JP2004532108A - Antibacterial polymer surface - Google Patents

Abstract

Disclosed is a germicidal composition comprising a polymeric compound immobilized on a substance. Medical devices comprising such a bactericidal composition are also disclosed. A method of covalently derivatizing a surface of a common substance with an antimicrobial polycation such as, for example, poly (vinyl-N-pyridinium bromide), wherein the first step of the method comprises the steps of: A method is disclosed that involves coating with a. Various commercial synthetic polymers derivatized in this way are bactericidal, that is, they deposit from either air or water and kill up to 99% of gram-positive and gram-negative bacteria that come into contact.

Description

BACKGROUND OF THE INVENTION
[0001]
The demand for healthy living is greater than ever, and there is a strong interest in substances that can kill harmful microorganisms. Such materials can also be used to coat and disinfect everyday items that are commonly touched by people, such as doorknobs, children's toys, computer keyboards, and telephones, so that bacterial infections are not transmitted. . Normal substances are not antimicrobial and need to be improved. For example, surfaces chemically modified with poly (ethylene glycol) and some other synthetic polymers can repel (but not kill) microorganisms (Bridgett, MJ, et ak, SP (1992) Biomaterials 13, 411-416. Arciola, CR, et al Alvergna, P., Cenni, E. & Pizzoferrato, A. (1993) Biomaterials 14, 1161-1164. Park, KD, Kim, YS, Han, DK, Kim. , YH, Lee, EHB, Suh, H. & Choi, KS (1998) Biomaterials 19, 851-859.).
[0002]
Alternatively, antimicrobials, such as antibiotics, quaternary ammonium compounds, silver ions or iodine, that can be gradually released into the surrounding solution over time and kill microorganisms, can soak into the material (Medlin , J. (1997) Environ. Health Persp. 105, 290-292; Nohr, RS & Macdonald, GJ (1994) J. Biomater. Sci., Polymer Edn. 5, 607-619 Shearer, AEH, et al (2000). ) Biotechnol. Bioeng. 67, 141-146.). Although these strategies have been demonstrated in aqueous solutions containing bacteria, they may not be effective against airborne bacteria without a fluid medium. This is especially true for releasable materials, which become ineffective when the leaching antimicrobial material is exhausted.
[0003]
Infections are a common complication of many invasive surgical, therapeutic and diagnostic procedures. Preventing infections is particularly difficult in procedures involving implantable medical devices. This is because the bacteria form a biofilm and the subject's immune system cannot remove the bacteria. Since such infections are difficult to treat with antimicrobial agents, the device often must be removed, causing pain to the patient and increasing medical costs.
[0004]
Any substance that is left implanted in the body has a surface that accumulates microorganisms that cause infectious diseases, particularly bacteria and sometimes fungi. This is understood as being caused by the formation of a biofilm. A biofilm is a type of deposit that occurs when microorganisms attach to a surface and secrete hydrated polymeric material around them. Microorganisms called cecils (adhered state) present in biofilms grow in a protected environment by blocking the attack of antibacterial substances. This cecil community produces non-cecil individuals, called planktonic (suspended), which multiply and spread rapidly. This planktonic organism causes invasive and contagious infections. It is this organism that is the target of antimicrobial therapy. With conventional processing methods, it is not possible to eliminate the cecil group rooted in the biofilm. It is understood that biofilms are often storage locations for infectious agents. Biofilm biology is described 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).
[0005]
Biofilms prefer to develop on internal surfaces or non-living tissue, and often occur on tissue that is not permeable or dying of medical devices and blood vessels. Biofilms have been identified in necrotic debris and bone grafts of dead bone, triggering an invasive infection that kills additional bone called osteomyelitis. Biofilms have also been identified in living poorly vascular tissues, such as native heart valves, causing a devastating infection called endocarditis, where the causative organism is located distally in the bloodstream. Not only can colonies form, but the heart valve itself can be destroyed. Infections involving implantable medical devices generally involve biofilms, and the cecils are the repository for invasive infections. The presence of microorganisms in the biofilm on the medical device indicates contamination of the foreign material. Induction of a clinically recognizable host response by a biofilm constitutes an infection.
[0006]
The onset of infection from contaminated sites is consistent with the natural history of biofilm growth and development. Biofilms grow slowly at one or more sites, with one or more microorganisms forming colonies. The biofilm development pattern consists of the first step in which the microorganisms attach to the solid surface, the step of forming microcolonies attached to the surface, and the last step in which the microcolonies differentiate into a mature biofilm encapsulated in exopolysaccharide. included. Since the planktonic cells are released from the biofilm in a programmed natural pattern of detachment, the biofilm is a site of multiple recurrent acute invasive infections. Antimicrobial agents typically treat infections caused by planktonic cells, but cannot kill biofilm-protected cecil organisms.
[0007]
Cecil microorganisms also cause local symptoms, release antigens, and stimulate the production of antibodies that activate the immune system that attacks the biofilm and surrounding areas. The protection of antibodies and host immunity causes the organisms that enter the biofilm to elicit antibodies and associated immune responses, but has no effect on killing these organisms. The cytotoxic products of the cells of the host whose immunity has been activated may be directed to the host's own tissues. This phenomenon is observed in the oral cavity, and the oral biofilm may cause inflammation of the tissues around the teeth, resulting in periodontitis. This phenomenon causes local inflammation around the implantable medical device and can also cause bone resorption due to loose orthopedic and dental implants.
[0008]
Although host defenses may protect invasive infections at the border by controlling the spread of planktonic organisms, this favorable equilibrium assumes that the immune system is intact. Many patients in hospitals are immunocompromised and once biofilms are established, they are more susceptible to invasive infections. Patients in need of implantable medical devices, regardless of duration, can be immunocompromised as well. If the immune system is not functioning properly, the host is at greater risk of forming contaminated biofilms around medical devices and the risk of planktonic organisms invading surrounding tissues and systems . When planktonic organisms cause a complete infection, immunocompromised hosts are more likely to lose control and control, and can even die.
[0009]
Despite protection by antimicrobial treatment and host defenses, biofilms typically repeat the infection, producing small local symptoms. Once established, biofilms can only be removed surgically. If the foreign body becomes contaminated with microorganisms, the only way to eliminate local and systemic infections would be to remove the contaminated foreign body. If the substance to be removed is essential for health, a similar substance will need to be replaced in the same location. Due to the residual microorganisms in the area, the replacement material will be particularly susceptible to infection.
[0010]
The difficulties associated with eliminating infections with biofilms are well recognized, and a number of techniques have been developed to treat surfaces or fluids that bathe the surfaces to prevent or reduce biofilm formation. Biofilms adversely affect medical systems and other systems essential to public health, such as water supply and food processing facilities. Numerous techniques have been proposed for treating the surface of organic or inorganic substances that inhibit biofilm formation. For example, antimicrobial agents (see, e.g., U.S. Patent Nos. For example, various methods have been utilized for coating with U.S. Patent 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 such techniques, contamination of medical devices and the resulting invasive infections continue to be a problem.
[0011]
Infectious organisms are ubiquitous in the healthcare environment, despite active efforts to maintain sterility. The presence of such organisms can infect inpatients and healthcare professionals. Such infections are called nosocomial infections and often involve abnormal organisms that are more infectious than those encountered outside the hospital. In addition, hospital-acquired infections are more likely to involve organisms that have developed resistance to many antimicrobials. Although cleaning and antimicrobial control are routine, infectious organisms can easily colonize various surfaces in the medical environment, especially those exposed to humid environments or impregnated in fluids. Form. Even barriers such as gloves, aprons, and shields can spread the infection to the wearer or others in the medical setting. Despite sterilization and cleaning, various metallic or non-metallic materials in the medical environment can retain dangerous organisms wrapped in biofilms and transmit them to other hosts.
[0012]
Any substance used to reduce the formation of biofilms in a medical setting must be safe for the user. Some biocidal substances can also damage host tissues when used in amounts sufficient to inhibit biofilms. Introduction of an antimicrobial substance into a localized area of tissue may induce the formation of resistant organisms, which may form biofilms, whose platonic microbes may be similar to the aforementioned antimicrobial substances. It becomes resistant. In addition, no anti-biofilm or anti-fouling material must interfere with the healthful features of the medical device. Some, such as operability, flexibility, waterproofness, tensile strength, or compression durability of a particular operator, and have properties that do not change with substances added for antimicrobial effects Select a substance.
[0013]
As a further problem, substances added to the surface of implantable devices to inhibit contamination and biofilm formation may have thrombogenic properties. Some of the implants are themselves thrombogenic. For example, it has been shown that contact with metal, glass, plastic or other similar surfaces can induce clotting. Therefore, heparin compounds known to have anticoagulant effects have been applied to some medical devices prior to implantation. However, only a small number of medical products that combine antibacterial and antithrombotic effects are known. This combination would be particularly useful for treating medical devices placed in the bloodstream, such as heart valves, artificial pump devices ("artificial hearts" or left ventricular assist devices), artificial blood vessels, and vascular stents. . In such a background, the formation of a thrombus can block the blood flow in the conduit, and can break down into fragments called emboli, which are transported downstream, blocking the circulation of distal tissues or organs. is there.
[0014]
Biofilm formation has important implications for public health. Drinking water supply systems are known to have hidden biofilms, despite the fact that their environment often contains fungicides. Any system that has an interface where the surface meets the fluid has the potential for biofilm formation. Water cooling towers for air conditioners are well known for having public health risks due to the formation of biofilms, and have been demonstrated with a sudden outbreak of infectious diseases such as legionellosis. It cannot be protected by turbulence of the fluid generated on the surface. Biofilms form in the channels through which running water or other fluids pass, alter flow characteristics, and may act to transport planktonic organisms downstream. Industrial fluid processing operations have experienced mechanical barriers, impedance of heat transfer processes, and biological degradation of industrial products using fluids, all due to biofilms. Biofilms have also been identified in flow channels, such as hemodialysis tubes, and in distribution pipes. Biofilms have also been found to biofoul certain municipal water tanks, privately owned wells, and drip irrigation systems, and have not been affected by chlorination up to 200 ppm.
[0015]
Biofilms are also a problem in food processing environments. Food processing involves fluids, solid materials, and combinations thereof. For example, milk processing facilities have fluid conduits and areas of fluid retention on surfaces. Currently, cleaning of milking and milk processing equipment uses the interaction of mechanical, thermal, and chemical processes with in-flush, in-place cleaning methods. Furthermore, the dairy products themselves are also sterilized. When making cheese, crystals of calcium lactate may form on cheddar cheese due to biofilms. Meat processing and packaging facilities are also prone to biofilm formation. May act on non-metallic and metallic surfaces. In meat processing facilities, biofilms have been detected on rubber "fingers", plastic curtains, belt conveyors, eviscerators, and stainless steel surfaces. Managing biofilm and microbial contamination in food processing also requires the use of substances that do not affect the taste, texture, or beauty of the product, which hinders it.
[0016]
Therefore, there is a need to provide a general surface with bactericidal properties. A general surface coating / derivatization procedure has been developed that can be applied to most materials, regardless of their nature.
Summary of the Invention
[0017]
One aspect of the present invention relates to an antimicrobial surface comprising a covalently attached amphiphilic compound. In some embodiments, the surfaces of the present invention act to remove contacting airborne organisms. In some embodiments, the surface of the present invention acts to remove contacting organisms in water. In some embodiments, the surface of the present invention acts to inhibit biofilm formation.
[0018]
In some embodiments, the covalently attached amphiphilic compound is a polymer comprising ammonium ions. In a preferred embodiment, the molecular weight of the polymer is at least 10,000 g / mol, more preferably 120,000 g / mol, and most preferably 150,000 g / mol.
[0019]
In some embodiments, the surface of the present invention is glass. In some other embodiments, the surface of the present invention is plastic. In one embodiment, the surface of the present invention is an amino-bearing glass.
[0020]
In some embodiments, the present invention is a composition comprising a substance and a compound immobilized on the substance, wherein the immobilized compound is a polymer, wherein the polymer is water-insoluble. Not relevant to the composition. In some embodiments, the immobilized compound is a polycation. In some embodiments, the immobilized compound is a water-soluble lipophilic polycation. In some embodiments, the immobilized compound is covalently linked to the solid substance. In some embodiments, the immobilized compound comprises poly (N-alkylvinylpyridine) or poly (N-alkylethyleneimine).
[0021]
In some embodiments, the compound covalently attached to the surface is of Formula I
Embedded image
(Formula I)
In the formula
R is each independently hydrogen, alkyl, alkenyl, alkynyl, acyl, aryl, carboxylate, alkoxycarbonyl, aryloxycarbonyl, carboxamide, alkylamino, acylamino, alkoxyl, acyloxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino ) Represents alkyl, thio, alkylthio, thioalkyl, (alkylthio) alkyl, carbamoyl, urea, thiourea, sulfonyl, sulfonate, sulfonamide, sulfonylamino, or sulfonyloxy;
R ′ each independently represents alkyl, alkylidentether to the surface, or acyltether to the surface,
Z independently represents Cl, Br, or I,
n is an integer of about 1500 or less,
It is represented by an equation.
[0022]
In certain embodiments, the present invention comprises poly (4-vinyl-N-alkylpyridinium bromide) or poly (methacryleuoxidodecylpyridinium bromide) (MDPB) covalently attached to a glass surface. In another preferred embodiment, the present invention comprises an N-alkylated poly (4-vinylpyridine) covalently attached to a glass surface.
[0023]
In some embodiments, the organism that is killed on the surface of the present invention is a bacterium. In some embodiments, the bacterium that is killed is a Gram-positive bacterium. In another embodiment, the killing bacterium is a gram negative bacterium.
[0024]
In some embodiments, the present invention relates to a method of sterilizing a surface. In some embodiments, the surface to be sterilized includes amino functionality. In some embodiments, a method of disinfecting a surface includes derivatizing a surface having amino functionality to form a covalent bond with a polymer containing quaternary amine groups on the surface.
[0025]
In some embodiments, the surface to be sterilized does not have amino functionality and a SiO 2_TwoAmino functionality is added to the surface by applying a nanoparticle layer and forming a Si-OH group by a hydroxylation reaction, followed by treatment with an aminated material to form a surface containing amino functionality. In some embodiments, the aminated material is 3-aminopropyltriethoxysilane.
[0026]
In some embodiments, the present invention relates to an antimicrobial article comprising a polymer covalently bonded on its surface, wherein the covalently bonded polymer comprises a quaternary ammonium group. In some embodiments, the antimicrobial article is a household item. Some implementations
In some embodiments, the antimicrobial article is a medical device.
[0027]
A. Overview
In general, the present invention relates to preventing the 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 situations affecting human or animal health that are involved in contamination. Pollution events involve recognition between organisms and surfaces, attachment of organisms to surfaces, and subsequent activity of organisms. As understood herein, biofilm formation is a type of contamination. Biofilms that affect health generally include infectious microorganisms.
