ultrathin combustion CVD layers on zinc coated
steel. Appl. Surf. Sci. 179, 79-87 (2001). Tiller, H. J., Goebel,
R., Magnus, B., Garschke, A. & Musil, R. A new concept of
metal-resin adhesion using an intermediate layer of silicon oxide
(SiO.sub.x)-carbon. Thin Solid Films 169, 159-168 (1989). Tiller,
H. J., Kaiser, W. D., Kleinert, H. & Goebel, R. The Silicoater
method improves bond strength and resistance to aging. Adhaesion
33, 27-31 (1989). Tiller, H. J., Goebel, R. & Gutmann, N.
Silicate coupling layer for metal to polymer adhesion improvement.
Physical-chemical fundamentals and technological importance.
Makromol. Chem., Macromol. Symp. 50, 125-135 (1991).
[0141] The visual appearance of the HDPE surface did not change
after the SiO.sub.2 coating procedure. The degree of crosslinking
of such a SiO.sub.2 layer is lower than that of glass, and numerous
Si--OH groups are formed on hydration by water adsorbed from the
environment after the deposition process. Schinkinger, B., Petzold,
R., Tiller, H.-J. & Grundmeier, G. Chemical structure and
morphology of ultrathin combustion CVD layers on zinc coated steel.
Appl. Surf. Sci. 179, 79-87 (2001). Because of these Si--OH groups,
the coated HDPE surface is expected to be hydrophilic, in contrast
to the very hydrophobic unmodified polymer. To quantify this
difference, we measured the contact angle formed by an aqueous drop
on a surface, a common method of determining the water affinity of
materials. David, J. The idea of water repellency. Text. Inst. Ind.
4, 293-5 (1966). To this end, 0.1 mL of distilled water was placed
on the HDPE surface. The angle between the core of the resultant
water drop and the surface under it was estimated to be some
120.degree., indicative of a hydrophobic material. When the same
amount of distilled water was placed onto the SiO.sub.2-coated
HDPE, the water spread on the surface and the contact angle was
less than 10.degree. reflecting the surface's hydrophilic
character. Independently, the presence of the SiO.sub.2 layer on
the HDPE slide was confirmed by the silicon and oxygen signals
detected in the X-ray photoelectron spectrum.
[0142] Next, amino groups were introduced into the SiO.sub.2-coated
HDPE slide surface by reacting it with 3-aminopropyltriethoxysilane
(step #2 in FIG. 4), a standard procedure for the amination of
glass surfaces. Bisse, E., Scholer, A. & Vonderschmitt, D. J. A
new method for coupling glucose dehydrogenase to glass tubes
activated with titanium tetrachloride. FEBS Lett. 138, 316-318
(1982). The NH.sub.2-functionalized HDPE slide was subsequently
alkylated with 1,4-dibromobutane to introduce bromoalkyl groups
(step #3 in FIG. 4), which were then reacted with PVP in presence
of 1-bromohexane (step #4 in FIG. 4). Under the conditions used,
only a few pyridine groups of the PVP chain (approximately 1,500
pyridine groups per chain) are alkylated by the surface-bound
bromoalkyls, with the majority being alkylated by 1-bromohexane.
Tiller, J. C., Liao, C.-J., Lewis, K. & Klibanov, A. M.
Designing surfaces that kill bacteria on contact. Proc. Natl. Acad.
Sci. U.S.A. 98, 5981-5985 (2001). The number of the pyridinium
groups on the resultant hexyl-PVP-derivatized polyethylene slides,
titrated spectrophotometrically with fluorescein, was determined to
be 8.2.+-.1.9 nmol/cm.sup.2, which is similar to that observed for
hexyl-PVP-modified NH.sub.2-glass. Tiller, J. C., Liao, C.-J.,
Lewis, K. & Klibanov, A. M. Designing surfaces that kill
bacteria on contact. Proc. Natl. Acad. Sci. U.S.A. 98, 5981-5985
(2001).
[0143] The generality of the foregoing coating/derivatization
approach was confirmed by modifying two other industrial
polyolefins, low-density polyethylene (LDPE) and polypropylene
(PP), as well as the polyamide nylon and the polyester
poly(ethylene terephthalate) (PET). All these synthetic polymers
were successfully derivatized as shown in FIG. 4 and exhibited
surface densities of the pyridinium groups similar to that of
HDPE-8.5.+-.1.8 (LDPE), 7.2.+-.2.1 (PP), 8.0.+-.1.1 (nylon), and
7.5.+-.0.9 (PET) nmol/cm.sup.2. All hexyl-PVP-derivatized slides,
starting with HDPE, were subsequently examined with respect to
their ability to kill airborne bacteria on contact.
[0144] To simulate natural deposition of airborne bacteria, a
suspension in distilled water of the ubiquitous pathogen
Gram-positive bacterium Staphylococcus aureus was sprayed onto a
slide surface and the latter was allowed to dry. Xiong, Y., Yeaman,
M. R. & Bayer, A. S. Linezolid: A new antibiotic. Drugs Today
36, 529-539 (2000). The slide was then incubated under nutrient
agar overnight as described earlier, and the number of viable
bacterial cells was determined by colony count. Tiller, J. C.,
Liao, C.-J., Lewis, K. & Klibanov, A. M. Designing surfaces
that kill bacteria on contact. Proc. Natl. Acad. Sci. U.S.A. 98,
5981-5985 (2001). As seen in FIG. 5a, left, numerous readily
distinguishable bacterial colonies grew on the unmodified HDPE
slide. The number of colonies grown on the SiO.sub.2-coated or
NH.sub.2-functionalized HDPE slides (FIG. 4) was essentially the
same indicating that these modifications are not toxic to S.
aureus. In sharp contrast, when sprayed onto a hexyl-PVP-modified
HDPE slide, 96.+-.3% of the deposited bacterial cells became
non-viable (FIG. 5a, right, and Table 3).
3TABLE 3 The ability of various commercial synthetic polymers
derivatized by hexyl-PVP to kill airborne bacteria on contact.
Percentage of bacteria killed Bacterium Type HDPE LDPE PP nylon PET
S. aureus Gram (+) 96 .+-. 3 97 .+-. 1 90 .+-. 3 92 .+-. 2 95 .+-.
1 E. coli Gram (-) 97 .+-. 1 96 .+-. 2 98 .+-. 1 98 .+-. 2 95 .+-.
1
[0145] Suspensions of bacteria (10.sup.6 cells/mL for S. aureus and
10.sup.5 cells/mL for E. coli) in distilled water were sprayed on a
hexyl-PVP-modified polymer slide, and the latter was air dried for
2 min and incubated under 0.7% agar in a bacterial growth medium
overnight; thereafter, the colonies were counted. The number of
viable cells obtained in the same manner with the corresponding
unmodified polymer slides was used as a standard (i.e., 0% of the
bacteria killed). Abbreviations for the polymer materials used are
as follows: LDPE--low-density polyethylene, HDPE--high-density
polyethylene, PP--polypropylene, and PET--poly(ethylene
terephthalate). All experiments were performed at least in
duplicate, and the errors given indicate the standard
deviations.
[0146] LDPE, PP, nylon, and PET derivatized with hexyl-PVP were
analogously tested for their ability to kill airborne S. aureus. As
seen in Table 3, in all cases the number of bacterial colonies
formed plunged 10 to 30-fold compared to those grown on the
corresponding unmodified slides.
[0147] Next, we tested the killing efficiency of the
hexyl-PVP-derivatized surfaces for a representative Gram-negative
bacterium, Escherichia coli. As seen in FIG. 5b, left, many E. coli
colonies grown on a HDPE slide after spraying, while less dense
than those of S. aureus grown under the same conditions, are still
distinguishable. The surface derivatization with hexyl-PVP slashed
their number by 97.+-.3% (FIG. 5b, right, and Table 3). Inspection
of the remaining data in the last line of Table 3 reveals that the
hexyl-PVP modification of LDPE, PP, nylon, and PET slides likewise
afforded a 20 to 50-fold drop in the viable E. coli cells,
following their deposition from the airborne state, when compared
to the corresponding unmodified slides.
[0148] To test whether the hexyl-PVP chains immobilized using our
methodology can leach from the slide surface (and possibly only
then kill the deposited bacteria), modified HDPE slide was placed
into a polystyrene Petri dish, and an aqueous suspension of S.
aureus cells was sprayed on it, as well as on the
(non-antibacterial) polystyrene surface. After air drying and
incubation under growth agar, only a few (4.+-.2 per cm.sup.2)
colonies grew on the hexyl-PVP-modified HDPE slide, while a far
greater number of colonies (85.+-.5 per cm.sup.2) grew on the
surface of the surrounding Petri dish, even in the immediate
proximity of the slide. The lack of inhibition zones around the
hexyl-PVP-slides, characteristic of a release of bactericidal
agents, indicates that the immobilized polycation does not leach
from the slide, i.e., that the airborne bacteria are indeed killed
on contact with the slide's surface. Kawabata, N. & Nishiguchi,
M. Antibacterial activity of soluble pyridinium-type polymers.
Appl. Environ. Microbiol. 54, 2532-2535 (1988).
[0149] Another important question was whether the
hexyl-PVP-derivatized slides can also kill waterborne bacteria. To
experimentally simulate a flowing aqueous solution, a polymer slide
was placed vertically in a bacterial suspension in PBS, pH 7.0, and
the resulting system was gently agitated at 37.degree. C. After a
2-h incubation, the slide was washed with the sterile buffer to
remove non-adhered bacteria and then incubated in sterile PBS under
the conditions outlined above for 1 h to allow the tentatively
adhered bacteria to either detach or adhere irreversibly. Wiencek,
K. M. & Fletcher, M. Bacterial adhesion to hydroxyl- and
methyl-terminated alkanethiol self-assembled monolayers. J.
Bacteriol. 177, 1959-1966 (1995). After rinsing the slide with PBS,
it was immediately covered with a layer of solid growth agar and
incubated overnight at 37.degree. C. This way, all genuinely
attached bacterial cells are captured on the slide surface, and
those of them still viable can proliferate and form colonies.
[0150] As seen in FIG. 6a, left, S. aureus cells allowed to adhere
to an HDPE slide form numerous easily distinguishable colonies.
Neither SiO.sub.2-- nor NH.sub.2-- modified HDPE slides exhibited
an appreciable decline in the number of bacterial colonies grown
after the adhesion. However, as seen in FIG. 6a, right, the number
of S. aureus colonies grown on a hexyl-PVP-modified HDPE slide
plunged 98.+-.1% (see also Table 4).
4TABLE 4 The ability of hexyl-PVP covalently attached to various
commercial polymer slides to kill waterborne bacteria on contact.
Percentage of bacteria killed Bacterium Type HDPE LDPE PP nylon PET
S. aureus Gram (+) 98 .+-. 1 99 .+-. 1 97 .+-. 2 97 .+-. 1 99 .+-.
1 E. coli Gram (-) 97 .+-. 2 98 .+-. 2 n.d. 99 .+-. 1 98 .+-. 1
[0151] Suspensions of bacteria (2.times.10.sup.6 cells/mL for S.
aureus and 4.times.10.sup.6 cells/mL for E. coli) in PBS, pH 7.0,
was allowed to adhere to the hexyl-PVP-modified polymer surfaces
under shaking at 37.degree. C. for 2 h. The slides were rinsed with
sterile PBS, pH 7.0, shaken in sterile PBS at 37.degree. C. for 1
h, rinsed again with the PBS, incubated under 1.5% agar in a
bacterial growth medium overnight, and the colonies were counted.
N.d. stands for "not determined" because adhered bacterial cells
grow but do not form distinguishable colonies on the control slide.
For other conditions, see the footnotes to Table 3.
[0152] It was further demonstrated that hexyl-PVP-derivatized LDPE,
PP, nylon, and PET also can kill waterborne bacteria on contact.
The data in Table 4 show that upon the derivatization (FIG. 4) the
number of viable adhered S. aureus cells drops by 97 to 99% for all
polymer slides. Likewise, the modified polymer slides were found to
be effective against waterborne E. coli. As seen in FIG. 6b, left,
E. coli cells adhered to an unmodified HDPE slide from an aqueous
suspension grow a number of distinguishable colonies under the
growth agar cover. The colonies on LDPE, nylon, and PET looked
similar, while no colonies were distinguishable on PP. Upon the
derivatization of HDPE with hexyl-PVP, the number of viable adhered
E. coli cells plummeted 97.+-.2% (FIG. 6b, right, Table 4). The
other modified polymers also displayed drastically lower numbers of
adhered viable E. coli cells compared to the corresponding
unmodified slides, with killing efficiencies of 98 to 99% observed
for LDPE, PET, and nylon (Table 4, last line).
[0153] Since the experimental conditions for the determination of
bactericidal properties of the hexyl-PVP-modified slides against
waterborne bacteria were different from those for airborne ones, we
again tested for release of the polycation from a slide into the
medium. To this end, a hexyl-PVP-modified HDPE slide was incubated
for 2 h in sterile PBS with shaking at 37.degree. C., removed, and
S. aureus cells were added to the remaining solution. The resulting
suspension was again shaken at 37.degree. C. for 2 h, and the
number of viable bacterial cells was determined by spreading it on
growth agar, followed by incubation at 37.degree. C. overnight. The
number of bacterial colonies observed, (42.+-.4).multidot.10.sup.4
per mL, coincided with that in the control
[(48.+-.7).multidot.10.sup.4 per mL]. Hence there is no appreciable
release of an antibacterial agent from the hexyl-PVP-modified HDPE
slide during the bacterial adhesion experiment, and the waterborne
bacteria must indeed be killed on contact with the derivatized
surface.
[0154] Mechanism of Attacking Bacteria
[0155] Tethered amphipathic polycations described in this study and
soluble cationic antimicrobials probably share a similar mechanism
of attacking bacteria. Polycations, such as polymyxin B and
antimicrobial cationic peptides of animals, displace the divalent
cations that hold together the negatively charged surface of the
lipopolysaccharide network, thereby disrupting the outer membrane
of Gram-negative bacteria like P. aeruginosa and E. coli (Vaara, M.
(1992) Microbiol Rev. 56, 395-411). This in itself might be
sufficient for a lethal outcome. It is also possible that, having
destroyed the outer membrane permeability barrier, the cationic
groups of the tethered polymers further penetrate into the inner
membrane, producing leakage. Such "self-promoted penetration" with
the subsequent damage of the inner membrane has been described for
polymyxin The action of immobilized polycations against the
Gram-positive bacteria S. aureus and S. epidermidis probably
requires penetration of the cationic groups across the thick cell
wall to reach the cytoplasmic membrane. Bactericidal action of
amphipathic cationic antiseptics, such as benzalkonium chloride or
biguanidine chlorhexidine, against Gram-positive bacteria is due
primarily to the disruption of the cytoplasmic membrane (Denton, G.
W. (2001) in Disinfection, Sterilization, and Preservation, ed.
Block, S. S. (Lippincott Williams & Wilkins, Philadelphia)).
The cell wall of S. aureus is some 30 nm thick (Friedrich, C. L.,
Moyles, D., Beveridge, T. J. & Hancock, R. E. (2000)
Antimicrob. Agents Chemother. 44, 2086-2092.); since the estimated
average length of the N-hexylated PVP (Method A) is 19 nm, with
some obviously being shorter and others longer, the latter could
penetrate the cell wall.
[0156] Medical Applications
[0157] Such surface modifications can be readily performed with a
number of other materials. A simple periodic washing would remove
the dead deposited cells and rejuvenate such surfaces.
[0158] Any object placed in the body from outside it is susceptible
to biological contamination with microorganisms and subsequent
biofilm formation. Therefore, compounds according to the present
invention can prevent such objects from becoming contaminated with
microorganisms in the first place. Further, these technologies can
be used to produce natural and synthetic materials resistant to
contamination that are especially suited for replacing those
objects that have already sustained infection, or that are intended
for being placed in those anatomic sites where infections can be
particularly devastating.
[0159] Naturally derived processed materials commonly are
positioned in the body in order to provide a structure for ingrowth
of the patient's own tissues. Examples include demineralized bone
materials and hydroxyapatite. These materials are destined to be
infiltrated or replaced entirely by the patient's tissue, during
which time the exogenous material retains the desired shape or
structural support in the affected area. These materials themselves
are non-living and avascular. Colonization of these materials with
microorganisms and biofilm formation can require their removal. If
the material is removed, the shape or the structure that it is
maintaining is destroyed and the progress made by tissue in growth
is in vain. Application of compounds of the invention to these
materials can enhance their resistance to biofilm formation and its
consequences.
[0160] Certain naturally derived processed materials will be
determined by artisans in these fields to especially suitable for
the application or incorporation of compounds of the invention. A
material can be contacted with the claimed compounds in a variety
of ways including immersion and coating. In forms where the
material has interstices, an AF compound can reside therein as a
liquid or as a gel. Fibrillar preparations can permit the fibers to
be coated with the compound. Solid articles such as reconstructive
blocks of hydroxyapatite can be painted with a coating of the
compound for additional protection. These temporary means of
application are appropriate for these materials because they only
reside in the body temporarily, to be resorbed or replaced.
[0161] Implantable medical devices, using artificial materials
alone or in combination with naturally-derived materials, can be
treated with compounds either by surface coating or by
incorporation. Metals may be suitably treated with surface coats
while retaining their biological properties. In certain embodiments
of the present invention, metals may be treated with paints or with
adherent layers of polymers or ceramics that incorporate the
compounds of the invention. Certain embodiments treated in this
manner may be suitable for orthopedic applications, for example,
pins, screws, plates or parts of artificial joints. Methods for
surface treatment of metals for biological use are well-known in
the relevant arts. Other materials besides metals can be treated
with surface coats of compounds according to the present invention
as the medical application requires.
[0162] Implantable devices may comprise materials suitable for the
incorporation of the instant claimed compounds. Embodiments whose
components incorporate compounds of the invention can include
polymers, ceramics and other substances. Materials fabricated from
artificial materials can also be destined for resorption when they
are placed in the body. Such materials can be called bioabsorbable.
As an example, polyglycolic acid polymers can be used to fabricate
sutures and orthopedic devices. Those of ordinary skill in these
arts will be familiar with techniques for incorporating agents into
the polymers used to shape formed articles for medical
applications. AF agents can also be incorporated into glues,
cements or adhesives, or in other materials used to fix structures
within the body or to adhere implants to a body structure. Examples
include polymethylmethacrylate and its related compounds, used for
the affixation of orthopedic and dental prostheses within the body.
The presence of the compounds of the instant invention can decrease
biofilm formation in those structures in contact with the glue,
cement, or adhesive. Alternatively, a compound of the invention can
coat or can permeate the formed article. In these compositions, the
formed article allows diffusion of the compound, or functional
portion thereof, into the surrounding environment, thereby
preventing fouling of the appliance itself. Microcapsules bearing
compounds can also be imbedded in the material. Materials
incorporating compounds are adaptable to the manufacture of a wide
range of medical devices, some of which are disclosed below. Other
examples will be readily apparent to those practitioners of
ordinary skill in the art.
[0163] In one embodiment, compounds of the invention can be applied
to or incorporated in certain medical devices that are intended to
be left in position permanently to replace or restore vital
functions. As one example, ventriculoatrial or ventriculoperitoneal
shunts are devised to prevent cerebrospinal fluid from collecting
in the brain of patients whose normal drainage channels are
impaired. As long as the shunt functions, fluid is prevented from
accumulating in the brain and normal brain function can continue.
If the shunt ceases to function, fluid accumulates and compresses
the brain, with potentially life-threatening effect. If the shunt
becomes infected, it causes an infection to enter the central
portions of the brain, another life-threatening complication. These
shunts commonly include a silicone elastomer or another polymer as
part of their fabrication. Silicones are understood to be
especially suited for combination with compounds according to the
present invention.
[0164] Another shunt that has life-saving import is a dialysis
shunt, a piece of polymeric tubing connecting an artery and a vein
in the forearm to provide the kidney failure patient a means by
which the dialysis equipment can cleanse the bloodstream. Even
though this is a high-flow conduit, it is susceptible to the
formation of biofilms and subsequent infection. If a shunt becomes
infected, it requires removal and replacement. Since dialysis may
be a lifelong process, and since there are a limited number of
sites where shunts can be applied, it is desirable to avoid having
to remove one through infectious complications. Imbedding or
otherwise contacting the compounds of the invention with the shunt
material can have this desired effect.
[0165] Heart valves comprising artificial material are understood
to be vulnerable to the dangerous complication of prosthetic valve
endocarditis. Once established, it carries a mortality rate as high
as 70%. Biofilms are integrally involved in the development of this
condition. At present, the only treatment for established
contamination is high-dose antibiotic therapy and surgical removal
of the device. The contaminated valve must be immediately replaced,
since the heart cannot function without it. Because the new valve
is being inserted in a recently contaminated area, it is common for
prosthetic valve endocarditis to affect the replacement valve as
well. Artificial heart valves comprised of the compounds of the
invention may reduce the incidence of primary and recurrent
prosthetic valve endocarditis. Compounds of the invention can be
applied to the synthetic portions or the naturally-derived portions
of heart valves.
[0166] Pacemakers and artificial implantable defibrillators
commonly comprise metallic parts in combination with other
synthetic materials. These devices, which may be coated with a
polymeric substance such as silicone are typically implanted in
subcutaneous or intramuscular locations with wires or other
electrical devices extending intrathoracically or intravascularly.
If the device becomes colonized with microorganisms and infected,
it must be removed. A new device can be replaced in a different
location, although there are a finite number of appropriate
implantation sites on the body. Devices comprising the compounds of
the invention may inhibit contamination and infection, or
substantially reduce the risk thereof.
[0167] Devices implanted into the body either temporarily or
permanently to pump pharmacological agents into the body can
comprise metallic parts in combination with other synthetic
materials. Such devices, termed infusion pumps, can be entirely
implanted or can be partially implanted. The device may be
partially or entirely covered with a polymeric substance, and may
comprise other polymers used as conduits or tubes. Incorporating AF
agents according to the present invention into the coating
materials imposed upon these devices or into the materials used for
the devices themselves, their conduits or their tubing may inhibit
their contamination and infection.