[0028]
In health-related environments, contamination can also cause biofilm formation. It is understood that biofilm formation results in local contamination of the affected area, which can lead to invasive local and systemic infections. Microorganisms can either 1) invade or contact host cells and cause direct cell death, 2) release endotoxins or exotoxins that kill cells at a distance, release enzymes that degrade tissue components, Or can damage blood vessels and cause ischemic necrosis 3) Host cells that, despite being directed at invaders, can cause additional tissue damage including suppuration, scarring, and hypersensitivity reactions Can cause tissue damage in at least three ways: Infections, whether local or systemic, are clinically identifiable when microorganisms invade host cells and damage tissues or trigger host defense mechanisms, or both To cause various symptoms. Common local symptoms can include pain, tenderness, swelling, and impaired function. Common systemic symptoms can include fever, malaise, and hyperactive neovascular effects. Large amounts of infectious agents entering the bloodstream can be rapidly fatal.
[0029]
If the infection is caused by a biofilm in the body that surrounds the surrounding material, whether it is a naturally-occurring substance or a foreign substance, the infection cannot be controlled unless the substance is removed. Often cannot. If the substance is naturally occurring, such as poorly vascular or ischemic tissue, it is surgically removed by a process called debridement. If the substance is a foreign substance such as a medical device, it is completely removed. In some cases, the margins of the tissue must be removed with the contaminants to ensure maximum contaminant removal. If the material to be removed is essential for health, it may be necessary to substitute a similar item in the same location. The substitute will be particularly susceptible to infection due to the microorganisms remaining at the site.
[0030]
The present invention relates to an antimicrobial surface comprising an amphiphilic compound that can be covalently bonded to a surface. In some embodiments, the surface acts to remove contacting airborne organisms, such as bacteria. In a preferred embodiment, the compound is an amphiphilic polycation.
[0031]
B. Definition
For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
[0032]
As used herein, the term "biological" refers to any bacteria, fungi, viruses, protozoa, parasites, or other infectious agents capable of causing disease in human or non-human animals.
[0033]
As used herein, "contacting" means any method of providing a compound of the present invention to a surface and protecting against biological contaminants. "Contacting" may include spraying, wetting, impregnating, dipping, coating, bonding or adhering, or other methods of providing a compound of the invention to a surface.
[0034]
"Coating" means any temporary, semi-permeable or permeable layer, coating, or surface. The coating may be a gas, vapor, liquid, paste, semi-liquid, or solid. Furthermore, the coating can be applied in liquid form and solidifies into a hard coating. Examples of coatings include brighteners, surface cleaners, caulks, adhesives, finishes, paints, waxes, polymerizable compositions (phenolic resins, silicone polymers, chlorinated rubbers, mixtures of coal tar and epoxy, epoxy Resins, polyamide resins, vinyl resins, elastomers, acrylic acid polymers, fluoropolymers, polyesters and polyurethanes, including latex). Silicone resins, silicone polymers (eg, RTV polymers) and silicone thermoset rubbers are suitable coatings for use in the present invention, for example, Encyclopedia of Polymer Science and Engineering (1989).15: 204 et seq. The coating may be abradable or soluble, so that the rate of dissolution of the substrate can control the rate at which the AF agent is delivered to the surface. The coating may also be a non-ablative coating, which depends on the principle of diffusion to deliver the AF agent to the surface. The non-ablative coating may be porous or non-porous. Coatings containing an AF agent that freely diffuses into the polymer binder are called "monolithic" coatings. The coating may be resilient to provide flexibility to the surface to be coated, such as swelling or shrinking.
[0035]
A "component" is a portion of a device that is structurally integrated with the device. The component is applied to the surface of the device, is contained within the entity of the device, is retained inside the device, or is otherwise such that the portion becomes an integral part of the structure of the device. May be any arrangement. For example, a silicone coating surrounding the mechanical part of the pacemaker is a component of the pacemaker. The component may be a lumen of the device, the lumen performing a function essential to the overall function of the device. The lumen of the tissue dilator inlet is a component of the tissue dilator. A component may also mean a reservoir or a discrete area within a device that is specifically adapted to deliver fluid to the surface of the device. The storage portion within the implantable drug delivery device is a component of the device.
[0036]
By "delivery system" is meant any system or device or component capable of delivering an antifouling compound disclosed herein to a surface that inhibits biofilm formation. Typical delivery systems include encapsulation of the substance, incorporation of the substance into the body of the product, and suitable substances so that a sufficient amount of the substance can reach the target surface and inhibit the biofilm. The insertion of the substance into a substrate or pore of the substrate. The delivery system may include a coating. The delivery system may include a mechanical substance adapted for delivery of the antifouling compound to the surface. Other mechanisms, including delivery systems, will be apparent to those skilled in the relevant art.
[0037]
By "bandage" is meant any bandage or coating applied to a lesion or used to prevent or treat an infection. Examples include chronic wounds (such as pressure ulcers, venous stasis ulcers and burns) or wound dressings for acute wounds, and percutaneous devices such as intravenous or subclavian catheters to reduce catheter sepsis due to bacterial invasion. Includes bandages. For example, the compositions of the present invention can be applied to a percutaneous puncture site, or can be incorporated into an adhesive dressing material that is applied directly to the insertion site.
[0038]
The phrase "effective amount" refers to the number of organisms attached to a defined area (cells / mm^Two) Means that the amount of antifouling compound disclosed herein can be significantly reduced from the number attached to the untreated surface. Particularly preferred is an amount that reduces the number of organisms attached to the surface by at least half. More preferably, it is an amount that reduces the number of organisms attached to the surface by at least 1/4, and even more preferably by 1/6. It is believed that an effective amount of the antifouling compound disclosed herein inhibits biofilm formation and inhibits the growth of organisms on defined surfaces. The term "inhibit" as used for the effect of an antifouling compound on a surface includes any effect that significantly reduces the number of organisms that bind to it.
[0039]
The term "health-related environment" is understood to include all environments in which activities involving the restoration or maintenance of human health are performed. The health-related environment may be a medical environment in which activities intended to restore human health directly or indirectly are performed. Operating rooms, doctor's offices, hospital rooms, and medical equipment factories are all examples of a medical environment. Other health related environments may also include places or dwellings where industrial activities take place where activities to maintain human health take place. Such activities include food processing, water purification, and public health.
[0040]
An “implant” is any object that is intended to be placed in a human body and is not living tissue. Implants include objects of natural origin that have been processed to kill living tissue. In one example, a bone graft can be processed to remove living cells and its shape can be maintained for use as a template for bone ingrowth from a host. In another example, naturally occurring corals can be processed into hydroxyapatite preparations and applied to the body for some orthopedic and dental treatments. An implant can also be an article that includes an artificial component. The term "implant" can apply to the entire range of medical devices intended for placement in the human body.
[0041]
As used herein, the term "infectious microorganism" or "infectious agent" refers to bacteria (such as Staphylococcus sp. (Eg, Staphylococcus aureus, Staphylococcus epidermidis) (E. faecalis), Pseudomonas spp. Fungi), gram-negative and gram-positive organisms such as Escherichia (E. coli), Proteus (P. mirabilis), fungi (including Candida albicans), viruses, and protozoa Mean disease.
[0042]
"Medical device" means a non-naturally occurring object that is inserted or implanted into a subject or applied to a surface of a subject. Medical devices may be made from a variety of biocompatible materials that are not normally found in the human body, including metals, ceramics, polymers, gels, and fluids. Examples of polymers useful for making medical devices include silicone, rubber, latex, plastic, polyanhydride, polyester, polyorthoester, polyamide, polyacrylonitrile, polyurethane, polyethylene, polytetrafluoroethylene, polyethylenetetraphthalate. And polymers such as polyphazene. Medical devices may be made using some type of naturally occurring material or may be treated with a naturally occurring material. In one example, a heart valve can be made by combining a treated pig heart valve with an attached device using artificial materials. Medical devices include any combination of man-made materials selected for a particular feature of a component. For example, a hip implant may include a weight bearing metal shaft, a ceramic prosthesis, and a polymeric adhesive to secure the structure to surrounding bone. The implantable device is intended to be completely implanted in the body without protruding a part of the structure outside the body (for example, a heart valve). An insertion device is a device that is partially embedded in the body but has a portion intended to be taken out of the body. The medical device may be intended for short-term or long-term retention at the location where it is placed. For example, hip implants are intended for decades of use. Conversely, the tissue dilator may only be required for a few months, after which it is removed. Insert devices tend to have a shorter dwell time than implantable devices, in part because of the greater opportunity for contact with potentially colonizing microorganisms.
[0043]
The term "soluble" means the ability to be unraveled or dissolved.
[0044]
The term “surface” or “surface (s)” can mean any surface of any material, including glass, plastic, metal, polymers, and the like. Surfaces may be made from one or more materials, including coated surfaces.
[0045]
Biofilm formation with health implications includes all aspects related to health, including those found in the medical environment and those in industrial or residential environments that are involved in health-critical functions such as nutrition, hygiene, and prevention of disease. It may include surfaces in the environment.
[0046]
Surfaces of articles adapted for use in a medical environment can be sterilized using gas poisoning techniques such as autoclaving, exposure to biocides, irradiation, or ethylene oxide exposure. Surfaces found in the medical environment include the inner and outer surfaces of various devices or instruments intended for disposable or repeated use. Examples include scalpels, needles, scissors, other instruments used in invasive surgery or therapeutic or diagnostic methods, artificial blood vessels, catheters and other instruments for transporting or removing fluids to patients, artificial hearts Implantable medical devices, including catheters, artificial kidneys, orthopedic pins, plates, and implants, urinary and bile tubes, endotracheal tubes, peripherally inserted central venous catheters, dialysis catheters, long-term tunnels Central venous catheters, peripheral venous catheters, short-term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urethral catheters, peritoneal catheters), urinary devices (long-term urine devices, tissue-coupled urine devices, artificial urethral sphincter, urethra) Dilator, shunt (ventricular shunt, arteriovenous shunt) Prostheses (including breast implants, artificial penis, artificial blood vessels, heart valves, artificial joints, artificial larynx, otolaryngological implants), vascular catheter ports, wound drains, hydrohead shunts, pacemakers, and implants. Includes the entire spectrum of medically adapted articles, such as devices including self-contained defibrillators. Other examples will be readily apparent to one skilled in the art.
[0047]
Surfaces found in the medical environment also include the inner and outer surfaces of components of medical devices, components of equipment worn or carried by healthcare personnel. Such surfaces include medical devices, tubes, and cans used for respiratory treatment, including for medical procedures or for administering oxygen, administering nebulized soluble drugs, and administering anesthetics. Also included are countertops and fixtures used for preparation. Also included are surfaces that have a purpose as biological shields against infectious organisms in medical settings, such as gloves, aprons, and face shields. Materials commonly used for biological shields may be made of latex or non-latex. Vinyl is commonly used as a material for non-latex surgical gloves. Other such surfaces may include the handles and cables of medical or dental instruments that are not to be sterilized. In addition, such surfaces may include the non-sterile exterior surfaces of tubes and other instruments found where blood or bodily fluids or other potentially dangerous biological materials are commonly encountered.
[0048]
Surfaces in contact with liquids are particularly prone to biofilm formation. As an example, storage containers and tubes used to deliver humidified oxygen to a patient may have a biofilm inhabited by the infectious agent. Dental water supplies also have water systems used for dentistry and water reservoirs where aerosolized water is continuously contaminated, as well as the potential for biofilms on their surfaces.
[0049]
Sprays, aerosols, and nebulizers are highly effective at distributing biofilm fragments to a potential host or another environment. It is understood that preventing biofilm formation on surfaces where biofilm fragments may be spread by sprays, aerosols, or nebulizers in contact with the surface is of particular importance to health.
[0050]
Other health-related surfaces include the inner and outer surfaces of articles involved in water purification, water storage, and water supply, and articles involved in food processing. Health-related surfaces can further include the inner and outer surfaces of daily necessities involved in providing nutrition, hygiene, or disease prevention. Examples include household food processing appliances, child care products, tampons and toilets.
[0051]
The term "gram-positive bacteria" is an art-recognized term characterized by having a polysaccharide and / or teichoic acid along with peptidoglycan as part of the structure of the cell wall, resulting in a blue-violet reaction by Gram staining. It is characterized by Representative gram-positive bacteria include Actinomyces, Bacillus anthracis, Bifidobacterium, Botulinum, Welsh, Clostridium, Tetanus, Diphtheria, Corynebacterium jacium, Enterococcus faecalis, Enterococcus・ Fecesium, erysipelas swine, Eubacterium, Gardnerella basinalis, Gemera morbirollum, Lactobacillus, Mycobacterium abscessus, Mycobacterium avium complex, Mycobacterium kerone, Mycobacterium fortium , Mycobacterium hemophilium, Mycobacterium kansasii, Mycobacterium lepra, Mycobacterium marinum, Mycobacterium scrofuraceum, Mycobacterium smear Matisse, Mycobacterium terre, Mycobacterium ulcerans, Mycobacterium ulcerans, Nocardia, Peptococcus niger, Peptococcus, Proprionibacterium, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus coney, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus sapropititicus, Staphylo Coccus schleiferi, Staphylococcus similis, Staphylococcus weneri, Staphylococcus xylosus, Streptococcus agalactie (B Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus mirelli, Streptococcus mitio, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus sp. And Streptococcus sanguis.
[0052]
The term "gram-negative bacteria" is an art-recognized term that is a bacterium characterized by the presence of a bilayer surrounding each cell of the bacterium. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella basilformis, Bordetella, Borrelia Burgdorferi, Branhamera Catarrhalis, Brucella, Campylobacter, Chlamydia pneumoniae, Chlamydia sittasi, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter, Eikenella colodense, Enterobacter erogenes, Escherichia coli, Flavobacterium Meningosepticum, Fusobacterium, Haemophilus influenza, Haemophilus, Helicobacter Lori, Klebsiella, Legionella, Leptospira, Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella maltosida, Pregiomonas sigeroides, Prevotius, Proteus・ Letgeri, Pseudomonas aeruginosa, Pseudomonas, Rickettsia plowaceki, Rickettsia rickettii, Locarimea, Salmonella, Salmonella typhi, Serratia marcesens, Shigella, Treponema carateum, Syphilis, Treponema paridum endemica, Treponema pallidum Includes Parthenue, Beyonella, Vibrio cholerae, Vibrio vulnificus, Yersinia enterocortica, and Yersinia pestis.
[0053]
An amphipathic molecule or compound is an art-recognized term that is a molecule or compound in which one end of the molecule or compound is polar and the other end is nonpolar.
[0054]
The term "polar" is recognized in the art. Polar compounds contain substances with an asymmetric charge distribution. Generally, non-polar substances dissolve non-polar molecules, and polar substances dissolve polar molecules. For example, the polar substance water dissolves other polar substances. The amphiphilic compound has a part that is soluble in an aqueous solvent and a part that is insoluble in an aqueous solvent.
[0055]
The term "ligand" refers to a compound that binds to a receptor site.
[0056]
The term "heteroatom" as used herein refers to an atom that is any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. The term "electron withdrawing group" is art-recognized and refers to the tendency of a substituent to attract a valence electron of an adjacent atom, i.e., that the substituent is electrically negative relative to the adjacent atom. means. The degree of electron withdrawing ability is quantified by Hammett sigma (σ) constant. This known constant is, for example, J. March,Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259. The value of the Hammett constant is generally negative (NH_TwoIn the case of σ [P] = − 0.66), in the case of an electron-withdrawing group, the value is positive (in the case of a nitro group, σ [P] = 0.78), where σ [P] indicates 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.