[0168] Equally lifesaving are the various vascular grafting
prostheses and stents intended to bypass blocked arteries or
substitute for damaged arteries. Vascular grafting prostheses, made
of Teflon, dacron, Gore-tex.RTM., expanded polytetrafluoroethylene
(e-PTFE), and related materials, are available for use on any major
blood vessel in the body. Commonly, for example, vascular grafting
prostheses are used to bypass vessels in the leg and are used to
substitute for a damaged aorta. They are put in place by being sewn
into the end or the side of a normal blood vessel upstream and
downstream of the area to be bypassed or replaced, so that blood
flows from a normal area into the vascular grafting prosthesis to
be delivered to other normal blood vessels. Stents comprising
metallic frames covered with vascular grafting prosthesis fabric
are also available for endovascular application, to repair damaged
blood vessels.
[0169] Suture material used to anchor vascular grafting prostheses
to normal blood vessels or to sew vessels or other structures
together can also harbor infections. Sutures used for these
purposes are commonly made of prolene, nylon or other
monofilamentous nonabsorbable materials. An infection that begins
at a suture line can extend to involve the vascular grafting
prosthesis. Suture materials comprising a compound of the invention
would have increased resistance to infection.
[0170] A general principle of surgery is that when a foreign object
becomes infected, it most likely needs to be removed so that the
infection can be controlled. For example, when sutures become
infected, they may need to be surgically removed to allow the
infection to be controlled. Any area where surgery is performed is
susceptible to a wound infection. Wound infections can penetrate to
deeper levels of the tissues to involve foreign material that has
been used as part of the operation. As an example, hernias are
commonly repaired by suturing a plastic screening material called
mesh in the defect. A wound infection that extends to the area
where the mesh has been placed can involve the mesh itself,
requiring that the mesh be removed. Surgical meshes comprising a
compound of the invention can have increased resistance to
infection. Surgical meshes are made of substances like
Gore-tex.RTM., teflon, nylon and Marlex.RTM.. Surgical meshes are
used to close deep wounds or to reinforce the enclosure of body
cavities. Removing an infected mesh can leave an irreparable
defect, with life-threatening consequences. Avoiding infection of
these materials is of paramount importance in surgery. Materials
used for meshes and related materials can be formulated to include
the claimed compounds of the instant invention.
[0171] Certain implantable devices intended to restore structural
stability to body parts can be advantageously treated with the
instant claimed compounds. A few examples follow, and others will
be readily identified by ordinary skilled artisans. Implantable
devices, used to replace bones or joints or teeth, act as
prostheses or substitutes for the normal structure present at that
anatomic site. Metallics and ceramics are commonly used for
orthopedic and dental prostheses. Implants may be anchored in place
with cements like polymethylmethacrylate. Prosthetic joint surfaces
can be fabricated from polymers such as silicones or teflon. Entire
prosthetic joints for fingers, toes or wrists can be made from
polymers.
[0172] Medical prostheses comprising compounds of the invention
would be expected to have reduced contamination and subsequent
local infection, thereby obviating or reducing the need to remove
the implant with the attendant destruction of local tissues.
Destruction of local tissues, especially bones and ligaments, can
make the tissue bed less hospitable for supporting a replacement
prosthesis. Furthermore, the presence of contaminating
microorganisms in surrounding tissues makes recontamination of the
replacement prosthesis easily possible. The effects of repeated
contamination and infection of structural prosthetics is
significant: major reconstructive surgery may be required to
rehabilitate the area in the absence of the prosthesis, potentially
including free bone transfers or joint fusions. Furthermore, there
is no guarantee that these secondary reconstructive efforts will
not meet with infectious complications as well. Major disability,
with possible extremity amputation, is the outcome from
contamination and infection of a structural prosthesis.
[0173] Certain implantable devices are intended to restore or
enhance body contours for cosmetic or reconstructive applications.
A well-known example of such a device is the breast implant, a gel
or fluid containing sac made of a silicone elastomer. Other
polymeric implants exist that are intended for permanent cosmetic
or reconstructive uses. Solid silicone blocks or sheets can be
inserted into contour defects. Other naturally occurring or
synthetic biomaterials are available for similar applications.
Craniofacial surgical reconstruction can involve implantable
devices for restoring severely deformed facial contours in addition
to the techniques used for restructuring natural bony contours.
These devices, and other related devices well-known in the field,
are suitable for coating with or impregnation with sulfate ester AF
agents to reduce their risk of contamination, infection and
subsequent removal.
[0174] Tissue expanders are sacs made of silicone elastomers
adapted for gradual filling with a saline solution, whereby the
filling process stretches the overlying tissues to generate an
increased area of tissue that can be used for other reconstructive
applications. Tissue expanders can be used, for example, to expand
chest wall skin and muscle after mastectomy as a step towards
breast reconstruction. Tissue expanders can also be used in
reconstructing areas of significant skin loss in burn victims. A
tissue expander is usually intended for temporary use: once the
overlying tissues are adequately expanded, they are stretched to
cover their intended defect. If a tissue expander is removed before
the expanded tissues are transposed, though, all the expansion
gained over time is lost and the tissues return nearly to their
pre-expansion state. The most common reason for premature tissue
expander removal is infection. These devices are subjected to
repeated inflations of saline solution, introduced percutaneously
into remote filling devices that communicate with the expander
itself. Bacterial contamination of the device is thought to occur
usually from the percutaneous inflation process. Once contamination
is established and a biofilm forms, local infection is likely.
Expander removal, with the annulment of the reconstructive effort,
is needed to control the infection. A delay of a number of months
is usually recommended before a new tissue expander can be inserted
in the affected area. The silicone elastomer used for these devices
is especially suitable for integrating with sulfate ester AF
agents. Use of these agents in the manufacture of these articles
may reduce the incidence of bacterial contamination, biofilm
development and subsequent local infection.
[0175] Insertable devices include those objects made from synthetic
materials applied to the body or partially inserted into the body
through a natural or an artificial site of entry. Examples of
articles applied to the body include contact lenses and stoma
appliances. An artificial larynx is understood to be an insertable
device in that it exists in the airway, partially exposed to the
environment and partially affixed to the surrounding tissues. An
endotracheal or tracheal tube, a gastrostomy tube or a catheter are
examples of insertable devices partially existing within the body
and partially exposed to the external environment. The endotracheal
tube is passed through an existing natural orifice. The tracheal
tube is passed through an artificially created orifice. Under any
of these circumstances, the formation of biofilm on the device
permits the ingress of microorganisms along the device from a more
external anatomic area to a more internal anatomic area. The ascent
of microorganisms to the more internal anatomic area commonly
causes local and systemic infections.
[0176] As an example, biofilm formation on soft contact lenses is
understood to be a risk factor for contact-lens associated corneal
infection. The eye itself is vulnerable to infections due to
biofilm production. Incorporating an antifouling agent in the
contact lens itself and in the contact lens case can reduce the
formation of biofilms, thereby reducing risk of infection. Sulfate
ester AF agents can also be incorporated in ophthalmic preparations
that are periodically instilled in the eye.
[0177] As another example, biofilms are understood to be
responsible for infections originating in tympanostomy tubes and in
artificial larynxes. Biofilms further reside in tracheostomy tubes
and in endotracheal tubes, permitting the incursion of pathogenic
bacteria into the relatively sterile distal airways of the lung.
These devices are adaptable to the incorporation or the topical
application of sulfate ester AF agents to reduce biofilm formation
and subsequent infectious complications.
[0178] As another example, a wide range of vascular catheters are
fabricated for vascular access. Temporary intravenous catheters are
placed distally, while central venous catheters are placed in the
more proximal large veins. Catheter systems can include those
installed percutaneously whose hubs are external to the body, and
those whose access ports are buried beneath the skin. Examples of
long-term central venous catheters include Hickman catheters and
Port-a-caths. Catheters permit the infusion of fluids, nutrients
and medications; they further can permit the withdrawal of blood
for diagnostic studies or the transfusion of blood or blood
products. They are prone to biofilm formation, increasingly so as
they reside longer within a particular vein. Biofilm formation in a
vascular access device can lead to the development of a blood-borne
infection as planktonic organisms disseminate from the biofilm into
the surrounding bloodstream. Further, biofilm formation can
contribute to the occlusion of the device itself, rendering it
non-functional. If the catheter is infected, or if the obstruction
within it cannot be cleared, the catheter must be removed.
Commonly, patients with these devices are afflicted with serious
medical conditions. These patients are thus poorly able to tolerate
the removal and replacement of the device. Furthermore, there are
only a limited number of vascular access sites. A patient with
repeated catheter placements can run out of locations where a new
catheter can be easily and safely placed. Incorporation of sulfate
ester AF agents within catheter materials or application of these
agents to catheter materials can reduce fouling and biofilm
formation, thereby contributing to prolonged patency of the devices
and minimizing the risk of infectious complications.
[0179] As another example, a biliary drainage tube is used to drain
bile from the biliary tree to the body's exterior if the normal
biliary system is blocked or is recovering from a surgical
manipulation. Drainage tubes can be made of plastics or other
polymers. A biliary stent, commonly fabricated of a plastic
material, can be inserted within a channel of the biliary tree to
keep the duct open so that bile can pass through it. Biliary sludge
which forms as a result of bacterial adherence and biofilm
formation in the biliary system is a recognized cause of blockage
of biliary stents. Pancreatic stents, placed to hold the pancreatic
ducts open or to drain a pseudocyst of the pancreas, can also
become blocked with sludge. Biofilms are furthermore implicated in
the ascent of infections into the biliary tree along a biliary
drainage tube. Ascending infections in the biliary tree can result
in the dangerous infectious condition called cholangitis.
Incorporation of compounds of the invention in the materials used
to form biliary drainage tubes and biliary stents can reduce the
formation of biofilms, thereby decreasing risk of occlusions and
infections.
[0180] As another example, a peritoneal dialysis catheter is used
to remove bodily wastes in patients with renal failure by using
fluids instilled into and then removed from the peritoneal cavity.
This form of dialysis is an alternative to hemodialysis for certain
renal failure patients. Biofilm formation on the surfaces of the
peritoneal dialysis catheter can contribute to blockage and
infection. An infection entering the peritoneal cavity is termed a
peritonitis, an especially dangerous type of infection. Peritoneal
dialysis catheters, generally made of polymeric materials like
polyethylene, can be coated with or impregnated with sulfate ester
AF agents to reduce the formation of biofilms.
[0181] As yet another example, a wide range of urological catheters
exist to provide drainage of the urinary system. These catheters
can either enter the natural orifice of the urethra to drain the
bladder, or they can be adapted for penetration of the urinary
system through an iatrogenically created insertion site.