[0057]
The term "alkyl" refers to the radical of a saturated fatty acid group, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In a preferred embodiment, a straight or branched chain alkyl has up to 30 carbon atoms in its backbone (eg, when straight chain, C₁To C₃₀, C for branched chain_ThreeTo C₃₀), More preferably 20 or less. 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.
[0058]
Unless otherwise specified, the term "lower alkyl" used in the present specification is the above-mentioned alkyl group, but has 1 to 10 carbon atoms, and more preferably has 1 to 10 carbon atoms in its skeleton structure. Has 6 carbon atoms. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyl. In a preferred embodiment, the substituents indicated herein as alkyl are lower alkyl.
[0059]
As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (eg, an aromatic or heteroaromatic group).
[0060]
"Alkenyl" and "alkynyl" refer to unsaturated fatty acid groups that are similar in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond, respectively.
[0061]
The term `` aryl, '' as used herein, refers to 0-4 heteroatoms, such as, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Includes 5, 6, and 7-membered monocyclic aromatic groups. These aryl groups having heteroatoms in the ring structure are sometimes referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring includes, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether , Alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic group, -CF<sub>Three</sub>Or one or more substituents described above such as —CN. The term "aryl" refers to a heterocyclic ring system having two or more rings that share two or more carbons in two adjacent rings (the ring is a "fused ring"). Also included are heterocyclic ring systems wherein at least one ring in the system is aromatic, for example, another ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl.
[0062]
The terms ortho, meta, and para apply to 1,2-, 1,3-, and 1,4-disubstituted benzenes, respectively. For example, 1,2-dimethylbenzene and orthodimethylbenzene are synonyms.
[0063]
The term "heterocyclyl" or "heterocyclyl group" means a 3- to 10-membered ring structure, more preferably a 3- to 7-membered ring, wherein the ring structure contains 1 to 4 heteroatoms. The heterocycle may be a heterocycle. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxatiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole , Indole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carbolin, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenaldazine, phenothiazine, flazan, phenoxazine, Pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lacto , Lactams such as azetidinones and pyrrolidinones, sultams, as well as sultone. Heterocyclyl rings include, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, Ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic group, -CF<sub>Three</sub>Or one or more positions may be substituted with the substituents described above, such as -CN.
[0064]
The term “heterocycle” or “heterocyclic group” refers to two or more rings in which two adjacent rings share two or more carbons (eg, cycloalkyl, cycloalkenyl, cyclo Alkynyl, aryl and / or heterocyclyl). Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each ring of the heterocycle is, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio , Sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic group, -CF<sub>Three</sub>Or -CN or the like.
[0065]
As used herein, the term "carbocycle" means an aromatic or non-aromatic ring in which each atom of the ring is carbon.
[0066]
As used herein, the term "nitro" refers to -NO<sub>Two</sub>The term "halogen" means -F, -Cl, -Br, or -I; the term "sulfhydryl" means -SH;
"Hydroxyl" refers to -OH, and the term "sulfonyl" refers to -SO<sub>Two</sub>-Means.
[0067]
The terms "amine" and "amino" are art-recognized and include, for example, the general formula
Embedded image
A group that can be represented by
In the general formula, R<sub>9</sub>, R<sub>Ten</sub>And R ’<sub>Ten</sub>Is a group that independently represents a group permitted by the law of valence,
And both substituted and unsubstituted amines.
[0068]
The term "acylamino" is art-recognized and has the general formula
Embedded image
A group that can be represented by
In the general formula, R<sub>9</sub>Is as described above, and R '<sub>11</sub>Is hydrogen, alkyl, alkenyl, or &#8209; (CH<sub>Two</sub>)<sub>m</sub>-R<sub>8</sub>Where m and R<sub>8</sub> Is as described above,
Means a group.
[0069]
The term "amide" is art-recognized as an amino-substituted carbonyl and has the general formula
Embedded image
Including a group represented by
In the general formula, R<sub>9</sub>, R<sub>Ten</sub>Is as described above. Preferred embodiments of the amide will not include imides, which may be unstable.
[0070]
The term "alkylthio" refers to an alkyl group as defined above having an attached sulfur radical. In a preferred embodiment, the `` alkylthio '' groups are -S-alkyl, -S-alkenyl, -S-alkynyl, and -S &#8209; (CH_Two)_m-R₈Where m and R₈Is as described above. Representative alkylthio groups include methylthio, ethylthio, and the like.
[0071]
The term "carbonyl" is art-recognized and has the general formula
Embedded image
A group that can be represented by
In the general formula, X is a bond or represents oxygen or sulfur;₁₁Is hydrogen, alkyl, alkenyl, &#8209; (CH_Two)_m-R₈Or a pharmaceutically acceptable salt;₁₁Is hydrogen, alkyl, alkenyl or &#8209; (CH_Two)_m-R₈Where m and R₈Is as described above,
Groups. X is oxygen and R₁₁ Or R ’₁₁Where is not hydrogen, the formula represents an "ester". X is oxygen and R₁₁Is as described above, the group herein refers to a carboxy group, especially R₁₁When is hydrogen, the formula represents a "carboxylic acid." X is oxygen and R '₁₁Where is hydrogen, the formula represents a "formate". Generally, when the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. X is sulfur and R₁₁And R ’₁₁If is not hydrogen, the formula represents a "thiol ester." X is sulfur and R₁₁When is hydrogen, the formula represents a "thiolcarboxylic acid." X is sulfur and R '₁₁Where is hydrogen, the formula represents a "thiol formate." On the other hand, if X is a bond and R₁₁Where is not hydrogen, the above formula represents a "ketone" group. X is a bond and R₁₁Where is hydrogen, the formula represents an "aldehyde" group.
[0072]
The terms "alkoxyl" or "alkoxy" as used herein, as described above, refer to an alkyl group 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. Thus, an alkyl substituent that makes an alkyl an ether is -O-alkyl, -O-alkenyl, -O-alkynyl, -O- (CH_Two)_m-R₈Or is similar to alkoxy.
[0073]
The term "sulfonate" is art-recognized and has the general formula
Embedded image
A group that can be represented by
In the general formula, R₄₁Is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl;
Groups.
[0074]
The terms trifryl, 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 are referred to as trifluoromethanesulfonate, p-toluenesulfonate, methanesulfonate, and nonamate, respectively. By fluorobutane sulfonic acid ester functional groups and by molecules containing said groups.
[0075]
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl, and methanesulfonyl, respectively. A more comprehensive list of abbreviations used by organic chemists in the art is given in the first volume of the Journal of Organic Chemistry, and this list is generallyStandard List of AbbreviationsIn the table titled. Abbreviations included in the above list, and all abbreviations used by organic chemists of skill in the art, are hereby incorporated by reference.
[0076]
The term "sulfate" is recognized in the art and has the general formula
Embedded image
A group that can be represented by
In the general formula, R₄₁Is as described above,
Group.
[0077]
The term "sulfonylamino" is art-recognized and has the general formula
Embedded image
And a group that can be represented by
[0078]
The term "sulfamoyl" is art-recognized and has the general formula
Embedded image
And a group that can be represented by
[0079]
As used herein, the term "sulfonyl" refers to a compound of the general formula
Embedded image
A group that can be represented by
In the general formula, R₄₄Is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Means a group.
[0080]
As used herein, the term "sulfoxide" refers to a compound of the general formula
Embedded image
A group represented by the formula:₄₄Represents a group selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, and aryl.
[0081]
For example, it is possible to make similar substitutions on alkenyl and alkynyl groups to produce aminoalkenyl, aminoalkynyl, amidoalkenyl, amidoalkynyl, iminoalkenyl, iminoalkynyl, thioalkenyl, thioalkynyl, carbonyl-substituted alkenyl or alkynyl. it can.
[0082]
As described herein, the definition of each expression, for example, alkyl, m, n, etc., is defined as if it appears more than once in any structure, or elsewhere in the structure. It is intended to be independent.
[0083]
"Substituted" and "substituted with" refer to such substitutions according to the allowed valencies of the substituted atoms and substituents, as a result of the substitutions which occur spontaneously, for example, by rearrangement, cyclization, removal, etc. It is intended to include the implicit condition that a stable compound that does not cause the above is obtained.
[0084]
As used herein, the term "substituted" is intended to include all allowed substituents on organic compounds. In a broad aspect, acceptable substituents include acyclic and cyclic, branched or unbranched, carbocyclic or heterocyclic, aromatic and non-aromatic substituents of organic compounds. Specific substituents include, for example, those described above. Acceptable substituents may be one or more of the same or different suitable organic compounds. For purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and / or any of the acceptable substituents of the organic compounds described herein that satisfy the valence of the heteroatom. . The present invention is not intended to be limited in any way by the permissible substituents of organic compounds.
[0085]
As used herein, the term "protecting group" refers to a temporary substituent that protects a potentially reactive functional group from unwanted chemical transformations. Examples of such protecting groups include carboxylic acids, silyl ethers of alcohols, acetals and ketals of aldehydes and ketones. The field of protecting group chemistry has been reviewed (Greene, T.W .; Wuts, P.G.M.Protective Groups in Organic Synthesis, 2^nd ed .; Wiley: New York, 1991).
[0086]
Contemplated equivalents of the aforementioned compounds include those compounds which otherwise correspond to the aforementioned compounds and which have the same properties (eg, function as an analgesic) as the general properties of the aforementioned compounds. And compounds that have one or more simple variations of substituents that do not adversely affect the efficiency of binding of the compound to the opioid receptor. In general, the compounds of the present invention employ readily available starting materials, reagents and conventional synthetic procedures, for example, by methods illustrated in the general reaction schemes described below, or by modifications thereof. May be prepared. In these reactions, variants which are known per se but are not mentioned here can also be used.
[0087]
For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements described in the inner cover of CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87.
[0088]
C. Compound
One aspect of the present invention relates to an antimicrobial surface comprising a covalently attached amphiphilic compound. In some embodiments, the surfaces of the present invention act to remove contacting airborne organisms. In some embodiments, the surface of the present invention acts to remove contacting organisms in water. In some embodiments, the surface of the present invention acts to inhibit biofilm formation.
[0089]
In some embodiments, the covalently bound amphiphilic compound is a polymer comprising ammonium ions. In a preferred embodiment, the molecular weight of the polymer is at least 10,000 g / mol, more preferably 120,000 g / mol, and most preferably 150,000 g / mol.
[0090]
In some embodiments, the surface of the present invention is glass. In some other embodiments, the surface of the present invention is plastic. In one embodiment, the surface of the present invention is an amino-containing glass.
[0091]
In some embodiments, the compound covalently attached to the surface is of Formula I:
Embedded image
(Formula I)
In the formula
R is each independently hydrogen, alkyl, alkenyl, alkynyl, acyl, aryl, carboxylate, alkoxycarbonyl, aryloxycarbonyl, carboxamide, alkylamino, acylamino, alkoxyl, acyloxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl, (alkyl Amino) alkyl, thio, alkylthio, thioalkyl, (alkylthio) alkyl, carbamoyl, urea, thiourea, sulfonyl, sulfonate, sulfonamide, sulfonylamino, or sulfonyloxy;
R ′ each independently represents alkyl, alkylidene tether to the surface, or acyl tether to the surface;
Z independently represents Cl, Br, or I,
n is an integer of about 1500 or less,
It is represented by an equation.
[0092]
In a preferred embodiment, the present invention comprises poly (4-vinyl-N-alkylpyridinium bromide) or poly (methacryloyloxidedecylpyridinium bromide) (MDPB) covalently attached to a glass surface. In another preferred embodiment, the present invention comprises an N-alkylated poly (4-vinylpyridine) covalently attached to a glass surface.
[0093]
In some embodiments, the surface killed organism of the present invention is a bacterium. In some embodiments, the bacterium that is killed is a Gram-positive bacterium. In another embodiment, the killing bacterium is a gram negative bacterium.
[0094]
Modification of surfaces containing reactive amino groups
In one embodiment, the surface is modified by graft copolymerization. In this case (method A), the surface is NH 3_TwoPrepared by acylating glass slides with an acylating agent to introduce double bonds. In one embodiment, the acylating agent is acryloyl chloride. This is followed by copolymerization with a copolymerizing agent such as, for example, 4-vinylpyridine. Immobilized polymers such as polyvinyl pyridine and PVP are sprayed with a suspension of raw S. aureus on their_TwoIt was observed that approximately the same number of Staphylococcus aureus survived as with the glass slide.
[0095]
The final step in creating the surface is to introduce a positive charge into the polymer chains attached to the glass. For example, the PVP pyridine ring may be N-alkylated with seven straight chain alkyl bromides (chain length from propyl to hexadecyl). This method is illustrated in Example 1. This method tends to produce linear polymers that are attached to the surface in a substantially straight line.
[0096]
The resulting slide surface was examined for its ability to kill contacting S. aureus sprayed on the surface. As can be seen in FIG. 2, the propylated, butylated, hexylated, and octylated immobilized PVP chains are effective in significantly reducing the number of viable bacterial cells, the most effective being hexyl PVP. It could be reduced by 94 ± 4% (right part of FIG. 1). In contrast, immobilized PVP N-alkylated with decyl bromide to hexadecyl, and the unalkylated chains were completely ineffective (FIG. 2).
[0097]
Behavioral patterns may correlate with the appearance of alkylated PVP slides. The unalkylated, decyl, dodecyl, and hexadecyl PVP modified slides were all turbid (absorbance A at 400 nm).₄₀₀Octyl PVP-modified slides are much less turbid (A₄₀₀= 0.03), propyl, butyl, and hexyl PVP-modified slides are clear (A₄₀₀= 0.002). The turbidity reflects the aggregates of the polymer formed on the surface, such aggregates clearly being unable to interact with the bacterial cells. The non-alkylated immobilized PVP chain and the PVP chain modified with a long alkyl group are liable to adhere to each other due to hydrophobic interaction, while the immobilized PVP chain modified with a short alkyl group is opposite in this case. Interaction is not strong enough to overcome the electrostatic repulsion of positively charged polymers.
[0098]
Using hexyl PVP (referred to herein as prepared by Method A), the surface of this surface against another gram-positive bacterium, S. epidermidis, and two gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa. Investigate the bactericidal effect of. The former two formed colonies of the same size as Staphylococcus aureus (left part of FIG. 1) when sprayed on an NH2 glass slide, but the colonies of Pseudomonas aeruginosa were larger but distinguishable. As can be seen in Table 1 (method A), the number of colonies formed by all three bacteria after spraying on hexyl PVP slides was reduced by a factor of 100 compared to unmodified NH2 glass.
[Table 1]
[0099]
In another embodiment, the surface is prepared by alkylating the surface with an alkyl halide group. This reactive alkyl group is then used for polymer attachment. The polymer chain was further alkylated to increase the positive charge. In this way, mainly branched and cyclic polymers covalently bonded to the surface are obtained.
[0100]
For example, NH_TwoGlass slides were alkylated with 1,4-dibromobutane to introduce reactive bromobutyl groups. This reactive bromobutyl group is later used for coupling PVP. In order to increase the positive charge of the bound PVP chain, this chain was further N-alkylated with hexyl bromide (optimized in method A, see FIG. 2). After spraying S. aureus cells and culturing by air drying, the resulting hexyl PVP slides (meaning those prepared by method B herein) are basically shown on the right side of FIG. It looked similar to what you had. NH_TwoCompared to glass slides, 94 ± 3% of the deposited Staphylococcus aureus cells were killed (first row, rightmost column in Table 1).
[0101]
The molecular weight of the immobilized polymer has been found to be important for the antimicrobial properties of the surface. For example, a hexyl PVP slide prepared by Method B using a short PVP chain, with 60,000 g / mol instead of 160,000 g / mol, kills only 62 ± 8% of the deposited S. aureus cells. Was.
[0102]
Examination of the remaining data in Table 1 reveals that Method B resulted in a lethal slide surface against S. epidermidis, Pseudomonas aeruginosa, and E. coli as well as the slide surface obtained in Method A. It is.
[0103]
The aforementioned substances, which kill bacteria by the release of germicidal substances, are eventually depleted of active substances because they are released into the surrounding solution. To examine whether the antimicrobial polymer immobilized using the method of the present invention leaches from the glass surface, place the hexyl PVP slide prepared in Methods A and B on a polystyrene petri dish and place it in an aqueous suspension of Staphylococcus aureus. The suspension was similarly sprayed onto the dish and non-antimicrobial polystyrene. When air dried and incubated on growth agar, a few (approximately 3 / cm) were found on both hexyl PVP glass slides.^Two), But a much larger number of colonies (60 ± 10 colonies / cm^Two) Was observed to have grown. Since there was no zone of inhibition around the hexyl PVP slide that would typically be seen if the germicide was released (23), the immobilized germicidal polymer hexyl PVP did not leach out of the slide, meaning that bacteria It is suggested that death occurs only on contact with the surface of the slide.
[0104]
To examine the specificity of the ability to kill airborne bacteria, suspensions of Staphylococcus aureus cells were sprayed on dry surfaces of various common materials, including metals, synthetic and natural polymers, and ceramics. The number of surviving colonies in all cases was determined by NH_TwoCompared to the number on the glass. As can be seen in Table 2, none of the substances investigated significantly reduced the amount of viable bacterial cells after spraying.
[Table 2]
[0105]
Surface modification without active amino groups
Since it would be desirable to render various objects sterilizable, a surface derivatization approach that could be applied to any material was developed. Conventional commercial synthetic polymers that do not themselves exhibit antimicrobial activity, namely polyolefins, polyamides, and polyesters, were used to test this approach. Slides made of high density polyethylene (HDPE) were selected as the first target. This polymer, like many other substances, does not have reactive groups suitable for easy chemical modification. Therefore, we decided to coat it with an ultra-thin silica layer by the 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 do this, a pen torch containing a compressed mixture of 0.6% tetramethylsilane and 7: 3 propane-butane (Pyrosil®) was used (FIG. 4, step 1). When this mixture burns in air, tetramethylsilane is oxidized to 2-5 nm SiO_TwoNanoparticles are formed, which cover the surface to which the flame hits. Tiller, HJ, Goebel, R., Magnus, B., Garschke, A. & Musil, R. A new concept of metal-resin adhesion using an intermediate layer of silicon oxide (SiOx) -carbon.Thin Solid Films 169, 159 -168 (1989). The resulting high-density, approximately 100 nm thick, chemically glass-like SiO_TwoThe layer (polysiloxane) can then be easily chemically modified to a homogeneous state, irrespective of the nature of the raw materials. 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, HJ, Goebel, R., Magnus, B., Garschke, A. & Musil, R. A new concept of metal-resin adhesion using an intermediate layer of silicon oxide (SiOx) -carbon.Thin Solid Films 169, 159-168 (1989) .Tiller, HJ, Kaiser, WD, Kleinert, H. & Goebel, R. The Silicoater method improves bond strength and resistance to aging.Adhaesion 33, 27-31 (1989) .Tiller, HJ, 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).
[0106]
HDPE surface appearance is SiO_TwoIt did not change after application of the coating. Such SiO_TwoThe degree of cross-linking of the layer is lower than that of glass, and many Si-OH groups are formed by hydration with water absorbed 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) . In contrast to the unmodified polymer being very hydrophobic, the coated HDPE surface is expected to be hydrophilic due to this Si-OH group. To quantify this difference, the contact angle formed by a water drop on the surface was measured. This is a common method of determining the hydrophilicity of a substance. David, J. The idea of water repellency. Text. Inst. Ind. 4, 293-5 (1966). To do this, 0.1 mL of distilled water was placed on the HDPE surface. The angle between the nucleus of the obtained droplet and the surface below it was estimated to be about 120 °, indicating a hydrophobic substance. Equivalent amount of distilled water to SiO_TwoWhen spread on the coated HDPE, the water spread over the surface and the contact angle was less than 10 °, reflecting the hydrophilic nature of the surface. Independently, HDPE slide-shaped SiO_TwoThe layer detected silicon and oxygen signals in the X-ray photoelectron spectrum and confirmed its presence.
[0107]
Next, the SiO_TwoThe amino group was introduced on the surface of the coated HDPE slide by reacting 3-aminopropyltriethoxysilane with the amino group (FIG. 4, step 2). This is a standard method for 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). Then NH_TwoFunctionalized HDPE slides were alkylated with 1,4-dibromobutane to introduce bromoalkyl groups (FIG. 4, step 3) and reacted with PVP in the presence of 1-bromohexane (FIG. 4, step 4). Under the conditions used, the pyridine group on the PVP chain is only slightly alkylated by bromoalkyl attached to the surface (about 1,500 pyridine groups per chain) and mostly 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). To determine the number of pyridinium groups on hexyl PVP derivatized polyethylene slides, the fluorescence was titrated spectrophotometrically to 8.2 ± 1.9 nmol / cm^TwoAnd hexyl PVP-modified NH_TwoIt is the same as when measuring with 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).
[0108]
The generality of the coating / derivatization approach described above modifies two other industrial polyolefins, low density polyethylene (LDPE) and polypropylene (PP), and polyamide nylon and polyester poly (ethylene terephthalate) (PET). And confirmed. As shown in FIG. 4, all of these synthetic polymers were successfully derivatized and the surface density of pyridinium groups was 8.5 ± 1.8 (LDPE), 7.2 ± 2.1 (PP), 8.0 ± 1.1 (nylon), and 7.5 ± 1.8. 0.9 (PET) nmol / cm^TwoShowed the same value as HDPE. All hexyl PVP derivatized slides then began to be tested from HDPE for the ability to kill contacting airborne bacteria.
[0109]
To simulate the natural deposition of airborne bacteria, a suspension of the ubiquitous pathogen Staphylococcus aureus, a gram-positive bacterium, in distilled water was sprayed on the slide surface and the slide was dried. . Xiong, Y., Yeaman, M.R. & Bayer, A.S.Linezolid: A new antibiotic. Drugs Today 36, 529-539 (2000). The slides were incubated overnight on nutrient agar as described above and the number of viable bacterial cells was counted by colony counting. 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, a number of easily distinguishable bacterial colonies were grown on unmodified HDPE slides. SiO_TwoCoating or NH_TwoThe number of colonies grown on functionalized HDPE slides (FIG. 4) was essentially the same, indicating that this modification is not toxic to S. aureus. Conversely, when sprayed onto hexyl PVP-modified HDPE slides, 96 ± 3% of the deposited bacterial cells were non-viable (FIG. 5a right, and Table 3).
[0110]
[Table 3]
[0111]
Bacterial distilled water suspension (10 Staphylococcus aureus⁶Cells / mL, 10 for E. coli^FiveCells / mL) were sprayed onto hexyl PVP-modified polymer slides, the slides were air-dried for 2 minutes and incubated overnight in 0.7% agar in bacterial growth medium. Thereafter, colonies were counted. The number of viable cells of the corresponding unmodified polymer slide counted in the same way (ie 0% dead bacteria) was used as a standard. Abbreviations of the used polymer substances are as follows. LDPE-low density polyethylene, HDPE-high density polyethylene, PP-polypropylene, and PET-poly (ethylene terephthalate). All experiments were performed at least twice and the error obtained indicates the standard deviation.
[0112]
Hexyl PVP-derivatized LDPE, PP, nylon and PET were similarly tested for their ability to kill airborne Staphylococcus aureus. As can be seen in Table 3, in all cases the number of bacterial colonies formed was reduced by a factor of 10 to 1/30 compared to that grown on the corresponding unmodified slide.
[0113]
Next, the hexyl PVP derivatized surface was tested for its ability to kill E. coli, a typical gram-negative bacterium. As seen on the left of FIG. 5b, many of the E. coli colonies sprayed and grown on HDPE slides are distinctive, though less dense than S. aureus grown under the same conditions. On surfaces derivatized with hexyl PVP, the number was reduced by 97 ± 3% (FIG. 5b right and Table 3). Examination of the remaining data listed in the last row of Table 3 shows that the modification of LDPE, PP, nylon and PET slides with hexyl PVP resulted in the surviving E. coli cells, after being deposited from the air, to the corresponding unmodified Similarly reduced to 1/20 to 1/50 compared to slides.
[0114]
To test whether hexyl PVP chains immobilized using the method of the present invention leach out of the slide surface (and may only kill deposited bacteria), a modified HDPE slide was placed on a polystyrene dish. In, an aqueous suspension of S. aureus cells was sprayed on the slide and polystyrene (non-antibacterial) surface. When air dried and incubated on growth agar, only a few colonies grew on hexyl PVP-modified HDPE slides (4 ± 2 cells / cm^TwoOn the other hand, far more colonies grew on the Petri dish surface around the slide (85 ± 5 / cm).^Two), The same was true of the slide. The lack of a zone of inhibition around the hexyl PVP slide, which is characteristic of the release of bactericidal substances, indicates that immobilized polycations have not leached out of this slide, indicating that bacteria in the air will die upon contact with the slide surface. ing. Kawabata, N. & Nishiguchi, M. Antibacterial activity of soluble pyridinium-type polymers. Appl. Environ. Microbiol. 54, 2532-2535 (1988).
[0115]
Another important question was whether hexyl PVP derivatized slides could also kill bacteria in water. To experimentally simulate the situation in flowing aqueous solution, the polymer slide was placed vertically in a bacterial PBS suspension, pH 7.0, and the resulting system was gently stirred at 37 ° C. After a 2 hour incubation, the slides were washed with sterile buffer to remove non-adhered bacteria and incubated in sterile PBS for approximately 1 hour under the conditions described above to irreversibly remove the temporarily adhered bacteria. Separated or attached. Wiencek, K.M. & Fletcher, M. Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers. J. Bacteriol. 177, 1959-1966 (1995). After washing the slides with PBS, they were immediately covered with a layer of solid growth agar and incubated overnight at 37 ° C. In this way, all truly bound bacterial cells are trapped on the slide surface, allowing surviving cells to proliferate and form colonies.
[0116]
As seen on the left of FIG. 6a, S. aureus cells attached to HDPE slides form many easily distinguishable colonies. SiO_Two-Or NH_Two-There was no significant decrease in the number of cell colonies that grew after attachment for any of the modified HDPE slides. However, as seen in Figure 6a right, the number of S. aureus colonies that grew on hexyl PVP-modified HDPE slides was reduced by 98 ± 1% (see also Table 4).
[0117]
[Table 4]
[0118]
Bacterial PBS suspension, pH 7.0 (2x10 for Staphylococcus aureus)⁶4x10 cells / mL and E. coli⁶Cells / mL) were allowed to adhere to the surface of the hexyl PVP-modified polymer under stirring at 37 ° C. for 2 hours. The slides were washed with sterile PBS, pH 7.0, shaken in sterile PBS for 1 hour at 37 ° C., washed again with PBS, and incubated overnight in 1.5% agar of bacterial growth medium to remove colonies. Was counted. N.d. means "cannot be measured". This is because the attached bacterial cells grow on the target slide but do not form distinguishable colonies. See the footnote in Table 3 for other conditions.
[0119]
It has further been demonstrated that hexyl PVP derivatized LDPE, PP, nylon, and PET can also kill bacteria in contacting water. The data in Table 4 show that when derivatized (FIG. 4), the number of viable S. aureus cells attached was reduced by 97-99% on all polymer slides. Similarly, the modified polymer slide was found to be effective against E. coli in water. As seen on the left of FIG. 6b, E. coli attached to unmodified HDPE from the aqueous suspension is growing a large number of distinct colonies under the cover of growth agar. The appearance of the colonies on LDPE, nylon, and PET was similar, but there were no distinguishable colonies on PP. When HDPE was derivatized with hexyl PVP, the number of surviving adherent E. coli cells was reduced to 97 ± 2% (FIG. 6b right, Table 4). Other modified polymers also showed a dramatic decrease in the number of viable adherent E. coli cells compared to the corresponding unmodified slides, with mortality observed with LDPE, PET and nylon of 98-99% ( Table 4, last row).
[0120]
Because the experimental conditions for determining the bactericidal properties of hexyl PVP-modified slides against bacteria in water differed from those in air, we again tested the release of polycations from slides into the culture. To do this, hexyl PVP-modified HDPE slides were incubated in sterile PBS with shaking at 37 ° C for 2 hours, removed, and S. aureus cells added to the remaining solution. The resulting suspension was shaken again at 37 ° C for 2 hours and the number of surviving bacterial cells was counted by spreading them on growth agar and then incubated overnight at 37 ° C. The number of cell colonies observed was (42 ± 2) 10^FourCounts / ml in control [(48 ± 7) 10^FourPcs / mL]. Thus, there is no apparent release of antimicrobial from hexyl PVP-modified HDPE slides during bacterial attachment experiments, and indeed bacteria in water must die upon contact with the derivatized surface.
[0121]
Mechanism of bacterial attack
The tethered amphiphilic polycations and soluble cationic antimicrobial agents described herein probably share a similar mechanism of bacterial attack. Polycations, such as polymyxin B and animal antimicrobial cationic peptides, displace divalent cations that both grip the negatively charged surface of the lipopolysaccharide network and replace the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. (Vaara, M. (1992) Microbiol Rev. 56, 395-411). This alone may be sufficient to produce a fatal result. Cationic groups of the tethered polymer may break through the permeation barrier of the outer membrane and then penetrate further into the inner membrane, possibly causing leakage. Such "self-promoted permeation" and subsequent damage to the intima has been described for polymyxin. The action of the immobilized polycation on the Gram-positive bacteria S. aureus and S. epidermidis probably requires the cationic groups to penetrate the thin cell membrane in order to reach the plasma membrane. The bactericidal action of amphiphilic cationic preservatives such as benzalkonium chloride or biguanidine chlorexidine on Gram-positive bacteria is mainly due to the disruption of the plasma membrane (Denton, GW (2001) in Disinfection, Sterilization, and Preservation , ed. Block, SS (Lippincott Williams & Wilkins, Philadelphia)). The thickness of the cell wall of S. aureus is about 30 nm (Friedrich, C.L., Moyles, D., Beveridge, T.J. & Hancock, R.E. (2000) Antimicrob. Agents Chemother. 44, 2086-2092.). This is because the estimated average length of N-hexylated PVP (Method A) is 19 nm, but some are clearly shorter and longer, and longer ones can penetrate the cell wall.
[0122]
Medical applications
Such surface modifications can easily be made to many other substances. A simple regular wash could remove dead and deposited cells and restore such a surface.