Nephrostomy tubes and suprapubic tubes represent examples of the
latter. Catheters can be positioned in the ureters on a
semipermanent basis to hold the ureter open; such a catheter is
called a ureteral stent. Urological catheters can be made from a
variety of polymeric products. Latex and rubber tubes have been
used, as have silicones. All catheters are susceptible to biofilm
formation. This leads to the problem of ascending urinary tract
infections, where the biofilm can spread proximally, carrying
pathogenic organisms, or where the sessile organisms resident in
the biofilm can propagate planktonic organisms that are capable of
tissue and bloodstream invasion. Organisms in the urinary tract are
commonly Gram-negative bacteria capable of producing
life-threatening bloodstream infections if they spread
systemically. Infections wherein these organisms are restricted to
the urinary tract can nonetheless be dangerous, accompanied by pain
and high fever. Urinary tract infections can lead to kidney
infections, called pyelonephritis, that can jeopardize the function
of the kidney. Incorporating sulfate ester AF agents can inhibit
biofilm formation and may reduce the likelihood of these infectious
complications.
[0182] A further complication encountered in urological catheters
is encrustation, a process by which inorganic compounds comprising
calcium, magnesium and phosphorous are deposited within the
catheter lumen, thereby blocking it. These inorganic compounds are
understood to arise from the actions of certain bacteria resident
in biofilms on catheter surfaces. Reducing biofilm formation by the
action of sulfate ester AF agents may contribute to reducing
encrustation and subsequent blockage of urological catheters.
[0183] Other catheter-like devices exist that can be treated with
AF agents. For example, surgical drains, chest tubes, hemovacs and
the like can be advantageously treated with materials to impair
biofilm formation. Other examples of such devices will be familiar
to ordinary practitioners in these arts.
[0184] Materials applied to the body can advantageously employ the
AF compounds disclosed herein. Dressing materials can themselves
incorporate the AF compounds, as in a film or sheet to be applied
directly to a skin surface. Additionally, AF compounds of the
instant invention can be incorporated in the glue or adhesive used
to stick the dressing materials or appliance to the skin. Stoma
adhesive or medical-grade glue may, for example, be formulated to
include an AF agent appropriate to the particular medical setting.
Stoma adhesive is used to adhere stoma bags and similar appliances
to the skin without traumatizing the skin excessively. The presence
of infectious organisms in these appliances and on the surrounding
skin makes these devices particularly appropriate for coating with
AF agents, or for incorporating AF agents therein. Other affixation
devices can be similarly treated. Bandages, adhesive tapes and
clear plastic adherent sheets are further examples where the
incorporation of an AF agent in the glue or other adhesive used to
affix the object, or incorporation of an AF agent as a component of
the object itself, may be beneficial in reducing skin irritation
and infection.
[0185] These above examples are offered to illustrate the
multiplicity of applications of compounds of the invention in
medical devices. Other examples will be readily envisioned by
skilled artisans in these fields. The scope of the present
invention is intended to encompass all those surfaces where the
presence of fouling has adverse health-related consequences. The
examples given above represent embodiments where the technologies
of the present invention are understood to be applicable. Other
embodiments will be apparent to practitioners of these and related
arts. Embodiments of the present invention can be compatible for
combination with currently employed antiseptic regimens to enhance
their antimicrobial efficacy or cost-effective use. Selection of an
appropriate vehicle for bearing a compound of the invention will be
determined by the characteristics of the particular medical use.
Other examples of applications in medical environments to promote
antisepsis will be readily envisioned by those of ordinary skill in
the relevant arts.
Exemplification
[0186] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Surface Derivatization
EXAMPLE 1
[0187] Method A
[0188] A NH.sub.2-glass slide (aminopropyltrimethoxysilane-coated
microscopic slides) was placed in 90 mL of dry dichloromethane
containing 1 mL of triethylamine. After cooling to 4.degree. C., 10
mL of acryloyl chloride was added, and the reaction mixture was
stirred in a cold room overnight and then at room temperature for 2
h. The acylated NH.sub.2-glass slides were rinsed with a
methanol/triethylamine mixture (1:1, v/v) and methanol. As judged
from the determination of the NH.sub.2 groups on the glass slide
surface before (6.6.+-.0.1.times.10.sup.-10 mol/cm.sup.2) and after
(3.3.+-.0.2.times.10.sup.-10 mol/cm.sup.2) the acryloylation using
the picric acid titration (17), some half of the surface-bound
amino groups reacted with acryloyl chloride. The glass-bonded
acryloyl moieties were then copolymerized with 4-vinylpyridine.
Perchloric acid (90 mL of a 20% solution in water) was degassed,
and 30 mg of Ce(SO.sub.4).sub.2 was added under argon. After 1 h of
stirring, an acryloylated glass slide was placed in this solution,
15 mL of freshly distilled 4-vinylpyridine was added under argon,
and the reaction mixture was stirred at room temperature for 3 h.
PVP not chemically attached to the slide was washed off with
pyridine, N,N-dimethylformide, and methanol. Immediately
thereafter, the slide with the attached PVP was placed in a 10%
(v/v) solution of an alkyl bromide in nitromethane. The reaction
mixture was then stirred at 75.degree. C. for 72 h, after which
time more than 90% of the pyridine rings were N-alkylated (19). The
resultant polycation-derivatized PVP-slide was rinsed with methanol
and distilled water and air dried.
EXAMPLE 2
[0189] Method B
[0190] A NH.sub.2-glass slide was immersed in a mixture containing
9 mL of 1,4-dibromobutane, 90 mL of dry nitromethane, and 0.1 mL of
triethylamine. After stirring at 60.degree. C. for 2 h, the slide
was removed, thoroughly rinsed with nitromethane, air dried, and
placed in a solution of 9 g of PVP (molecular weight of 60,000 or
160,000 g/mol) in 90 mL of nitromethane/hexyl bromide (10:1, v/v).
After stirring the reaction mixture at 75.degree. C. for 24 h, the
slide was rinsed with acetone, thoroughly washed with methanol (to
remove the non-attached polymer), and air dried. According to the
literature (14), more than 96% of the pyridine rings of PVP should
be N-alkylated under these conditions.
Surface Analysis
EXAMPLE 3
[0191] A chemically modified glass slide was immersed in a 1%
solution of fluorescein (Na salt) in distilled water for 5 min.
Under these conditions, the dye binds to quaternary amino groups
(20), but not to tertiary or primary ones (we found that
PVP-modified or NH.sub.2-glass slides do not adsorb fluorescein).
After rinsing with distilled water, a stained slide was placed in
25 mL of the 0.1% detergent cetyltrimethyl-ammonium chloride in
distilled water and shaken for 10 min to desorb the dye. The
absorbance of the of the resultant aqueous solution was measured at
501 nm (after adding 10% of a 100 mM aqueous phosphate buffer, pH
8.0). The independently determined extinction coefficient of
fluorescein in this solution was found to be 77 mM.sup.-1
cm.sup.-1.
[0192] From staining different hexyl-PVP-films (160,000 g/mol,
degree of alkylation >95%) with a known polymer content, the
stoichiometry of fluorescein binding was found to be approximately
1 dye molecule per 7 hexyl-PVP monomer units. The following amounts
of the attached hexyl-PVP were determined (assuming that more than
90% of the polymer is hexylated): 5.8.+-.3.0 .mu.g/cm.sup.2 (method
A); 2.8.+-.1.0 .mu.g/cm.sup.2 (method B, PVP with M.sub.w=160,000
g/mol); and 0.4.+-.0.05 .mu.g/cm.sup.2 (method B, PVP with
M.sub.w=60,000 g/mol). In the case of hexyl-PVP immobilized by
method A, the minimal chain length of the attached polycation was
estimated to be 61.+-.30 monomer units.
Antimicrobial Susceptibility Determination
EXAMPLE 4
[0193] A suspension (100 .mu.L) of Staphylococcus aureus (ATCC,
strain 33807), Staphylococcus epidermidis (wild type), Pseudomonas
aeruginosa (wild type), or Escherichia coli (ZK 605) in 0.1 M
aqueous PBS buffer (pH 7.0, approximately 10.sup.11 cells/mL) was
added to 50 mL of a yeast-dextrose broth (prepared as described by
Cunliffe et al. (21)) in a sterile Erlenmeyer flask. The suspension
was incubated at 37.degree. C. with shaking at 200 rpm for 6-8 h.
After centrifugation (2,700 rpm, 10 min), the bacterial cells were
washed with, and re-suspended in, distilled water at a
concentration of 10.sup.6 cells per mL (10.sup.7 in the case of E.
coli).
[0194] A bacterial suspension was then sprayed onto a glass slide
(or another surface) in a fume hood using a commercial
chromatography sprayer (VWR) (spray rate of approximately 10
mL/min). After drying for 2 min under air, the slide was placed in
a petri dish, and then growth agar (0.7% agar in a yeast-dextrose
broth, autoclaved, and cooled to 37.degree. C.) was added. The
petri dish was closed, sealed, and incubated at 37.degree. C.
overnight.
EXAMPLE 5
[0195] The bacteria were suspended in distilled water and sprayed
onto the surface of a slide to simulate the deposition of airborne
bacteria--a common method of spreading bacterial infections
generated, for example, by talking, sneezing, coughing, or just
breathing. To determine the number of viable bacterial cells on the
infected surface, the slide, following a 2-min air drying, was
incubated under growth agar; the bacteria able to proliferate form
countable colonies under these conditions. Infectious Gram-positive
bacterium Staphylococcus aureus was used for the initial studies.
Next, a NH.sub.2-glass slide was acylated with acryloyl chloride to
introduce double bonds, followed by copolymerization with
4-vinylpyridine. Such an immobilized PVP was found to afford
approximately the same number of viable S. aureus cells after
spraying the bacterial suspension onto its surface as a plain
NH.sub.2-glass slide. The final step was to introduce positive
charges into the PVP chains attached to glass. To this end, the
polymer's pyridine rings were N-alkylated by seven linear alkyl
bromides (with chain lengths varying from propyl to hexadecyl). The
resultant slides were examined with respect to their ability to
kill on contact S. aureus cells sprayed on them. As seen in FIG. 2,
propylated, butylated, hexylated, and octylated immobilized PVP
chains were effective in markedly reducing the number of viable
bacterial cells, with the most effective, hexyl-PVP affording a
94.+-.4% reduction. (See right portion of FIG. 1).
EXAMPLE 6
[0196] Using a glass slide surface modified with hexyl-PVP
(henceforth referred to as that prepared by method A), we then
tested the bactericidal effect of this surface toward another
Gram-positive bacterium, S. epidermidis, as well as two
Gram-negative bacteria, E. coli and P. aeruginosa. The first two
formed colonies of the same size as S. aureus (FIG. 1, left
portion) when sprayed on NH.sub.2-glass slides, whereas the
colonies of P. aeruginosa were larger but still distinguishable. As
seen in Table 1 (method A), the number of colonies all three
bacteria formed after spraying onto hexyl-PVP-slides dropped more
than 100-fold compared to the plain NH.sub.2-glass.
[0197] Suspensions (10.sup.6 cells/mL for the first 3 bacteria and
10.sup.7 cells/mL for the last one) of bacteria in distilled water
were sprayed on hexyl-PVP-modified glass surfaces, air dried for 2
min, incubated under 0.7% agar in a bacterial growth medium
overnight, and the colonies were counted. The number of viable
cells obtained in the same manner with commercial NH.sub.2-glass
slides was used as a standard (i.e., 0% of the bacteria
killed).