[0123]
Any substance placed from outside the body into the body is susceptible to biological contamination by bacteria and subsequent biofilm formation. Thus, the compounds according to the invention can protect such substances from microbial contamination from the beginning. In addition, such techniques make it possible to use natural and synthetic materials that are resistant to contamination, especially those that are already infected, or anatomical that can be particularly catastrophic when infected. It can also be used to make a substance suitable for exchange with a substance intended to be placed at a site.
[0124]
Naturally derived processing materials are generally placed in the body to provide a structure for in-growing the patient's own tissue. Examples include demineralized bone material, and hydroxyapatite. Such materials are to completely penetrate or displace the patient's tissue while exogenous material maintains the desired shape or structural support in the affected area. These materials themselves are non-living and avascular. If microorganisms colonize these materials to form biofilms, they may need to be removed. Removal of the material destroys the shape or structure that is being maintained and wastes the growth progression of the tissue. Applying the compounds of the present invention to these materials can enhance their resistance to biofilm formation and its effects.
[0125]
Some naturally occurring processing materials will be determined by those skilled in the art to be particularly suitable for application or incorporation of the compounds of the present invention. Certain substances can be contacted with the compounds claimed herein in various ways, including immersion and coating. If there are gaps in the material, the AF compound can enter there as a liquid or gel. Fibrous preparations can also be prepared by coating the fibers with the compound. Solid substances, such as reconstructed blocks of hydroxyapatite, can be coated with a coating of the compound for further protection. Temporary methods of such application are appropriate for these materials. Because it is only temporarily present in the body and is not reabsorbed or replaced.
[0126]
Implantable medical devices using only artificial materials or mixtures with naturally occurring materials can be treated with compounds by surface coating or by incorporation. It is believed that the metal is preferably treated with a surface coating while maintaining its biological properties. In some embodiments of the present invention, the metal may be treated with a paint or an adhesive layer of a polymer or ceramic incorporating the compound of the present invention. Some embodiments processed in this manner may be suitable for orthopedic applications, such as, for example, pins, screws, plates, or components of artificial joints. Methods for treating metal surfaces for biological applications are known in the relevant art. Other substances besides metals can be treated with surface coatings of the compounds according to the invention as required by medical applications.
[0127]
Implantable devices may include materials suitable for incorporation of the compounds of the present invention. Embodiments in which the compounds of the present invention are incorporated into components include polymers, ceramics, and other materials. Materials made from artificial materials can also be absorbed when placed in the body. Such a substance is called a bioabsorbable substance. For example, polyglycolic acid polymers can be used to make sutures and orthopedic devices. Those skilled in the art will be familiar with techniques for incorporating substances into polymers used to make moldings for medical applications. AF agents can also be incorporated into adhesives, cements, or adhesives, or other materials used to fix structures in the body or adhere implants to body structures. Examples include polymethyl methacrylate, and related compounds, used to attach orthopedic or dental prostheses to the body. The compounds of the present invention can reduce the formation of biofilms on structures that come into contact with adhesives, cements, or adhesives. Alternatively, the compounds of the present invention can coat or infiltrate moldings. In such a composition, the molding can diffuse the compound or a functional part thereof into the surrounding environment, thereby preventing contamination of the device. Compounds having microcapsules can also be embedded in the material. A part of the substance mixed with the compound can be applied to manufacture of a wide range of medical devices described later. Other examples will be readily apparent to the practitioner or skilled in the art.
[0128]
In certain embodiments, the compounds of the present invention may be applied or incorporated into a number of medical devices that are intended to be left in place permanently to replace or restore vital functions. In certain instances, a ventricular atrial or peritoneal shunt is employed to prevent cerebrospinal fluid collection in the brain of a patient with an impaired normal drainage tract. As long as the shunt functions, it can prevent fluid from accumulating in the brain and continue normal brain function. When the shunt stops functioning, fluid accumulates and compresses the brain, which can be life-threatening. When a shunt is infected, it invades the central part of the brain, which is also a life-threatening complication. The shunt generally includes a silicone elastomer or another polymer as part of the fabrication. Silicones are understood to be particularly suitable for combination with the compounds according to the invention.
[0129]
Another shunt of vital importance is the dialysis shunt, which is a polymer tube that connects the arteries and veins of the forearm and provides a means for dialysis equipment to flush the bloodstream to patients with renal failure. provide. Although this is a high flow conduit, biofilm formation and subsequent infection are likely. Once the shunt is infected, it must be removed and replaced. Because dialysis is a lifelong process and the number of sites to which the shunt can be applied is limited, it is desirable to avoid infectious complications when removing the shunt. Implantation of the compound of the present invention or other contact with the shunt material has this desirable effect.
[0130]
Heart valves containing artificial materials are understood to be prone to dangerous complications of artificial endocarditis. Once affected, mortality can reach 70%. Biofilms are completely involved in the development of this condition. Currently, the only treatments for established contamination are bolus therapy of antimicrobial agents and surgical removal of instruments. Contaminated valves must be replaced immediately. Because the heart will not work. The replacement valve also suffers from prosthetic valve endocarditis due to the insertion of a new valve into the recently contaminated area. Prosthetic heart valves made of the compounds of the present invention are believed to reduce the onset and recurrence of prosthetic valve endocarditis. The compounds of the present invention can be applied to synthetic or naturally occurring portions of heart valves.
[0131]
Pacemakers and implantable defibrillators generally include metal parts in combination with other synthetic materials. These devices, which may be coated with a polymeric material such as silicone, are typically implanted subcutaneously or intramuscularly using wires or other electrical devices that extend into the thorax or into blood vessels. If microorganisms colonize and infect the device, it must be removed. Although new devices can be replaced in different locations, there are only a limited number of suitable implantation sites in the body. Devices containing the compounds of the present invention are believed to inhibit or substantially reduce the risk of contamination and infection.
[0132]
Devices that temporarily or permanently implant a pharmacological substance into the body for delivery into the body can include metal parts in combination with other synthetic substances. Such devices are called drug infusion pumps and can be completely or partially implanted. The device may be partially or completely covered with a polymer material and may include other polymers as conduits or tubes. When the AF agent of the present invention is incorporated into the coating material imposed on these devices or the materials used in the devices themselves, the conduits or tubes are believed to inhibit contamination and infection.
[0133]
Equally necessary for life support are various vascular prostheses and stents aimed at bypassing occluded arteries or replacing damaged arteries. Artificial blood vessels made from Teflon, Dacron, Gore-Tex®, expanded polytetrafluoroethylene (e-PTFE), and related materials are available for use on any tumor blood vessel in the body. For example, artificial blood vessels are commonly used for bypassing leg blood vessels and replacing damaged aortas. The vascular prosthesis is inserted into place by sewing to the end or side of a normal blood vessel upstream or downstream of the area to be bypassed or replaced. Therefore, blood flows from the normal region to the artificial blood vessel and is transported to other normal blood vessels. Stents that include a metal frame covered with an artificial vascular material can also be applied intravascularly to repair damaged blood vessels.
[0134]
Suture materials used to secure the vascular graft to normal blood vessels and to suture blood vessels or other structures may also mask the infection. Sutures used for such purposes are typically made of prolene, nylon, or other monofilament non-absorbable materials. Infections starting with sutures can also spread to artificial blood vessels. Suture materials containing the compounds of the present invention will have a high resistance to infection.
[0135]
As a general principle of surgery, if a foreign body becomes infected, it is likely that it will be necessary to remove the foreign body so that the infection can be controlled. For example, if the suture becomes infected, it may need to be surgically removed to control the infection. Any treatment site is prone to wound infection. Wound infections can penetrate deep into tissues and involve foreign substances used as part of surgery. For example, hernias are commonly repaired by suturing a plastic screening material called a mesh to the lesion. Wound infections that spread to the area where the mesh is located also involve the mesh itself and the mesh must be removed. Surgical meshes containing the compounds of the present invention will have a high resistance to infection. Surgical mesh is made from materials such as Gore-Tex®, Teflon, nylon, and Marrex®. Surgical meshes are used to close deep wounds or to strengthen the surrounding of body cavities. Removal of the infected mesh leaves irreparable failures and can be life threatening. Avoidance of infection with such substances is of paramount importance in surgery. The materials used for the mesh and related materials can be made to include the claimed compounds of the present invention.
[0136]
Some implantable devices aimed at restoring structural stability to body parts can be advantageously treated with the compounds of the present invention. Some examples are described below and others will be readily identifiable by those skilled in the art. Implantable devices used to replace bones or joints or teeth serve as prostheses or replacements for normal structures at anatomical sites. Metals and ceramics are commonly used as orthopedic and dental prostheses. The implant may be fixed in place with a cement such as polymethyl methacrylate. The surface of the prosthesis can be made of a polymer such as silicone or Teflon. The entire prosthesis for fingers, toes, or wrists can be made of polymers.
[0137]
Medical prostheses containing the compounds of the present invention would be expected to reduce infection and subsequent local infection, obviating or reducing the need for implant removal with local tissue destruction. . Destruction of local tissue, particularly bone and ligaments, can render the tissue bed unsuitable for supporting a replacement prosthesis. In addition, the presence of contaminating microorganisms in surrounding tissues may facilitate recontamination of the replacement prosthesis. The consequences of repeated contamination and infection of structural prostheses are severe. Extensive reconstructive surgery may be required to repair areas without prostheses, which may include free bone grafts or arthrodesis. Furthermore, there is no guarantee that these secondary reconstruction operations will not result in infectious complications. In the unlikely event of amputation of any limb, the biggest obstacle is the consequences of structural prosthesis contamination and infection.
[0138]
Some implantable devices are intended to restore or enhance body contours for cosmetic or reconstructive purposes. A known example of such a device is a breast implant, which is a bag made of silicone elastomer containing gel or liquid. Other polymeric implants are intended for permanent cosmetic or remodeling purposes. Solid silicone blocks or sheets can also be inserted due to contour defects. Other naturally occurring or synthetic biomaterials are available for similar uses. Surgical reconstruction of the craniofacial surface may require implantable instruments to recover severely deformed facial contours, in addition to the techniques used to reconstruct the original bone contours. These devices and other related devices known in the art are suitable for coating or impregnating with sulfated AF agents to reduce the risk of contamination, infection, and subsequent removal.
[0139]
Tissue dilators are bags made of silicone elastomer adapted to slowly fill saline, and this filling process stretches the tissue over it, creating a large area of tissue that can be used for other reconstruction applications. Occurs. Tissue dilators are performed, for example, as a step in breast reconstruction after a mastectomy and can be used to dilate the skin and muscles of the chest wall. The tissue dilator can also be used to reconstruct areas of significant skin loss in burn patients. Tissue expanders are typically intended for temporary use. The covering tissue, once fully expanded, extends and covers the obstruction to be covered. If the tissue expander is removed before replacing the expanded tissue, all of the expansion obtained over time will be lost, and the tissue will almost return to its pre-expansion state. The most common reason for removing tissue dilators too early is infection. The device must be inflated multiple times with saline and introduced percutaneously into a remote filling device to connect with the dilator. Bacterial contamination of the device will usually result from the transdermal swelling process. Once contamination is established and a biofilm is formed, local infections are likely to occur. In order to control the infection, the reconstruction process must be stopped and the dilator removed. It is usually recommended that the reinsertion of a new tissue expander into the affected area be delayed for months. It is particularly preferable that the silicone elastomer used for these devices be mixed with a sulfated AF agent. The use of this sulfate AF agent in the manufacture of these articles would reduce bacterial contamination, biofilm formation, and the subsequent occurrence of local infection.
[0140]
Insertable devices include materials made from synthetic materials that are applied to the body or partially inserted into the body from natural or artificial entry sites. Examples of articles applied to the body include contact lenses and fistula appliances. An artificial larynx is understood as an insertable device. The device is in the respiratory tract, partially exposed outside the body, and partially secured to surrounding tissue. An endotracheal or organ tube, gastrostomy tube, or catheter is an example of an insertable device that is partially inside the body and partially exposed outside the body. The endotracheal tube passes through the existing natural opening. The tracheal tube passes through an artificially created opening. In any of these situations, when a biofilm is formed on the device, microorganisms can penetrate along the device from a more external anatomical site to a more internal anatomical site. Invasion of microorganisms further into internal anatomical sites generally causes local and systemic infections.
[0141]
For example, the formation of biofilms on soft contact lenses is understood as a risk factor for contact lens-related diaphragmatic infections. The eyes themselves are prone to infections due to biofilm formation. The incorporation of antimicrobial substances into the contact lens itself and the contact lens case can reduce biofilm formation and reduce the risk of infection. The sulfated AF agent can also be mixed periodically into ophthalmic preparations to be instilled.
[0142]
In another example, biofilms are understood to be responsible for infections from middle ear ventilation tubes and artificial larynx. Biofilms are also lurking in tracheostomy and endotracheal tubes, causing pathogenic bacteria to enter the distal airways of the relatively sterile lung. The device is compatible with the incorporation or topical application of sulfate AF agents to reduce biofilm formation and subsequent infectious complications.
[0143]
In another example, various vascular catheters are made to connect to blood vessels. The temporary venous catheter is placed distally while the central venous catheter is placed in the more proximal vena cava. Catheter systems may include percutaneously mounted hubs outside the body, and access ports implanted subcutaneously. Examples of long-term central venous catheters include Hickman catheters and Port-a-cath. Catheters allow the infusion of fluids, nutrients, and pharmaceuticals, as well as blood collection for diagnostic research or transfusion of blood or blood products. In such a catheter, a biofilm is easily formed, and the longer it stays in a specific vein, the more the biofilm is formed. The formation of a biofilm in a vascular connection device may release planktonic organisms from the biofilm into the surrounding bloodstream, leading to the development of blood-borne infections. In addition, the biofilm can occlude the device itself and cause a malfunction. If the catheter becomes infected or if the obstruction does not clear, the catheter must be removed. Generally, patients using this device suffer from severe medical symptoms. Such patients have limited ability to remove and replace instruments. In addition, the number of sites that access blood vessels is limited. Patients who repeat catheter placement may have exhausted the area where a new catheter can be easily and safely placed. The incorporation of the sulfated AF agent into the catheter material, or the application of the agent to the catheter material, reduces contamination and biofilm formation, thereby maintaining device patency and minimizing the risk of infectious complications Can be suppressed.
[0144]
In another example, a bile drainage tube is used to drain bile out of the body when the normal bile duct system is disrupted or recovers from surgical manipulation. The discharge tube may be made of plastic or another polymer. Bile duct stents are typically made of a plastic material and inserted into one passage of the bile duct, leaving the bile open and letting bile go through the bottom. Gall sand resulting from the formation of bacteria and biofilms attached to the biliary system has been found to cause biliary stent blockage. Pancreatic stents are placed to open the pancreatic duct or to drain the pancreatic pseudopustules, which may also be occluded by pancreatic sand. In addition, biofilms are involved in the spread of infection deeper into the bile duct along the bile drainage tube. When the bile duct spreads, it can cause a dangerous infection called cholangitis. Incorporation of the compounds of the present invention into the materials used to make bile drainage tubes and biliary stents can reduce biofilm formation and thus reduce the risk of occlusion and infection.
[0145]
In another example, a peritoneal dialysis catheter is used to remove excreta from the body of a patient with renal failure using a fluid that is infused from the peritoneal cavity and subsequently removed. This form of dialysis is an alternative to hemodialysis for some patients with renal failure. Formation of a biofilm on the surface of a peritoneal dialysis catheter can result in occlusion and infection. Infections that invade the abdominal cavity are called peritonitis and are one of the most dangerous infections. Peritoneal dialysis catheters are generally made of a polymeric material such as polyethylene, but can be coated or impregnated with a sulfated AF agent to reduce biofilm formation.
[0146]
In yet another example, there are a variety of urological catheters to provide urinary system drainage. The catheter may be inserted into the natural urethral opening to drain the bladder, or may be adapted to pass the urethral system through an iatomically created insertion site. Nephrostomy tubes and suprapubic bladder puncture tubes are examples of the latter. The catheter is placed semipermanently in the ureter to keep the ureter open. Such a catheter is called a ureteral stent. Urological catheters can be made from a variety of polymer products. Latex and rubber tubes have been used, but silicone has also been used. All catheters are prone to biofilm formation. This raises the problem of ascending urinary tract infections, where the biofilm spreads proximally and propagates pathogenic organisms, or the ability of planktonic organisms resident in the biofilm to invade tissues and bloodstreams. May increase some planktonic organisms. In general, organisms in the ureter are gram-negative bacteria that can spread fatally to cause fatal bloodstream infections. Even infections where such organisms are present exclusively in the ureter can be dangerous, with pain and high fever. Ureteral infections can cause a renal infection called pyelonephritis and can also compromise renal function. The incorporation of sulfated AF agents would suppress biofilm formation and reduce the likelihood of these infectious complications.
[0147]
A further complication encountered by urological catheters is encrustation, a process in which inorganic compounds, including calcium, magnesium, and phosphorus, deposit in the lumen of the catheter and occlude the lumen. It is understood that such inorganic compounds result from the action of some bacteria that are resident on the biofilm on the catheter surface. Reduction of biofilm formation by the action of sulfated AF agents is thought to contribute to a reduction in encrustation and a subsequent decrease in urological catheter occlusion.
[0148]
There are other catheter-like devices that can be treated with AF agents. For example, surgical drains, thoracic tubes, and hemovacs can be advantageously treated with substances that impair biofilm formation. Other examples of such devices will be familiar to those skilled in the art.
[0149]
Substances for body application can be used to advantage in the AF compounds disclosed herein. The dressing may itself be mixed with an AF compound and may be in the form of a film or sheet for direct application to the skin surface. Further, the AF compound of the present invention can be mixed into a pressure-sensitive adhesive or an adhesive for attaching a dressing or a device to be attached to the skin. For example, a fistula adhesive or medical grade adhesive may be formulated to include an AF agent appropriate for the particular medical context. Fistula adhesives are used to adhere fistula bags and similar devices to the skin without excessive damage to the skin. Due to the presence of infection products in such devices and on the surrounding skin, these devices are particularly suitable for coating with or incorporating AF agents. Other attachments can be treated similarly. Adhesive plasters, adhesive tapes, and transparent plastic adhesive sheets are further examples, including the incorporation of an AF agent into an adhesive or other adhesive used to secure the object, or an AF agent as a component of the object. Is believed to be effective in reducing skin irritation and infection.
[0150]
This above example is provided to illustrate the versatility of the compounds of the present application in medical devices. Other examples will be readily envisioned by those skilled in the art. The scope of the present invention is intended to include all surfaces where the presence of contamination has adverse health-related consequences. The above examples represent embodiments in which the techniques of the present invention are understood to be applicable. Other embodiments will be apparent to those skilled in the art and practitioners of the relevant art. Embodiments of the present invention may be compatible with currently used bactericidal therapies to enhance antimicrobial or cost-effective use. Selection of the appropriate excipient with the compound of the invention will be dictated by the characteristics of the particular medical application. Other examples of uses in the medical setting to promote antimicrobial properties will be readily envisioned by the relevant artisan.
[0151]
Now that the present invention has been schematically described, it will be more easily understood by referring to the following examples. However, the following examples have been included merely to illustrate certain aspects and embodiments of the present invention, and are not intended to limit the present invention.
[0152]
Surface derivatization
[0153]
Example 1
Method A
NH_TwoGlass slides (microscope slides coated with aminopropyltrimethoxysilane) were placed in 90 mL of anhydrous dichloromethane containing 1 mL of triethylamine. After cooling to 4 ° C., 10 mL of acryloyl chloride was added and the reaction mixture was stirred overnight in a cold room, then at room temperature for 2 hours. Acylated NH_TwoGlass slides were rinsed with a methanol / triethylamine mixture (1: 1, v / v) and methanol. Before this acryloylation (6.6 ± 0.1 × 10^-Tenmol / cm^Two) And after (3.3 ± 0.2 × 10^-Tenmol / cm^Two) NH on glass slide surface_TwoAs determined by picric acid titration of the groups (17), about half of the amino groups bound to the surface had reacted with acryloyl chloride. Next, the acryloyl moiety bonded to the glass was copolymerized with 4-vinylpyridine. Degas perchloric acid (90 mL of a 20% aqueous solution) and add 30 mg of Ce (SO_Four)_TwoWas added under argon. After stirring for 1 hour, 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 hours. PVP that did not chemically attach to the slide was washed off with pyridine, N, N-dimethylformamide, and methanol. Immediately thereafter, the slide with the PVP attached was placed in a 10% (v / v) nitromethane solution of alkyl bromide. Thereafter, the reaction mixture was stirred at 75 ° C. for 72 hours, and more than 90% of the pyridine rings had been N-alkylated (19). The resulting polycation-derivatized PVP-slide was rinsed with methanol and distilled water and air dried.
[0154]
Example 2
Method B
NH_Two-A glass slide was immersed in a mixture containing 9 mL of 1,4-dibromobutane, 90 mL of anhydrous nitromethane, and 0.1 mL of triethylamine. After stirring at 60 ° C. for 2 hours, the slide was removed, rinsed well with nitromethane, air-dried, and 90 g of 9 g of PVP (60,000 or 160,000 g / mol in molecular weight) nitromethane / hexyl bromide (10: 1, v / v) placed in solution. After stirring the reaction mixture at 75 ° C. for 24 hours, the slides were rinsed with acetone, rinsed well with methanol (to remove unattached polymer) and air dried. According to (14), under these conditions more than 96% of the pyridine ring of PVP should be N-alkylated.
[0155]
Surface analysis
Example 3
The chemically modified glass slide was immersed in a 1% distilled aqueous solution of fluorescein (Na salt) for 5 minutes. Under these conditions, the dye binds to quaternary amino groups (20) but not to tertiary or primary ones (we have modified PVP or NH_TwoGlass slide did not adsorb fluorescein). After rinsing with distilled water, the colored slides were placed in 25 mL of a 0.1% aqueous solution of the surfactant cetyltrimethyl-ammonium chloride and shaken for 10 minutes to desorb the dye. The absorbance of the resulting aqueous solution was measured at 501 nm (after adding 10% 100 mM aqueous phosphate buffer, pH 8.0). The absorbance of fluorescein in this solution, determined individually, is 77 mM^-1cm^-1Turned out to be.
[0156]
Various hexyl-PVP-films of known polymer content (160,000 g / mol, colored> 95% degree of alkylation showed that the stoichiometric ratio of fluorescein linkages was approximately 7 units per dye molecule. The following amount of hexyl-PVP attached was determined (assuming that more than 90% of the polymer was hexylated): 5.8 ± 3.0μg / cm^Two(Method A): 2.8 ± 1.0 μg / cm^Two(Method B, PVP with a molecular weight of 160,000 g / mol); and 0.4 ± 0.05 μg / cm^Two(Method B, PVP with a molecular weight of 60,000 g / mol). For hexyl-PVP immobilized in Method A, the minimum chain length of the attached polycation was estimated to be 61 ± 30 monomer units.
[0157]
Determination of antimicrobial susceptibility
Example 4
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, Almost 10¹¹Cells / mL), and 50 mL of yeast-dextrose broth (prepared as described by Cunliffe et al. (21)) in a sterile Erlenmeyer flask. added. The suspension was incubated at 37 ° C. for 6-8 hours with shaking at 200 rpm. After centrifugation (2,700 rpm, 10 minutes), wash the bacterial cells with distilled water and add⁶(For E. coli 10⁷).
[0158]
The bacterial suspension was then sprayed onto a glass slide (or other surface) in a fume hood using a commercial chromatography sprayer (VWR) at a spray rate of 10 mL / min. After drying under air for 2 minutes, the slides were placed in a Petri dish and growth agar (0.7% agar with yeast-dextrose broth, autoclaved and cooled to 37 ° C.) was added. The petri dish was capped, sealed, and incubated at 37 ° C. overnight.
[0159]
Example 5
The bacteria were suspended in distilled water and sprayed onto the surface of a slide to mimic the attachment of airborne bacteria, a common method of transmitting bacterial infections, such as talking, sneezing, coughing, or simply breathing. To determine the number of viable bacterial cells on the infected surface, slides after 2 minutes air drying were incubated on growth agar. Countable colonies are formed under these conditions if the bacteria are capable of growth. The infectious Gram-positive bacterium Staphylococcus aureus was used in the first experiment. Then, NH_Two-A glass slide was acylated with acryloyl chloride to introduce a double bond and subsequently copolymerized with 4-vinylpyridine. With such immobilized PVP, the viable S. aureus cell count after spraying the bacterial suspension onto_Two-It turns out to be about the same number as glass slides. The last step is to add a positive charge to the PVP chains attached to the glass. For this purpose, the pyridine ring of the polymer was N-alkylated with seven linear alkyl bromides (chain length from propyl to hexadecyl). The slides were tested for their ability to kill S. aureus cells sprayed on them upon contact. As can be seen in FIG. 2, the propylated, butylated, hexylated, and octylated immobilized PVP chains were significantly more effective at reducing viable bacterial cell numbers, with the most effective hexyl- PVP could result in a 94 ± 4% reduction (see right part of FIG. 1).
[0160]
Example 6
Using a glass slide surface modified with hexyl-PVP (hereinafter referred to as prepared in Method A), we determined the bactericidal effect of this surface by another gram-positive bacterium, S. epidermidis, or E. coli. And two Gram-negative bacteria such as P. aeruginosa. NH_TwoWhen sprayed on glass slides, the first two formed colonies of the same size as S. aureus (Fig. 1, left part), whereas the colonies of P. aeruginosa were larger but still discriminated. It was possible. As can be seen in Table 1 (method A), the number of colonies of the three bacteria formed after spraying on hexyl-PVP-slides was all normal NH._TwoIt was reduced by more than 100 times compared to glass.
[0161]
Suspension of bacteria in distilled water (the first three bacteria are 10⁶Cells / mL, and the last one is 10⁷Cells / mL) were sprayed onto a hexyl-PVP-modified glass surface, air-dried for 2 minutes, incubated on 0.7% agar in bacterial growth medium overnight, and colonies counted. Commercial NH_Two-The number of viable cells obtained in the same manner on glass slides was used as standard (ie 0% of the bacteria were killed).
[0162]
Example 7
The antimicrobial properties of a PVP-based polycation immobilized on a glass slide were alkylated with 1,4-dibromobutane to introduce a reactive bromobutyl group, which was then used to attach PVP. NH_Two-Analyzed by various methods using glass slides. The surface thus obtained was unable to kill S. aureus cells after spraying. These chains were further N-alkylated with hexyl bromide to increase the positive charge of the attached PVP chains. Hexyl-PVP-slides sprayed with S. aureus cells, air-dried and cultured (hereinafter referred to as prepared by method B) looked almost identical to those shown in the right part of FIG. NH_Two-94 ± 3% of the attached S. aureus cells were killed compared to glass slides (last column, line 1, Table 1).
[0163]
Suspension of bacteria in distilled water (the first three bacteria are 10⁶Cells / mL, and the last one is 10⁷Cells / mL) were sprayed onto a hexyl-PVP-modified glass surface, air-dried for 2 minutes, incubated on 0.7% agar in bacterial growth medium overnight, and colonies counted. Commercial NH_Two-The number of viable cells obtained in the same manner on glass slides was used as a standard (ie 0% of the bacteria die).
[0164]
Surface derivatization
Example 8
HDPE slides (7.5 × 2.5 cm, Polymer Plastics, Leno, Nevada) were sonicated in isopropyl alcohol for 5 minutes, rinsed with this solvent and dried at 80 ° C. for 30 minutes. The front (oxidized) portion of the flame of a hand-held burner (SurA Instruments, Jena, Germany) was then fanned against the slide surface for 15 seconds and the slide was stored under air overnight. The compressed gas mixture of this burner is 0.6% (v / v) tetramethylsilane mixed propane / butane (7: 3, v / v) (Pyrosir^{( R)}). Thus SiO_TwoThe HDPE slides coated with are then placed in a 20% solution of 3-aminopropyltriethoxysilane (Aldrich Chemical Co., Milwaukee, Wis.) In anhydrous toluene and incubated at room temperature with stirring for 3 hours. And 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). Next, this NH_TwoThe functionalized HDPE slide was treated with a mixture of 10 mL of 1,4-dibromobutane, 0.1 mL of triethylamine, and 90 mL of nitromethane for 5 hours at 60 ° C. with stirring and rinsed with nitromethane. Thereafter, a freshly prepared solution of 9 g of PVP (molecular weight 160,000 g / mol) in 81 mL of nitromethane and 10 mL of 1-bromohexane (all from Aldrich) was added. After 9 hours of stirring at 75 ° C., the slides were rinsed thoroughly with methanol and distilled water and allowed to air dry. Slides made of LDPE (Polymer Plastics), PP, nylon (Type 6/6) (Plastic Materials, Cleveland, Ohio) and PET (Wheaton, Millville, NJ) (all 7.5 x 2.5 cm in size) ) Was derivatized according to the same protocol, except that PET was treated at 60 ° C. instead of 75 ° C. in the last step.
[0165]
Surface analysis
Example 9
SiO_TwoX-ray photoelectron spectra of HDPE slides coated with A. were recorded using Kratos Axis Ultra Instruments (Craitos Analytical, New York, NY) using a 150 W A1 Kα monochromator source.
[0166]
Example 10
Hexyl-PVP-derivatized slides are placed in a 1% distilled aqueous solution of fluorescein (Sigma Chemical Co., St. Louis, Mo.), shaken for 5 minutes, rinsed thoroughly with water, and a 0.25% aqueous solution of cetyltrimethylammonium chloride (Aldrich). Company). After shaking for 5 minutes, 10% (v / v) 0.1 M aqueous phosphate buffer, pH 8.0, was added to this solution, the absorbance was measured at 501 nm, and the number of pyridinium groups on the surface was described first. Calculated as done. 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).
[0167]
Determination of antibacterial efficacy
Example 11
S. aureus (ATCC, strain 33807, AT, Manassas, VA) or E. coli (strain ZK650, provided by Dr. Gary Bonner of Harvard Medical School) in 0.1 M aqueous phosphate buffer, pH 7.0 (approximately 10¹¹ Cells / mL) were added to 50 mL of yeast-dextrose broth (prepared as described by Cunliffe et al.) In a sterile Erlenmeyer flask to culture the bacteria. Cunliffe, D., Smart, C.A., Alexander, C. & Vulfson, E.N. Bacterial adhesion at synthetic surfaces. Appl. Environ. Microbiol. L65, 4995-5002 (1999). The suspension was incubated at 37 ° C. for 6-8 hours with shaking at 200 rpm.