EXAMPLE 7
[0198] Antibacterial properties of a PVP-based polycation
immobilized onto glass slides were analyzed in different procedure
using a NH.sub.2-glass slide was alkylated with 1,4-dibromobutane
to introduce reactive bromobutyl groups, which were subsequently
used for the attachment of PVP. The resultant surface was not able
to kill S. aureus cells upon spraying. To increase the positive
charge of the attached PVP chains, the chains were further
N-alkylated them with hexyl bromide After S. aureus cells were
sprayed, air dried, and cultured, the resultant hexyl-PVP-slides
(henceforth referred to as those prepared by method B) looked
essentially the same as shown in the right portion of FIG. 1.
Compared to a NH.sub.2-glass slide, 94.+-.3% of the deposited S.
aureus cells were killed (the last column, 1st line, of Table
1).
[0199] Suspensions (10.sup.6 cells/mL for the first 3 bacteria and
10.sup.7 cells/mL for the last one) of bacteria in distilled water
were sprayed on hexyl-PVP-modified glass surfaces, air dried for 2
min, incubated under 0.7% agar in a bacterial growth medium
overnight, and the colonies were counted. The number of viable
cells obtained in the same manner with commercial NH.sub.2-glass
slides was used as a standard (i.e., 0% of the bacteria
killed).
Surface Derivatization
EXAMPLE 8
[0200] A HDPE slide (7.5.times.2.5 cm, Polymer Plastics Co., Reno,
Nev.) was sonicated in isopropyl alcohol for 5 min, rinsed with
that solvent, and dried at 80.degree. C. for 30 min. Next, the
front (oxidizing) part of the flame of a hand-held burner (SurA
Instruments GmbH, Jena, Germany) was fanned over the slide surface
for 15 s, and the slide was stored under air overnight. The
compressed gas mixture of the burner contained 0.6% (v/v)
tetramethylsilane in a propane/butane (7:3, v/v) mixture
(Pyrosil.RTM.). The SiO.sub.2-coated HDPE slide was then placed
into a 20% solution of 3-aminopropyltriethoxysilane (Aldrich
Chemical Co., Milwaukee, Wis.) in dry toluene, incubated with
stirring at room temperature for 3 h, rinsed with toluene and
methanol, and dried under air overnight. Bisse, E., Scholer, A.
& Vonderschmitt, D. J. A new method for coupling glucose
dehydrogenase to glass tubes activated with titanium tetrachloride.
FEBS Lett. 138, 316-318 (1982). The NH.sub.2-functionalized HDPE
slide was then treated with a mixture of 10 mL of
1,4-dibromobutane, 0.1 mL of triethylamine, and 90 mL of
nitromethane with stirring at 60.degree. C. for 5 h and rinsed with
nitromethane, followed by addition of a freshly prepared solution
of 9 g of PVP (molecular weight of 160,000 g/mol) in 81 mL of
nitromethane and 10 mL of 1-bromohexane (all from Aldrich). After 9
h of stirring at 75.degree. C., the slide was thoroughly rinsed
with methanol and distilled water, and dried under air. Slides (all
7.5.times.2.5 cm in size) of LDPE (Polymer Plastics Co.), PP, nylon
(type 6/6) (Plastic Material Co., Cleveland, Ohio), and PET
(Wheaton, Millville, N.J.) were derivatized following the same
protocol, except that PET in the last step was treated at
60.degree. C. instead of 75.degree. C.
Surface Analysis
EXAMPLE 9
[0201] The X-ray photoelectron spectrum of the SiO.sub.2-coated
HDPE slide was recorded using a Kratos Axis Ultra instrument
(Kratos Analytical, New York, N.Y.) using a 150 W Al K.alpha.
monochromator source.
EXAMPLE 10
[0202] A hexyl-PVP-derivatized slide was placed in a 1% fluorescein
(Sigma Chemical Co., St. Louis, Mo.) solution in distilled water,
shaken for 5 min, thoroughly rinsed with water, and placed in a
0.25% aqueous solution of cetyltrimethylammonium chloride
(Aldrich). After shaking for 5 min, 10% (v/v) of 0.1 M aqueous
phosphate buffer, pH 8.0, was added to the solution, the absorbance
was measured at 501 nm, and the number of pyridinium groups on the
surface was calculated as described earlier. Tiller, J. C., Liao,
C.-J., Lewis, K. & Klibanov, A. M. Designing surfaces that kill
bacteria on contact. Proc. Natl. Acad. Sci. U.S.A. 98, 5981-5985
(2001).
Antimicrobial Potency Determination
EXAMPLE 11
[0203] Bacteria were cultivated by adding a suspension (100 .mu.L)
of S. aureus (strain 33807, ATCC, Manassas, Va.) or E. coli (strain
ZK 650, provided by Dr. Gary Bonner, Harvard Medical School) in 0.1
M aqueous PBS buffer, pH 7.0 (approximately 10.sup.11 cells/mL) to
50 mL of a yeast-dextrose broth (prepared as described by Cunliffe
et al.) in a sterile Erlenmeyer flask. Cunliffe, D., Smart, C. A.,
Alexander, C. & Vulfson, E. N. Bacterial adhesion at synthetic
surfaces. Appl. Environ. Microbiol. 165, 4995-5002 (1999). The
suspension was incubated at 37.degree. C. with shaking at 200 rpm
for 6-8 h.
[0204] The ability of surfaces to kill airborne bacteria was tested
as described earlier. Tiller, J. C., Liao, C.-J., Lewis, K. &
Klibanov, A. M. Designing surfaces that kill bacteria on contact.
Proc. Natl. Acad. Sci. U.S.A. 98, 5981-5985 (2001). Bacterial cells
were centrifuged at 2,700 rpm for 10 min, washed with distilled
water, and re-suspended at a concentration of 10.sup.6 cells per mL
for S. aureus and 10.sup.5 for E. coli. The bacterial cell
concentration was assessed assuming that the optical density of 1.0
at 540 nm is equivalent to approximately 10.sup.9 cells per mL.
Hogt, A. H., Dankert, J. & Feijen, J. Adhesion of
coagulase-negative staphylococci to methacrylate polymers and
copolymers. J. Biomed. Mater. Res. 20, 533-545 (1986). A bacterial
suspension was then sprayed onto a polymer slide in a fume hood
using a commercial chromatography sprayer (VWR, Boston, Mass.)
(spraying rate of approximately 10 mL/min). After drying for 2 min
under air, the slide was placed in a Petri dish, and growth agar
(0.7% agar in yeast-dextrose broth, autoclaved, and cooled to
37.degree. C.) was added. The dish was closed, sealed, and
incubated at 37.degree. C. overnight. The grown bacterial colonies
were counted on a light box (Picker International, Cleveland, Ohio)
which magnifies the contrast between colonies and polymer slide. In
the case of nylon, the bacterial colonies were stained after the
incubation: 1 mg of Crystal Violet (Sigma) was dissolved in 100 mL
of distilled water and added onto the growth agar covering the
infected slide until the surface was completely wetted. This
procedure was repeated after 2 h; the stained colonies were visible
within 5 h.
[0205] When polymers were tested against waterborne bacteria,
bacterial cells were centrifuged at 2,700 rpm for 10 min, washed
twice with PBS at pH 7.0, re-suspended in the same buffer, and
diluted to 2.multidot.10.sup.6 cells per mL of PBS
(4.multidot.10.sup.6 in the case of E. coli). A plastic slide was
placed in 45 mL of this suspension, incubated with shaking at 200
rpm and 37.degree. C. for 2 h, washed three times by immersing the
slide in the sterile PBS, immediately covered with a slab cut out
of the solid growth agar (1.5% agar in the bacterial growth medium,
autoclaved, poured into a Petri dish, and dried under reduced
pressure at room temperature overnight), and incubated in a sealed
Petri dish at 37.degree. C. overnight. The bacterial colonies were
counted as described above.
EXAMPLE 12
[0206] Polymer Surfaces Derivatized with
poly(vinyl-N-hexylpyridinium) Kill Airborne, Waterborne, and
Deposited Bacteria
[0207] A facile methodology has been developed for covalently
derivatizing the surfaces of common materials with a designed
antibacterial polycation, poly(vinyl-N-pyridinium bromide), wherein
the first, key step involves surface coating with a nanolayer of
silica. Various commercial synthetic polymers derivatized in this
manner become bactericidal--they kill up to 99% of deposited,
whether through air or water, Gram-positive and Gram-negative
bacteria on contact.
[0208] We have discovered a novel, non-release strategy for
creating bactericidal surfaces which involves covalent coating with
long hydrophobic polycationic chains; critically, the latter must
be fine-tuned with respect to charge and hydrophobicity to resist,
by means of electrostatic repulsion, hydrophobic interchain
aggregation and yet be able to penetrate bacterial cell membranes.
As a result, glass slides covalently modified with
poly(vinyl-N-hexylpyridinium bromide) (hexyl-PVP) were found to
kill up to >99% of airborne bacteria. In this Example, we
demonstrate a greatly extended range of such bactericidal
materials, including common synthetic polymers. In doing so, a
general surface coating/derivatization procedure has been
discovered that will be applicable to most materials regardless of
their nature. Specifically, five representative, inherently
nonbactericidal, commercial polymers derivatized with hexyl-PVP
have been demonstrated to kill 90 to 99% of Gram-positive and
Gram-negative bacteria deposited, through either air or water, onto
their surfaces.
[0209] Materials
[0210] Fluorescein (Na salt) and Crystal Violet were purchased from
Sigma Chemical Co. 3-Aminopropyltriethoxysilane, 1-bromohexane,
cetyltrimethylammonium chloride, poly(4-vinylpyridine) (PVP)
(molecular weight of 160 kg/mol), and all other chemicals used in
this work (analytical grade or purer) were obtained from Aldrich
Chemical Co. and used without further purification. High-density
polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene
(PP), and nylon 6/6 were supplied by Polymer Plastics Co. (Reno,
Nev.), and poly(ethylene terephthalate) (PET) by Wheaton
(Millville, N.J.). A hand-held burner containing 0.6% (v/v)
tetramethylsilane in a propane/butane (7:3, v/v) mixture
(Pyrosil.RTM.) was from SurA Instruments GmbH (Jena, Germany).
[0211] Surface Derivatization
[0212] A HDPE slide (7.5.times.2.5 cm) was sonicated in isopropyl
alcohol for 5 min, rinsed with that solvent, and dried at
80.degree. C. for 30 min. Next, the front (oxidizing) part of the
flame of a hand-held burner was fanned over the slide surface for
15 s, and the slide was stored under air overnight. The
SiO.sub.2-coated HDPE slide was then placed into a 20% solution of
3-aminopropyltriethoxysilane in dry toluene, incubated with
stirring at room temperature for 3 h, rinsed with toluene and
methanol, and dried under air overnight (Bisse et al., 1982). The
NH.sub.2-functionalized HDPE slide was then treated with a mixture
of 10 mL of 1,4-dibromobutane, 0.1 mL of triethylamine, and 90 mL
of nitromethane with stirring at 60.degree. C. for 5 h and rinsed
with nitromethane, followed by addition of a freshly prepared
solution of 9 g of PVP in 81 mL of nitromethane and 10 mL of
1-bromohexane. After 9 h of stirring at 75.degree. C., the slide
was thoroughly rinsed with methanol and distilled water and dried
under air. Slides (all 7.5.times.2.5 cm in size) of LDPE, PP,
nylon, and PET were derivatized following the same protocol, except
that PET in the last step was treated at 60.degree. C. instead of
75.degree. C.