[0168]
The surface was tested for its ability to kill airborne bacteria as previously described. 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 are centrifuged at 2,700 rpm for 10 minutes, rinsed with distilled water, and for S. aureus, the concentration is 10 mL / mL.⁶Be a cell, and 10 for E. coli^FiveWas resuspended. The bacterial cell concentration is 1.0 at 540 nm with an optical density of about 10 per mL.⁹Scored assuming equal to individual cells. 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). The bacterial suspension was then sprayed onto the polymer slide in a fume hood using a commercially available chromatographic nebulizer (VWR, Boston, MA) at a spray rate of about 10 mL / min. After drying under air for 2 minutes, the slides were placed in a Petri dish and growth agar (0.7% agar with yeast-dextrose broth, autoclaved and cooled to 37 ° C.) was added. The dish was covered, sealed, and incubated at 37 ° C. overnight. Growing bacterial colonies were counted in a lightbox (Picker International, Cleveland, Ohio) that amplifies the contrast between the colonies and the polymer slides. In the case of nylon, bacterial colonies were stained after incubation. 1 mg of Crystal Violet (Sigma) was dissolved in 100 mL of distilled water and added to growth agar covering infected slides until the surface was completely painted. This procedure was repeated after 2 hours. Colored colonies became visible within 5 hours.
[0169]
When testing the polymer for aquatic bacteria, bacterial cells are centrifuged at 2,700 rpm for 10 minutes, washed twice with PBS at pH 7.0, resuspended in the same buffer, and 2 × 10 5 per mL of PBS.⁶Cells (4 x 10 for E. coli)⁶). Plastic slides were placed in 45 mL of this suspension, incubated for 2 hours with shaking at 200 rpm and 37 ° C., washed three times by immersing the slides in sterile PBS, and immediately set on solid growth agar (bacteria 1.5% agar in growth medium was autoclaved, poured into a Petri dish, dried overnight at room temperature under reduced pressure, covered with a slab cut out, and incubated overnight at 37 ° C in a sealed Petri dish. did. The bacterial colonies were counted as described above.
[0170]
Example 12
Polymer surface derivatized with poly (vinyl-N-hexylpyridinium) kills airborne, waterborne and attached bacteria
A simple method has been developed for covalently derivatizing the surface of commercially available materials with the designed antimicrobial polycation, poly (vinyl-N-pyridinium bromide). In this method, the first key step involves coating the surface with a nanolayer of silica. Many commercially available synthetic polymers derivatized in this manner become bactericidal. They kill up to 99% of contacted Gram-positive and Gram-negative bacteria, whether attached in the air or in water.
[0171]
We have discovered a novel non-releasing strategy to create a germicidal surface that involves covalent coating with long hydrophobic polycation chains. Importantly, the latter long-chain hydrophobic polycation chains must be fine-tuned to a charge and hydrophobicity that counteracts hydrophobic interchain coagulation by electrostatic repulsion and that can penetrate bacterial cell membranes. It must be. As a result, glass slides covalently modified with poly (vinyl-N-hexylpyridinium bromide) (hexyl-PVP) were found to kill more than 99% of airborne bacteria. In this example, we demonstrate a greatly expanded range of such germicidal materials, including conventional synthetic polymers. In doing so, a general surface coating / derivatization method was discovered that would be applicable to most materials, regardless of their nature. Specifically, five representative naturally non-bacterial commercial polymers derivatized with hexyl-PVP produce 90-99% of Gram-positive and Gram-negative bacteria attached to surfaces through air or water. Has been proven to sterilize.
[0172]
material
Fluorescein (Na salt) and crystal violet were purchased from Sigma Chemical Company. 3-Aminopropyltriethoxysilane, 1-bromohexane, cetyltrimethylammonium chloride, poly (4-vinylpyridine) (PVP) (molecular weight 160 kg / mol), and all other chemicals used in this study ( Analytical and pure) 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 are available from Polymer Plastics, Inc. (Reno, Nevada), and poly (ethylene terephthalate) (PET) Courtesy of Wheaton (Millville, NJ). A mixture of 0.6% (v / v) tetramethylsilane in propane / butane (7: 3, v / v) (Pyrosil^{( R)}) Was obtained from SurA Instruments (Jeva, Germany).
[0173]
Surface derivatization
HDPE slides (7.5 × 2.5 cm) were sonicated in isopropyl alcohol for 5 minutes, rinsed with this solvent and dried at 80 ° C. for 30 minutes. Next, the front (oxidized) portion of the flame of the hand-held burner was fanned against the slide surface for 15 seconds, and the slide was stored under air overnight. Next, the SiO_TwoPlace the HDPE slides coated with in a 20% solution of 3-aminopropyltriethoxysilane in anhydrous toluene, incubate with stirring for 3 hours at room temperature, rinse with toluene and methanol, and remove Dried overnight (Bisse et al., 1982). Next, this NH_Two-A functionalized HDPE slide was treated with a mixture of 10 mL of 1,4-dibromobutane, 0.1 mL of triethylamine, and 90 mL of nitromethane at 60 ° C. for 5 hours with stirring, rinsed with nitromethane, and then prepared. A solution of just 9 g of PVP in 81 mL of nitromethane and 10 mL of 1-bromohexane was added. After stirring at 75 ° C. for 9 hours, slides were rinsed thoroughly with methanol and distilled water and dried under air. LDPE, PP, nylon, and PET slides (all 7.5 × 2.5 cm) were derivatized according to the same protocol, except that PET was treated at 60 ° C. instead of 75 ° C. in the last step. .
[0174]
X-ray electron spectroscopy
SiO_TwoThe X-ray photoelectric spectra of HDPE slides coated with were recorded using Kratos Axis Ultra Instruments (Craitos Analytical, New York, NY) using a 150 W A1 Kα monochromator source.
[0175]
Titration of pyridinium groups on the surface
Hexyl-PVP-derivatized slides were placed in a 1% distilled aqueous solution of fluorescein, shaken for 5 minutes, rinsed thoroughly with water, and placed in a 0.25% aqueous solution of cetyltrimethylammonium chloride. After shaking for 5 minutes, 10% (v / v) 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 described first. Calculated as it is.
[0176]
Determination of antibacterial efficacy
S. aureus (ATCC, strain 33807, Manassas, VA) or E. coli (strain ZK650, provided by Dr. Gary Bonner, Harvard Medical School) was added to 0.1 M aqueous PBS buffer, pH 7.0 (approximately 10¹¹Cells (in cells / mL) and 50 μL of yeast-dextrose broth (prepared as described by Cunliffe et al., 1999) in a sterile Erlenmeyer flask. Was cultured. The suspension was shaken at 200 rpm and 37 ° C. for 6-8 hours.
[0177]
The ability of the surface to kill airborne bacteria was tested as previously described (Tiller et al., 2001). Bacterial cells are centrifuged at 2,700 rpm for 10 minutes, washed with distilled water, and S. aureus at 10⁶Individual cell concentration and for E. coli 10^FiveResuspended into individual pieces. The bacterial cell concentration is 1.0 at an optical density at 540 nm of about 10 per mL.⁹Was evaluated assuming equal to individual cells (Hogt et al., 1986). The bacterial suspension was then sprayed onto the polymer slides in a fume hood using a commercially available chromatographic nebulizer (VWR, Boston, Mass.), At a spray rate of about 10 mL / min. After drying for 2 minutes under air, the slides were placed in a Petri dish and growth agar (0.7% agar in yeast-dextrose broth, autoclaved and cooled to 37 ° C.) was added. The dish was covered, sealed, and incubated at 37 ° C. overnight. Growing bacterial colonies were counted in a light box (Picker International, Cleveland, Ohio) that amplifies the contrast between the colonies and the polymer slides. In the case of nylon, bacterial colonies were stained after incubation. 1 mg of crystal violet was dissolved in 100 mL of distilled water and added to growth agar over infected slides until the surface was completely painted. This procedure was repeated after 2 hours. Colored colonies became visible within 5 hours.
[0178]
When testing the polymer for aquatic bacteria, bacterial cells are centrifuged at 2,700 rpm for 10 minutes, washed twice with PBS at pH 7.0, resuspended in the same buffer, and 2 × 10 5 per mL of PBS.⁶Cells (4 x 10 for E. coli)⁶). Plastic slides were placed in 45 mL of this suspension, shaken at 200 rpm and 37 ° C. for 2 hours, washed three times by immersing the slides in sterile PBS, and immediately stiffened on solid agar (with bacterial growth medium). 1.5% agar was autoclaved, poured into a Petri dish, dried overnight at room temperature under reduced pressure, covered with slabs, and incubated overnight at 37 ° C. in a sealed Petri dish. The bacterial colonies were counted as described above.
[0179]
Results and discussion
Because it would be desirable to render a large number of diverse objects bactericidal, we selected a surface derivatization method that could be applied to any material. We have validated this method with common commercial synthetic polymers that exhibit no antimicrobial activity by themselves, such as polyolefins, polyamides, and polyesters. Slides made of high density polyethylene (HDPE) were selected as the first target. This polymer, like many other substances, lacks reactive groups suitable for simple chemical modification. We therefore decided to coat it with an ultra-thin silica layer by the combustion chemical vapor deposition technique (Schinkinger et al., 2001). We used a compressed mixture of 0.6% tetramethylsilane and 7: 3 propane-butane (Pyrosil) for this purpose (step 1 in FIG. 4).^(R)) Containing a pen-like torch. When this mixture burns in the air, the tetramethylsilane oxidizes, resulting in a 2- to 5-nm SiO coating the flamed surface._TwoParticles are formed (Tiller et al., 1989). The resulting dense ~ 100 nm thick SiO chemically similar to glass (polysiloxane)_TwoThe layers (Schinkinger et al., 2001; Tiller et al., 1989) can then be easily chemically modified in a uniform manner regardless of the nature of the bulk material (Tiller et al., 1989 and 1991).
[0180]
The visual appearance of the HDPE surface is this SiO_TwoIt did not change after the coating method. Such SiO_TwoThe degree of cross-linking of the layer is lower than that of glass, and numerous Si-OH groups are formed in the hydration of moisture 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 highly hydrophobic unmodified polymer. To quantify this difference, we measured the contact angle of a water droplet on a surface, a common method of determining the water affinity of a substance (David, 1966). 0.1 mL of distilled water was placed on the HDPE surface for this purpose. The angle between the center of the resulting drop and the surface below it was estimated to be around 120 degrees, indicating a hydrophobic substance. Add the same amount of distilled water to SiO_TwoWhen placed on HDPE coated with, water spread on the surface and the contact angle was less than 10 degrees, reflecting the hydrophilicity of the surface. Separately, SiO on HDPE slides_TwoThe presence of the layer was confirmed by a silicon signal and an oxygen signal detected by X-ray electron spectroscopy.
[0181]
Next, the standard method for aminating the glass surface, SiO_TwoThe amino groups were introduced to the surface of the HDPE slide coated with, by reacting the surface with 3-aminopropyltriethoxysilane (step 2 in FIG. 4) (Bisse et al., 1982). Then NH_TwoThe functionalized HDPE slide was alkylated with 1,4-dibromobutane to introduce a bromoalkyl group (step 3 in FIG. 4), which was then added to PVP with 1-bromohexane (step 4 in FIG. 4). Was reacted in the presence of Under the conditions used, very few of the pyridine groups (approximately 1,500) of the PVP chain are alkylated by bromoalkyl attached to the surface, and most of them are alkylated by 1-bromohexane (Tiller et al., 2001). The number of pyridinium groups on HDPE slides derivatized with hexyl-PVP was 8.2 ± 1.9 nmol / cm when titrated spectrophotometrically with fluorescein.^TwoNH modified with hexyl-PVP_TwoSimilar to that observed in glass (Tiller et al., 2001).
[0182]
By modifying the other two industrial polyolefins, low density polyethylene (LDPE) and polypropylene (PP), and polyamide nylon and polyester poly (ethylene terephthalate) (PET), the universal nanocoating / derivatization method described above Was confirmed. All of these synthetic polymers were successfully derivatized, as shown in FIG. 4, with surface densities of pyridinium groups similar to those of HDPE, -8.5 ± 1.8 (LDPE), 7.2 ± 2.1 (PP), 8.0 ± 1.8. 1.1 (nylon) and 7.5 ± 0.9 (PET) nmol / cm^Twoshowed that. Next, all hexyl-PVP-derivatized slides were tested for their ability to kill contacted airborne bacteria, starting with HDPE.
[0183]
To simulate the natural adhesion of airborne bacteria and the adherence of bacterial contacts, a suspension of the ubiquitous pathogen Gram-positive bacterium Staphylococcus aureus in distilled water (Xiong et al., 2000) was used. The latter was dried by spraying on the slide surface. The slides were then incubated overnight on nutrient agar as described previously (Tiller et al., 2001) and the number of viable bacterial cells determined by colony counting. As seen on the left side of FIG. 5a, a number of easily identifiable bacterial colonies grew on unmodified HDPE slides. SiO_TwoCoated with or NH_TwoThe number of colonies grown on HDPE slides functionalized with (FIG. 4) was essentially the same, indicating that these modifications were not toxic to S. aureus. Surprisingly in contrast, 96 ± 3% of the attached bacterial cells were killed or non-viable when sprayed onto hexyl-PVP-modified HDPE slides (FIG. 5a, right side and Table 1).
[0184]
[Table 5]
[0185]
Bacterial distilled water suspension (S. aureus 10⁶Cells / mL, and 10 for E. coli^FiveCells / mL) were sprayed onto hexyl-PVP-modified polymer slides, the latter air-dried for 2 minutes and incubated overnight in 0.7% agar with bacterial growth medium. Thereafter, colonies were counted. The number of viable cells obtained in the same manner on the corresponding unmodified polymer slide was used as a standard (ie 0% of the bacteria were killed). All experiments were performed at least in duplicate and the errors indicated indicate standard deviations.
[0186]
Hexyl-PVP-derivatized LDPE, PP, nylon, and PET were similarly tested for their ability to kill S. aureus, an airborne bacterium. As seen in Table 1, in each case, the number of bacterial colonies formed was reduced by a factor of 10 to 30 compared to that grown on the corresponding unmodified slide.
[0187]
Next, we tested the bactericidal efficiency of the hexyl-PVP-derivatized surface against Escherichia coli, a representative gram-negative bacterium. As seen on FIG. 5b, left, many E. coli colonies grown on HDPE slides after spraying were less dense, but still discernable, than those of S. aureus grown under the same conditions. is there. Surface derivatization with hexyl-PVP reduced these numbers by 97 ± 3% (FIG. 5b, right side, and Table 1). Examination of the remaining data in the last row of Table 1 shows that LDPE, PP, Nylon, and PET slides, when modified with hexyl-PVP, also have a 20 to 50 when compared to the corresponding unmodified slides. It can be seen that a reduction in viable E. coli cells by a factor of one is seen after their attachment from airborne state.