[0213] X-ray Photoelectron Spectroscopy
[0214] The X-ray photoelectron spectrum of the SiO.sub.2-coated
HDPE slide was recorded using a Kratos Axis Ultra instrument
(Kratos Analytical, N.Y.) using a 150 W Al K.alpha. monochromator
source.
[0215] Titration of Surface Pyridinium Groups
[0216] A hexyl-PVP-derivatized slide was placed in a 1% fluorescein
solution in distilled water, shaken for 5 min, thoroughly rinsed
with water, and placed in a 0.25% aqueous solution of
cetyltrimethylammonium chloride. After shaking for 5 min, 10% (v/v)
of 0.1 M aqueous phosphate buffer, pH 8.0, was added to the
solution, the absorbance was measured at 501 nm, and the number of
pyridinium groups on the surface was calculated as described
earlier.
[0217] Antimicrobial Potency Determination
[0218] Bacteria were cultivated by adding a suspension (100 .mu.L)
of S. aureus (strain 33807, ATCC, Manassas, Va.) or E. coli (strain
ZK 650, provided by Dr. Gary Bonner, Harvard Medical School) in 0.1
M aqueous PBS buffer, pH 7.0 (approximately 10.sup.11 cells/mL) to
50 mL of a yeast-dextrose broth (prepared as described by Cunliffe
et al., 1999) in a sterile Erlenmeyer flask. The suspension was
shaken at 200 rpm and 37.degree. C. for 6-8 h.
[0219] The ability of surfaces to kill airborne bacteria was tested
as described earlier (Tiller et al., 2001). Bacterial cells were
centrifuged at 2,700 rpm for 10 min, washed with distilled water,
and re-suspended at a concentration of 10.sup.6 cells per mL for S.
aureus and 105 for E. coli. The bacterial cell concentration was
assessed assuming that the optical density of 1.0 at 540 nm is
equivalent to approximately 10.sup.9 cells per mL (Hogt et al.,
1986). A bacterial suspension was then sprayed onto a polymer slide
in a fume hood using a commercial chromatography sprayer (VWR,
Boston, Mass.) (spraying rate of approximately 10 mL/min). After
drying for 2 min under air, the slide was placed in a Petri dish,
and growth agar (0.7% agar in yeast-dextrose broth, autoclaved, and
cooled to 37.degree. C.) was added. The dish was closed, sealed,
and incubated at 37.degree. C. overnight. The grown bacterial
colonies were counted on a light box (Picker International,
Cleveland, Ohio) which magnifies the contrast between colonies and
polymer slide. In the case of nylon, the bacterial colonies were
stained after the incubation: 1 mg of Crystal Violet was dissolved
in 100 mL of distilled water and added onto the growth agar
covering the infected slide until the surface was completely
wetted. This procedure was repeated after 2 h; the stained colonies
were visible within 5 h.
[0220] When polymers were tested against waterborne bacteria,
bacterial cells were centrifuged at 2,700 rpm for 10 min, washed
twice with PBS (pH 7.0), re-suspended in the same buffer, and
diluted to 2.multidot.10.sup.6 cells per mL of PBS
(4.multidot.10.sup.6 in the case of E. coli). A plastic slide was
placed in 45 mL of this suspension, shaken at 200 rpm and
37.degree. C. for 2 h, washed three times by immersing the slide in
the sterile PBS, immediately covered with a slab cut out of the
solid growth agar (1.5% agar in the bacterial growth medium,
autoclaved, poured into a Petri dish, and dried under reduced
pressure at room temperature overnight), and incubated in a sealed
Petri dish at 37.degree. C. overnight. The bacterial colonies were
counted as described above.
[0221] Results and Discussion
[0222] Since it would be desirable to make numerous diverse objects
bactericidal, we selected a surface derivatization approach
potentially applicable to any material. We validated this approach
with ordinary commercial synthetic polymers, namely polyolefins, a
polyamide, and a polyester, which by themselves exhibit no
antibacterial activity. A slide made of high-density polyethylene
(HDPE) was selected as the initial target. This polymer, as many
other materials, lacks reactive groups suitable for a facile
chemical modification. Therefore, we decided to coat it with an
ultrathin silica layer by a combustion chemical vapor deposition
technique (Schinkinger et al., 2001). To this end, we employed
(step #1 in FIG. 4) a pen-like torch containing a compressed
mixture of 0.6% tetramethylsilane with 7:3 propane-butane
(Pyrosil.RTM.). When this mixture burns in the air,
tetramethylsilane is oxidized to form 2-5-nm SiO.sub.2 particles
which cover a surface to which the flame is applied (Tiller et al.,
1989). The resulting dense, .about.100-nm thick SiO.sub.2 layer
(Schinkinger et al., 2001; Tiller et al., 1989) chemically
resembling glass (polysiloxane) can thereafter be readily
chemically modified in a uniform fashion regardless of the nature
of the bulk material (Tiller et al., 1989 and 1991).
[0223] The visual appearance of the HDPE surface did not change
after the SiO.sub.2 coating procedure. The degree of crosslinking
of such a SiO.sub.2 layer is lower than that of glass, and numerous
Si--OH groups are formed on hydration by water adsorbed from the
environment after the deposition process (Schinkinger et al.,
2001). Because of these Si--OH groups, the coated HDPE surface is
expected to be hydrophilic, in contrast to the very hydrophobic
unmodified polymer. To quantify this difference, we measured the
contact angle formed by an aqueous drop on a surface, a common
method of determining the water affinity of materials (David,
1966). To this end, 0.1 mL of distilled water was placed on the
HDPE surface. The angle between the core of the resultant water
drop and the surface under it was estimated to be some 1200,
indicative of a hydrophobic material. When the same amount of
distilled water was placed onto the SiO.sub.2-coated HDPE, the
water spread on the surface and the contact angle was less than
10.degree. reflecting the surface's hydrophilic character.
Independently, the presence of the SiO.sub.2 layer on the HDPE
slide was confirmed by the silicon and oxygen signals detected in
the X-ray photoelectron spectrum.
[0224] Next, amino groups were introduced into the SiO.sub.2-coated
HDPE slide surface by reacting it with 3-aminopropyltriethoxysilane
(step #2 in FIG. 4), a standard procedure for the amination of
glass surfaces (Bisse et al., 1982). The NH.sub.2-functionalized
HDPE slide was subsequently alkylated with 1,4-dibromobutane to
introduce bromoalkyl groups (step #3 in FIG. 4), which were then
reacted with PVP in the presence of 1-bromohexane (step #4 in FIG.
4). Under the conditions used, only a few (out of approximately
1,500) pyridine groups of the PVP chain are alkylated by the
surface-bound bromoalkyls, with the majority being alkylated by
1-bromohexane (Tiller et al., 2001). The number of the pyridinium
groups on the resultant hexyl-PVP-derivatized HDPE slides, titrated
spectrophotometrically with fluorescein, was determined to be
8.2.+-.1.9 nmol/cm.sup.2, which is similar to that observed for
hexyl-PVP-modified NH.sub.2-glass (Tiller et al., 2001).
[0225] The generality of the foregoing nanocoating/derivatization
approach was confirmed by modifying two other industrial
polyolefins, low-density polyethylene (LDPE) and polypropylene
(PP), as well as the polyamide nylon and the polyester
poly(ethylene terephthalate) (PET). All these synthetic polymers
were successfully derivatized as shown in FIG. 4 and exhibited
surface densities of the pyridinium groups similar to that of
HDPE-8.5.+-.1.8 (LDPE), 7.2.+-.2.1 (PP), 8.0.+-.1.1 (nylon), and
7.5.+-.0.9 (PET) nmol/cm.sup.2. All hexyl-PVP-derivatized slides,
starting with HDPE, were subsequently examined with respect to
their ability to kill airborne bacteria on contact.
[0226] To simulate natural deposition of airborne bacteria, as well
as contact deposition of bacteria, a suspension in distilled water
of the ubiquitous pathogen Gram-positive bacterium Staphylococcus
aureus (Xiong et al., 2000) was sprayed onto a slide surface and
the latter was allowed to dry. The slide was then incubated under
nutrient agar overnight as described earlier (Tiller et al., 2001),
and the number of viable bacterial cells was determined by colony
count. As seen in FIG. 5a, left, numerous readily distinguishable
bacterial colonies grew on the unmodified HDPE slide. The number of
colonies grown on the SiO.sub.2-coated or NH.sub.2-functionalized
HDPE slides (FIG. 4) was essentially the same indicating that these
modifications are not toxic to S. aureus. In striking contrast,
when sprayed onto a hexyl-PVP-modified HDPE slide, 96.+-.3% of the
deposited bacterial cells died, i.e., became non-viable (FIG. 5a,
right, and Table I).
5TABLE I The ability of various commercial synthetic polymers
derivatized by hexyl-PVP to kill airborne bacteria on contact.
Percentage of bacteria killed Bacterium Type HDPE LDPE PP nylon PET
S. aureus Gram (+) 96 .+-. 3 97 .+-. 1 90 .+-. 3 92 .+-. 2 95 .+-.
1 E. coli Gram (-) 97 .+-. 1 96 .+-. 2 98 .+-. 1 98 .+-. 2 95 .+-.
1 Suspensions of bacteria (10.sup.6 cells/mL for S. aureus and
10.sup.5 cells/mL for E. coli) in distilled water were sprayed on a
hexyl-PVP-modified polymer slide, and the latter was air dried for
2 min and incubated under 0.7% agar in a bacterial growth medium #
overnight; thereafter, the colonies were counted. The number of
viable cells obtained in the same manner with the corresponding
unmodified polymer slides was used as a standard (i.e., 0% of the
bacteria killed). All experiments were performed at least in #
duplicate, and the errors given indicate the standard
deviations.
[0227] LDPE, PP, nylon, and PET derivatized with hexyl-PVP were
analogously tested for their ability to kill airborne S. aureus. As
seen in Table I, in all cases the number of bacterial colonies
formed plunged 10 to 30-fold compared to those grown on the
corresponding unmodified slides.
[0228] Next, we tested the killing efficiency of the
hexyl-PVP-derivatized surfaces for a representative Gram-negative
bacterium, Escherichia coli. As seen in FIG. 5b, left, many E. coli
colonies grown on a HDPE slide after spraying, while less dense
than those of S. aureus grown under the same conditions, are still
distinguishable. The surface derivatization with hexyl-PVP slashed
their number by 97.+-.3% (FIG. 5b, right, and Table I). Inspection
of the remaining data in the last line of Table I reveals that the
hexyl-PVP modification of LDPE, PP, nylon, and PET slides likewise
afforded a 20 to 50-fold drop in the viable E. coli cells,
following their deposition from the airborne state, when compared
to the corresponding unmodified slides.
[0229] To test whether the hexyl-PVP chains immobilized using our
methodology can leach from the slide surface (and conceivably only
then kill the deposited bacteria), modified HDPE slide was placed
into a polystyrene Petri dish, and an aqueous suspension of S.
aureus cells was sprayed on it, as well as on the
(non-antibacterial) polystyrene surface. After air drying and
incubation under growth agar, only a few (4.+-.2 per cm.sup.2)
colonies grew on the hexyl-PVP-modified HDPE slide, while a far
greater number of colonies (85.+-.5 per cm.sup.2) grew on the
surface of the surrounding Petri dish, even in the immediate
proximity of the slide. The lack of inhibition zones around the
hexyl-PVP-slides, characteristic of release of bactericidal agents
(Kawabata and Nishiguchi, 1988), indicates that the immobilized
polycation does not leach from the slide, i.e., that the airborne
bacteria are indeed killed on contact with the slide's surface.
[0230] Another important question was whether the
hexyl-PVP-derivatized slides can also kill waterborne bacteria. To
experimentally simulate a flowing aqueous solution, a polymer slide
was placed vertically in a bacterial suspension in PBS, pH 7.0, and
the resulting system was gently agitated at 37.degree. C. After a
2-h incubation, the slide was washed with the sterile buffer to
remove non-adhered bacteria and then incubated in sterile PBS under
the conditions outlined above for 1 h to allow the tentatively
adhered bacteria to either detach or adhere irreversibly (Wiencek
and Fletcher, 1995). After rinsing the slide with PBS, it was
immediately covered with a layer of solid growth agar and incubated
overnight at 37.degree. C. This way, all genuinely attached
bacterial cells are captured on the slide surface, and those of
them still viable can proliferate and form colonies.
[0231] As seen in FIG. 6a, left, S. aureus cells allowed to adhere
to an HDPE slide form numerous easily distinguishable colonies.
Neither SiO.sub.2-- nor NH.sub.2-- modified HDPE slides exhibited
an appreciable decline in the number of bacterial colonies grown
after the adhesion. However, as seen in FIG. 6a, right, the number
of S. aureus colonies grown on a hexyl-PVP-modified HDPE slide
plunged 98.+-.1% (see also Table II).
6TABLE II The ability of hexyl-PVP covalently attached to various
commercial polymer slides to kill waterborne bacteria on contact.
Percentage of bacteria killed Bacterium Type HDPE LDPE PP nylon PET
S. aureus Gram (+) 98 .+-. 1 99 .+-. 1 97 .+-. 2 97 .+-. 1 99 .+-.
1 E. coli Gram (-) 97 .+-. 2 98 .+-. 2 n.d. 99 .+-. 1 98 .+-. 1
Suspensions of bacteria (2 .times. 10.sup.6 cells/mL for S. aureus
and 4 .times. 10.sup.6 cells/mL for E. coli) in PBS, pH 7.0, was
allowed to adhere to the hexyl-PVP-modified polymer surfaces under
shaking at 37.degree. C. for 2 h. The slides were rinsed with
sterile PBS, pH 7.0, shaken in it at # 37.degree. C. for 1 h,
rinsed again with it, incubated under 1.5% agar in a bacterial
growth medium overnight, and the colonies were counted. N.d. stands
for "not determined" because adhered bacterial cells grow but do
not form distinguishable colonies on the control slide. # For other
conditions, see the footnotes to Table I.
[0232] It was further demonstrated that hexyl-PVP-derivatized LDPE,
PP, nylon, and PET also can kill waterborne bacteria on contact.
The data in Table II show that upon the derivatization (FIG. 4) the
number of viable adhered S. aureus cells drops by 97 to 99% for all
polymer slides. Likewise, the modified polymer slides were found to
be effective against waterborne E. coli. As seen in FIG. 6b, left,
E. coli cells adhered to an unmodified HDPE slide from an aqueous
suspension grow a number of distinguishable colonies under the
growth agar cover. The colonies on LDPE, nylon, and PET looked
similar, while no colonies were distinguishable on PP. Upon the
derivatization of HDPE with hexyl-PVP, the number of viable adhered
E. coli cells plummeted 97.+-.2% (FIG. 6b, right, and Table II).
The other modified polymers also displayed drastically lower
numbers of adhered viable E. coli cells compared to the
corresponding unmodified slides, with killing efficiencies of
98-99% observed for LDPE, PET, and nylon (Table II, last line).
[0233] Since the experimental conditions for the determination of
bactericidal properties of the hexyl-PVP-modified slides against
waterborne bacteria were different from those for airborne ones, we
again tested, for release of the polycation from a slide into the
medium. To this end, a hexyl-PVP-modified HDPE slide was incubated
for 2 h in sterile PBS with shaking at 37.degree. C., removed, and
S. aureus cells were added to the remaining solution. The resulting
suspension was again shaken at 37.degree. C. for 2 h, and the
number of viable bacterial cells was determined by spreading it on
growth agar, followed by incubation at 37.degree. C. overnight. The
number of bacterial colonies observed, (42.+-.4).multidot.10.sup.4
per mL, coincided with that in the control
[(48.+-.7).multidot.10.sup.4]. Hence there is no appreciable
release of an antibacterial agent from the hexyl-PVP-modified HDPE
slide during the bacterial adhesion experiment, and the waterborne
bacteria must indeed be killed on contact with the derivatized
surface.
[0234] The bactericidal mechanism of the surface-attached
polycationic chains probably involves their penetration into the
bacterial membranes, leading to cell damage and death (Tiller et
al., 2001). To add a quantitative dimension to this model, HDPE
slides were derivatized with hexyl-PVP with varying surface
densities of the polycation, and the antibacterial efficiency
against airborne or waterborne S. aureus cells was measured as
outlined above. As seen in FIG. 7, the bactericidal effect is
expressed only when the surface density of the pyridinium groups
exceeds some 3 nmoles per cm.sup.2 for the airborne bacterium and 6
mmoles per cm.sup.2 for the waterborne one, with no additional
increase thereafter. The difference between the two curves is
presumably due to the fact that the sprayed bacterial cells, in
contrast to those in solution, have no possibility of detaching
from the surface and hence are killed at lower pyridinium group
densities. These observations show the critical dependence of the
material's ability to kill bacteria on the density of coverage of
its surface with the polycation.
[0235] In closing, we have developed a general method of
derivatizing surfaces with the antibacterial polymer hexyl-PVP
applicable to common materials. The hexyl-PVP-modified surfaces of
the synthetic polymers HDPE, LDPE, PP, nylon, and PET kill up to
99% of contact-deposited S. aureus and E. Coli cells. The
methodology should be useful in rendering numerous products
bactericidal both in dry and wet states; the surfaces could be
rejuvenated simply by periodic washings.
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EXAMPLE 13
[0255] Insights into Bactericidal Action of Surface-Attached
poly(vinyl-N-hexylpyridinium) Chains
[0256] The surface of polyethylene slides nanocoated with silica
and derivatized with long-chain poly(vinyl-N-hexylpyridinium)
becomes permanently bactericidal: it kills 90-99% of (both airborne
and waterborne) wild-type and antibiotic-resistant strains of the
human pathogen Staphylococcus aureus. The material created was
similarly lethal to strains expressing multidrug resistance pumps,
the only known mechanism of resistance to cationic antiseptics.
[0257] Creating inherently bactericidal materials that could be
used to make common objects would be a major step toward a
healthier living. Recently, we (Tiller et al. 2001 and 2002) have
discovered that when certain long-chain, hydrophobic polycations
are immobilized onto surfaces of glass or plastics, the latter
acquire the ability to efficiently kill bacteria on contact.
Specifically, various surfaces covalently coated with
poly(vinyl-N-hexylpyridinium) (hexyl-PVP) have been found to kill
the vast majority of bacteria, both Gram-positive and
Gram-negative, either deposited onto them through aerosols
(followed by drying) or allowed to attach from aqueous
solution.
[0258] In the present study, we have further explored this
potentially useful phenomenon. In particular, it has been
ascertained that polyethylene surface-derivatized with hexyl-PVP is
equally lethal against wild-type and mutant, including
antibiotic-resistant, strains of the ubiquitous pathogenic
bacterium Staphylococcus aureus. The dead cells can be readily
removed--and the surface fully rejuvenated--by washing with a
detergent. S. aureus cells collected during the logarithmic growth
phase are as susceptible to the bactericidal action as those in the
stationary phase.
[0259] Materials
[0260] The list of S. aureus strains used in this study is given in
Table 1. A hand-held burner containing 0.6% (v/v) tetramethylsilane
in a propane/butane mixture (7:3, v/v) (Pyrosil.RTM.) was from SurA
Instruments GmbH (Jena, Germany). High-density polyethylene sheets
were purchased from Polymer Plastic Co. Poly(4-vinylpyridine) (PVP)
(Mw 160,000 g/mol), 3-aminopropyltriethoxysilane,
1,4-dibromobutane, 1-bromohexane, and all other chemicals and
solvents were obtained from Aldrich Chemical Co. and used without
further purification.
7TABLE 1 Strains of Staphylococcus aureus used in this study.
Strain Description Reference 8325-4 Wild-type parent of 1758 Kaatz
et al. 1999 1758 norA Kaatz et al. 1999 982 Wild-type parent of
2355 Rouch et al. 1990 2355 QacA.sup.+, Kan.sup.r Rouch et al. 1990
ATCC 700698 Methicillin-resistant strain Hiramatsu 1997 ATCC BAA-38
Methicillin-resistant strain De Lencastre 2000 ATCC BAA-39
Methicillin-resistant strain De Lencastre 1997 ATCC 33807 No
pyrogenic exotoxin C a a Obtained from ATCC.
[0261] Surface Modification
[0262] A polyethylene slide (7.5.times.2.5 cm) was ultrasonicated
in isopropyl alcohol for 5 min and dried at 80.degree. C. The front
(oxidizing) part of the flame of a hand-held burner was fanned over
the slide surface for 15 s, and the slide was stored under air
overnight. The SiO.sub.2-coated slide was aminated with a 20%
solution of 3-aminopropyltriethoxysilane in dry toluene at room
temperature for 3 h. The aminated slide was immersed in a solution
containing 9 mL of 1,4-dibromobutane, 90 mL of dry nitromethane,
and 0.1 mL of triethylamine with stirring at 60.degree. C. for 5 h,
followed by placing in a freshly prepared solution of 9 g of PVP,
10 mL of 1-bromohexane, and 81 mL of nitromethane. After stirring
at 75.degree. C. for 9 h, the slide was thoroughly rinsed with
methanol and distilled water, and dried under air.
[0263] Antibacterial Efficiency Determination
[0264] Bacteria were grown in yeast-dextrose broth (Cunliffe et al.
1999) at 37.degree. C. with aeration at 200 rpm for 6-8 hours. The
inoculum from an overnight culture was transferred into 0.1 M PBS
(approximately 10.sup.11 cells/mL) and then introduced into the
growth medium at a 1:500 dilution.
[0265] The airborne bacterial suspension was prepared as described
earlier (Tiller et al. 2001). The bacterial cells were centrifuged
at 5,160.times.g for 10 min and washed with distilled water twice.