[0188]
To determine if hexyl-PVP chains immobilized using our method can leach out of the slide surface (and perhaps only by doing so, kill attached bacteria), modify the modified HDPE slides into polystyrene Petri dishes. And an aqueous suspension of S. aureus cells was sprayed thereon or onto a (non-antibacterial) polystyrene surface. Very little (1 cm) on hexyl-PVP-modified HDPE slides when incubated on air dried and growth agar^TwoPer colony was growing, while a much larger number (1 cm)^Two85 ± 5) had grown on the surface of the surrounding petri dish, even to the immediate vicinity of the slide. The absence of a stop zone (Kawabata and Nishiguchi, 1988), which is characteristic of the release of bactericidal substances, is not present around the hexyl-PVP-slide, which means that immobilized polycations do not leach out of the slide, i.e. It suggests that it is sterilized when it comes into contact with the slide surface.
[0189]
Another important question is whether hexyl-PVP-derivatized slides can also kill waterborne bacteria. To simulate the flowing aqueous solution experimentally, a polymer slide was placed vertically in a suspension of bacterial PBS, pH 7.0, and the resulting system was gently stirred at 37 ° C. After a 2 hour incubation, the slides were washed with sterile buffer to remove non-adherent bacteria, and then incubated in sterile PBS for 1 hour under the conditions outlined above to test adherent bacteria. Irreversibly detached or attached (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 ° C. By this method, all truly attached bacterial cells have been captured on the slide surface, and those that are still viable can proliferate and form colonies.
[0190]
As seen in FIG. 6a, left, S. aureus cells attached to HDPE slides form a large number of easily distinguishable colonies. SiO_Two-Or NH_TwoNone of the-modified HDPE slides showed a significant decrease in the number of bacterial colonies growing after attachment. However, as shown in FIG. 6a, right, the number of S. aureus colonies growing on HDPE slides modified with hexyl-PVP was reduced by 98 ± 1% (see also Table 2).
[0191]
[Table 6]
[0192]
Suspension of bacteria in PBS, pH 7.0 (2 x 10 for S. aureus⁶Cells / mL, and 4 × 10 for E. coli⁶Cells / mL) were allowed to adhere to the hexyl-PVP modified polymer surface with shaking at 37 ° C. for 2 hours. The slides were rinsed with sterile PBS, pH 7.0, which was shaken at 37 ° C. for 1 hour, rinsed again, incubated overnight in 1.5% agar with bacterial growth medium, and colonies counted. N.d. represents "not determined" because the attached bacterial cells grow but do not form discernable colonies on control slides. See the footnote in Table 1 for other conditions.
[0193]
Furthermore, it was demonstrated that LDPE, PP, nylon, and PET derivatized with hexyl-PVP can kill contacted aqueous bacteria. The data in Table 2 show that derivatization (FIG. 4) reduces the number of viable S. aureus cells attached by 97-99% for all polymer slides. Similarly, the modified polymer slides have been found to be effective for aqueous E. coli. As seen in FIG. 6b, left, E. coli cells attached to unmodified HDPE slides from the aqueous suspension grow a large number of identifiable colonies under the cover of growth agar. Colonies on LDPE, nylon, and PET were similar, but there were no discernable colonies on pp. When HEPE is derivatized with hexyl-PVP, the number of viable E. coli cells attached drops sharply by 97 ± 2% (FIG. 6b, right side, and Table 2). The other modified polymer also dramatically reduces the number of viable E. coli cells attached, as compared to the corresponding unmodified slide, and kills 98-99% for LDPE, PET, and nylon. Efficiency was observed (Table 2, last row).
[0194]
Since the experimental conditions to determine the bactericidal properties of hexyl-PVP-modified slides against aquatic bacteria were different from those against airborne bacteria, we again tested the release of polycations from the slides into the medium. For this purpose, hexyl-PVP modified HDPE slides were incubated for 2 hours in sterile PBS at 37 ° C. with shaking, removed, and S. aureus cells were added to the remaining solution. The resulting suspension was again shaken for 2 hours at 37 ° C. and the number of viable bacterial cells was determined by growing it on growth agar and incubating at 37 ° C. overnight. (42 ± 4) × 10 per mL^FourThe number of bacterial colonies observed was the control ([48 ± 7) × 10^Four]. Therefore, during bacterial attachment experiments, little release of antimicrobial was observed from the hexyl-PVP modified HDPE slides, and the aqueous bacteria would have been killed when they actually came into contact with the derivatized surface.
[0195]
The bactericidal mechanism of the polycation chain attached to the surface probably involves a step in which it penetrates the bacterial membrane and causes cell damage and death (Tiller et al., 2001). To add a quantitative aspect to this model, HDPE slides were derivatized with hexyl-PVP at various surface densities of polycations and measured for antimicrobial efficacy against airborne or aqueous S. aureus cells as described above. did. As can be seen in FIG. 7, the surface pyridinium group density was 1 cm for airborne bacteria.^TwoAbout 3 nanomoles per liter, and 1 cm for aqueous ones^TwoOnly when it exceeds 6 nanomoles per bactericidal effect appears and there is no subsequent increase. The difference between these two curves is probably due to the fact that the sprayed bacterial cells can detach from the surface, as opposed to those in solution, and are therefore killed even at lower pyridinium group densities. Would. These observations indicate that the bactericidal capacity of a material is critically dependent on the density of its surface coverage by polycations.
[0196]
Finally, we have developed a general method for derivatizing surfaces with the antimicrobial polymer hexyl-PVP, which can be applied to ordinary materials. Hexyl-PVP modified surfaces of the synthetic polymers HDPE, LDPE, PP, nylon, and PET kill up to 99% of contact-attached S. aureus and E. coli cells. This method should be useful in rendering many products sterilizable, both dry and wet. Regular cleaning could easily restore the surface.
[0197]
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alkanethiol self-assembled monolayers.J Bacteriol 177: 1959-1966.
Xiong Y,
Yeaman MR, Bayer AS. 2000.Linezolid: A new antibiotic.Drugs Today 36: 529-539.
[0198]
Example 13
Consideration of bactericidal action of poly (vinyl-N-hexylpyridinium) chain attached to surface
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 wild-type (both airborne and waterborne) and antibiotic-resistant strains of the human pathogenic Staphylococcus aureus. The materials produced are also lethal to strains expressing multidrug resistance pumps, the only known mechanism of resistance to cationic preservatives.
[0199]
The ability to make naturally sterile materials that can be used in everyday products would be a major step forward for a healthier life. Recently we (Tiller et al. 2001 and 2002) found that when certain long-chain hydrophobic polycations were immobilized on glass or plastic surfaces, the latter acquired the ability to efficiently kill contacted bacteria. . Specifically, the various surfaces covalently coated with poly (vinyl-N-hexylpyridinium) (hexyl-PVP) adhered via aerosol (and then dried) or from aqueous solution Regardless, most bacteria, both gram positive and gram negative, were found to kill.
[0200]
In this study we further explored this potentially useful phenomenon. Specifically, polyethylene surfaces derivatized with hexyl-PVP should be equally lethal to wild-type and mutant ubiquitous pathogenic bacteria, Staphylococcus aureus, including those resistant to antibiotics. Was confirmed. Dead cells can be easily removed and the surface can be completely recovered by washing with detergent. S. aureus cells recovered in logarithmic growth phase, as well as in stationary phase, are susceptible to this bactericidal action.
[0201]
material
Table 3 lists the S. aureus strains used in this study. 0.6% (v / v) Tetramethylsilane in propane / butane mixture (7: 3, v / v) (Pyrosil^{( R )}) Hand held burners were obtained from SurA Instruments (Jena, Germany). High density polyethylene sheets were purchased from Polymer Plastics. Poly (4-vinylpyridine) (PVP) (MW 160,000 g / mol), 3-aminopropyltriethoxysilane, 1,4-dibromobutane, 1-bromohexane, and all other chemicals and solvents Obtained from Chemical Company and used without further purification.
[0202]
[Table 7]
[0203]
Surface modification
Polyethylene slides (7.5 × 2.5 cm) were sonicated in isopropyl alcohol for 5 minutes and dried at 80 ° C. The front (oxidized) portion of the flame of the hand-held burner was rubbed against the slide surface for 15 seconds and the slide was stored under air overnight. SiO_TwoThe slides coated with were aminated with a 20% solution of 3-aminopropyltriethoxysilane in anhydrous toluene at room temperature for 3 hours. After immersing the aminated slide in a solution containing 9 mL of 1,4-dibromobutane, 90 mL of anhydrous nitromethane, and 0.1 mL of triethylamine at 60 ° C. for 5 hours with stirring, it was added to 9 g of the slide. Placed in a freshly prepared solution of PVP, 10 mL of 1-bromohexane, and 81 mL of nitromethane. After stirring at 75 ° C. for 9 hours, slides were rinsed thoroughly with methanol and distilled water and dried under air.
[0204]
Determination of antibacterial efficiency
Bacteria were grown in yeast-dextrose broth (Cunliffe et al. 1999) for 6-8 hours at 37 ° C with aeration at 200 rpm. The inoculum from the overnight culture was transferred to 0.1 M PBS (approximately 10¹¹(Cells / mL), and then introduced into the growth medium at a dilution of 1: 500.
[0205]
Airborne bacterial suspensions were prepared as previously described (Tiller et al. 2001). The bacterial cells were centrifuged at 5,160 xg for 10 minutes and washed twice with distilled water. Concentration in distilled water 10⁶Cells / mL of the bacterial suspension were sprayed on the surface of the slide at a rate of approximately 10 mL / min in a fume hood. After drying under air for 2 minutes, the slides were placed in a Petri dish and growth agar (0.7% agar in yeast-dextrose broth, autoclaved and cooled to 37 ° C.) was added. The Petri dish was sealed and incubated at 37 ° C. overnight. The growing bacterial colonies were counted in a light box.
[0206]
A suspension of aqueous bacteria was prepared as follows: bacterial cells were centrifuged at 5,160 xg for 10 minutes, washed twice with PBS pH 7.0, resuspended in the same buffer, 2 × 10⁶Diluted to cells / mL. Slides were immersed in 45 mL of the suspension and incubated for 2 hours at 37 ° C. with shaking at 200 rpm, then rinsed three times with sterile PBS, which was incubated for 1 hour. The slides were immediately covered with solid growth agar (1.5% agar in yeast-dextrose broth, autoclaved, poured into petri dishes and dried under vacuum overnight at room temperature). Thereafter, bacterial colonies were counted.
[0207]
Synthesis of monomers and polymers containing pyridinium
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 ° C. for 9 hours. Then the solvent was removed under reduced pressure. The N-hexylpyridinium bromide thus obtained was washed four times with hexane and dried under vacuum overnight.
[0208]
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 ° C. for 9 hours. After cooling to room temperature, the solvent was removed and 100 mL of toluene was added to the residue. Toluene-insoluble poly (4-vinyl-N-methylpyridinium iodide) was collected by filtration, washed with toluene and acetone, and dried.
[0209]
Results & Discussion
We have answered several questions regarding the mechanism and utility of bactericidal action on surfaces derivatized with poly (vinyl-N-alkylpyridinium) chains (Tiller et al. 2001 and 2002). For example, a 7.5 × 2.5 cm slide cut from a large sheet of commercially available high density polyethylene was coated with a nanolayer of silica and then coated with 160,000-g / mol hexyl- as described previously (Tiller et al. 2002). PVP was covalently attached. The antimicrobial activity of the prepared slides was tested for common pathogenic S. aureus in two different characteristics (referred to as "airborne" and "aqueous"). For airborne bacteria, slides were dried with an aqueous suspension of bacterial cells, dried, plated on growth agar, incubated at 37 ° C, and counted for bacterial colonies. For aqueous systems, slides were immersed in an aqueous suspension of bacterial cells, where they were incubated at 37 ° C, washed, covered with solid growth agar, and incubated again at 37 ° C before counting the number of bacterial colonies.
[0210]
When wild-type S. aureus cells were attached to the surface of unmodified high-density polyethylene slides by the method of airborne bacteria and cultured as described above, then 308 ± 16 colonies 3.75 cm^TwoWas detected in the front part of. The same experiment was performed on silica-coated slides and also with amination, and the corresponding numbers of bacterial colonies were 339 ± 2 and 228 ± 2, respectively. All three types of bacterial cells attached to different surfaces survived at a high rate. In contrast, applying the same procedure to slides derivatized with hexyl-PVP, only 14 ± 1 bacterial colonies were found at 3.75 cm^TwoWas observed in the front part, ie 5 ± 1% compared to unmodified slides. Similarly, for aqueous S. aureus cells, only 4 ± 2% of the colonies were counted on the hexyl-PVP-fixed surface compared to the original polyethylene slide. These germicidal efficiencies of 95% + are the same as those previously discovered for hexyl-PVP modified surfaces (Tiller et al. 2001 and 2002).
[0211]
Next, we addressed the question of the durability of hexyl-PVP surface protection described above. For this purpose, after counting colonies grown from living S. aureus cells, the slides derivatized with hexyl-PVP were washed with 0.1 M aqueous cetyltrimethylammonium chloride and then rinsed with distilled water. Was completely removed. The slides thus washed were reused for attachment to either airborne or aqueous S. aureus. The percentage of colonies formed was 4 ± 1% and 2 ± 1%, respectively, when compared to that of unmodified polyethylene slides that were washed identically. Thus, washing with detergent has no effect on the bactericidal efficacy of the immobilized hexyl-PVP.
[0212]
In our previous studies both here and elsewhere (Tiller et al. 2001 and 2002), the bacterial cells used in the experiments were all in the stationary phase of their growth curves. We were interested in establishing whether cells in the log phase of growth were equally susceptible to the antibacterial effects of hexyl-PVP chains attached to the surface. Thus, we fermented S. aureus and observed cell concentration as a function of time. The resulting data was a classic sigmoidal curve (Ingraham et al. 1983), which under our conditions reached a stationary phase completely after 6 hours (see Methods). Instead of recovering the bacterial cells after 6-8 hours as before, we recovered just after 4 hours, ie during their exponential growth phase. When these airborne and waterborne cells were attached to slides derivatized with hexyl-PVP, the resulting bacterial efficiency (when compared to the same cells on unmodified polyethylene slides) was 5 ± 2% and 2 ± 1%, the same values observed for cells in the stationary phase (see above).
[0213]
Multidrug resistant (MDR) bacterial strains are a major threat to human health (Levy 1998, Lewis et al. 2001). With this in mind, we tested whether immobilized hexyl-PVP was also effective against such strains. For this purpose, in addition to the wild-type strain of S. aureus previously used (ATCC 33807), we have three separate 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 (penicillin, tetracycline, imipenem, cefaclor, oxacillin, tobramycin, cephalexin, cefuroxime, gentamicin, amoxicillin, clindamycin, erythromycin, (Resistance to cefamandole) was examined. As seen in FIG. 8, polyethylene slides coated with hexyl-PVP are similarly lethal to these bacterial strains, regardless of airborne or waterborne bacteria, and their bactericidal efficiencies are in each case reduced. Well over 90%. The immobilized hexyl-PVP chains penetrate the bacterial cell wall / membrane and presumably exert their bactericidal effect by causing autolysis in this way (Tiller et al. 2001).
[0214]
Like all other bacterial studies (