A bacterial suspension at a concentration of 10.sup.6 cells/mL in
distilled water was sprayed at a rate of approximate 10 mL/min onto
the surface of a slide in a fume hood. After drying for 2 min under
air, the slide was placed in a Petri dish, and growth agar (0.7%
agar in the yeast-dextrose broth, autoclaved, and cooled to
37.degree. C.) was added. The Petri dish was sealed and incubated
at 37.degree. C. overnight. The grown bacterial colonies were
counted on a light box.
[0266] The waterborne bacterial suspension was prepared as follows:
bacterial cells were centrifuged at 5,160.times.g for 10 min,
washed twice with PBS at pH 7.0, re-suspended in the same buffer,
and diluted to 2.times.10.sup.6 cells/mL. A slide was immersed in
45 mL of the suspension and incubated with shaking at 200 rpm at
37.degree. C. for 2 h, then rinsed three times with sterile PBS,
and incubated in it for 1 h. The slide was immediately covered with
a layer of solid growth agar (1.5% agar in the yeast-dextrose
broth, autoclaved, poured into a Petri dish, and dried under
reduced pressure at room temperature overnight). The bacterial
colonies were then counted.
[0267] Synthesis of Pyridinium-Containing Monomer and Polymer
[0268] Pyridine (10 mL), hexyl bromide (18 mL), and triethylamine
(0.1 mL) were dissolved in 122 mL of toluene, and the solution was
stirred at 75.degree. C. for 9 h. The solvent was then removed
under reduced pressure. N-Hexylpyridinium bromide thus obtained was
washed with hexane four times and dried overnight under vacuum.
[0269] Poly(4-vinylpyridine) (5.25 g), methyl iodide (13 mL), and
triethylamine (0.1 mL) were dissolved in 87 mL of nitromethane, and
the solution was stirred at 75.degree. C. for 9 h. After cooling to
room temperature, the solvent was removed and 100 mL of toluene was
added to the residue. Poly(4-vinyl-N-methylpyridinium iodide),
insoluble in toluene, was recovered by filtration, washed with
toluene and acetone, and dried.
[0270] Results & Discussion
[0271] We have answered certain questions concerning the mechanism
and practicalities of the bactericidal effect of surfaces
derivatized with poly(vinyl-N-alkylpyridinium) chains (Tiller et
al. 2001 and 2002). For example, 7.5.times.2.5 cm slides cut out of
a large sheet of commercial high-density polyethylene were coated
with a nanolayer of silica, followed by the covalent attachment of
160,000-g/mol hexyl-PVP, as previously described (Tiller et al.
2002). The antibacterial activity of the resultant slides was
tested against the common pathogen S. aureus in two distinct
modalities (referred to as "airborne" and "waterborne"). In the
airborne case, an aqueous suspension of the bacterial cells was
sprayed onto a slide, followed by drying, overlaying with growth
agar, incubation at 37.degree. C., and counting the number of
bacterial colonies. In the waterborne case, a slide was immersed in
an aqueous suspension of the bacterial cells, incubated there at
37.degree. C., washed, covered with solid growth agar, and
incubated at 37.degree. C. again, followed by counting the number
of bacterial colonies.
[0272] When wild-type S. aureus cells were deposited onto the
surface of an unmodified high-density polyethylene slide via the
airborne method, followed by cultivation as described above,
308.+-.16 colonies were subsequently detected in a 3.75 cm.sup.2
frontal area. The same experiment was performed with the slide
coated with silica and with that also aminated; the corresponding
numbers of bacterial colonies were 339.+-.2 and 228.+-.2,
respectively. Thus the bacterial cells deposited onto all three
different surfaces remain highly viable. In contrast, when the
identical procedure was applied to the hexyl-PVP-derivatized slide,
merely 14.+-.1 bacterial colonies were observed in a 3.75-cm.sup.2
frontal area, i.e., 5.+-.1% compared to the unmodified slide.
Likewise, with waterborne S. aureus cells, only 4.+-.2% of colonies
were counted on the immobilized hexyl-PVP surface compared to the
original polyethylene slide. These 95%+killing efficiencies are
analogous to those discovered previously (Tiller et al. 2001 and
2002) for surfaces modified with hexyl-PVP.
[0273] Next, we addressed the question of the durability of the
aforementioned hexyl-PVP surface protection. To this end, after the
colonies grown from the surviving S. aureus cells were counted,
they were removed by thoroughly washing the hexyl-PVP-derivatized
slides with 0.1 M cetyltrimethylammonium chloride in water,
followed by rinsing with distilled water. The resultant washed
slides were re-used for the deposition of either airborne or
waterborne S. aureus. The percentages of the colonies formed, as
compared to those on the identically washed unmodified polyethylene
slides, were 4.+-.1% and 2.+-.1%, respectively. Thus washing with
the detergent has no effect on the bactericidal potency of
immobilized hexyl-PVP.
[0274] In our studies thus far, both herein and elsewhere (Tiller
et al. 2001 and 2002), bacterial cells used in all experiments were
in the stationary phase of their growth curve. It was of interest
to establish whether the cells in the logarithmic phase of growth
would be equally susceptible to the antibacterial action of the
surface-attached hexyl-PVP chains. Consequently, we carried out a
fermentation of S. aureus whereby the cell concentration was
monitored as a function of time. The resultant data yielded a
classical sigmoidal curve (Ingraham et al. 1983), with the
stationary phase fully reached after 6 h under our conditions (see
Methods). Instead of collecting the bacterial cells after 6-8 h as
before, we did so after just 4 h, i.e., during their logarithmic
growth phase. When these cells, airborne or waterborne, were
deposited onto hexyl-PVP-derivatized slides, the killing
efficiencies obtained (compared to the same cells on the unmodified
polyethylene slides) were 5.+-.2% and 2.+-.1%, respectively, i.e.,
identical to the values observed for the cells in their stationary
phase (see above).
[0275] Multidrug resistant (MDR) bacterial strains pose a major
threat to human health (Levy 1998, Lewis et al. 2001). With this in
mind, we tested whether immobilized hexyl-PVP would be effective
against such strains. Toward this end, in addition to the
heretofore used wild strain of S. aureus (ATCC 33807), we explored
three different antibiotic-resistant strains (Kluytmans et al.
1997)--ATCC 700698 (resistant to methicillin), ATCC BAA-38
(resistant to methicillin, penicillin, streptomycin, and
tetracycline), and ATCC BAA-39 (resistant to penicillin,
tetracycline, imipenem, cefaclor, oxacillin, tobramycin,
cephalexin, cefuroxime, gentamicins, amoxicillin, clindamycin,
erythromycin, and cephamandole). As seen in FIG. 8, polyethylene
slides coated with hexyl-PVP were similarly lethal to these
bacterial strains, whether airborne or waterborne--the killing
efficiencies in all instances well exceeded 90%. Immobilized
hexyl-PVP chains likely exert their bactericidal effect by
penetrating the bacterial cell wall/membrane and perhaps causing
autolysis (Tiller et al. 2001).
[0276] Like all other bacteria studied (Lewis 1994, Lewis et al.
2001), S. aureus possesses several MDR pumps that expel various
toxic compounds from the cell. For example, the NorA MDR pump
protects the cells from numerous amphipathic cations including the
common disinfectant benzalkonium chloride (Ng et al. 1994).
Consequently, the mutant of S. aureus with a knockout in the norA
gene coding for the MDR pump has a substantially greater
sensitivity to such compounds (Hsieh et al. 1998). Since hexyl-PVP,
like benzalkonium, is a hydrophobic quaternary ammonium cation, we
decided to test the sensitivity of the norA mutant strain to
immobilized hexyl-PVP. The results of this experiment are depicted
in FIG. 9A. One can see that the killing efficiencies against the
pump-lacking mutant were somewhat higher than of its pump-competent
parent--99.+-.1% for both airborne and waterborne cells vs.
95.+-.2% and 97.+-.1%, respectively.
[0277] We carried out similar experiments with a S. aureus strain
with an additional MDR pump (denoted QacA.sup.+) expressed from a
natural transmissible plasmid compared to its parent. This strain
had a slightly higher resistance to immobilized hexyl-PVP than its
pump-deficient parent (FIG. 2B)--92.+-.3% and 94.+-.3% for the
airborne and waterborne mutant, respectively, vs. 96.+-.2% and
98.+-.1% for its parent.
[0278] The very small difference in observed susceptibilities to
hexyl-PVP between strains lacking/overexpressing MDRs suggest that
the polymeric form of the antiseptic is not effectively extruded by
the pump. Alternatively, N-hexylpyridinium may not be a substrate
for MDRs. In order to distinguish between these possibilities,
cells were treated with an aqueous solution of N-hexylpyridinium
bromide, and the minimal inhibitory concentration (MIC) was
determined. That compound turned out to be a very weak
antimicrobial, with an MIC of 4 mg/mL against wild-type S. aureus
(Table 2). This value is more than 1,000 times above that of the
conventional antiseptic benzalkonium chloride (Table 2). There was
a difference in strain susceptibilities to N-hexylpyridinium
depending on their MDR status. Thus the MIC of the norA strain was
0.5 mg/mL, and that of the QacA.sup.+ strain was above 4 mg/mL.
This qualitatively resembles the difference in susceptibilities of
these strains to benzalkonium chloride and shows that while
N-hexyl- pyridinium is a weak antimicrobial, it is a reasonable
substrate for MDRs. When water-soluble
poly(vinyl-N-methylpyridinium iodide) was tested instead (note that
hexyl-PVP itself, used in its immobilized form in our studies thus
far, is not soluble in water and therefore could not be used in MIC
studies), the MIC value was found to be 75 .mu.g/mL, i.e.,
considerably lower than that of the monomeric precursor
N-hexylpyridinium bromide. There was no difference in the MIC
values of this compound among the strains tested. It thus appears
that polymerization of an antiseptic has two consequences for its
properties--increases its potency and makes it insensitive to the
action of MDR pumps.
8TABLE 2 The minimal inhibitory concentration (MIC) for quaternary
ammonium containing monomers and polymer. N- Poly(vinyl-
Benzalkonium Hexylpyridinium N-methyl- chloride Bromide pyridinium
iodide) MIC (.mu.g/mL) MIC (.mu.g/mL) MIC (.mu.g/mL) ATCC 33807 2.5
4,000 75 ATCC 700698 2.5 >4,000 75 ATCC BAA-38 2.5 >4,000 75
ATCC BAA-39 2.5 4,000 75 8325-4 2.5 4,000 75 norA 0.6 500 75 982
2.5 4,000 75 QacA.sup.+ 5.0 >4,000 75
[0279] Development of a resistance is a major concern in
introducing any new antimicrobial. Extrusion by MDRs is the only
known mechanism of resistance to hydrophobic cationic antiseptics
(Lewis 2001, Severina et al. 2002). Our findings suggest that
resistance to surface-attached hexyl-PVP is unlikely to develop
through this mechanism.
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[0297] Incorporation by Reference
[0298] All publications and patents mentioned herein, are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
[0299] Equivalents
[0300] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *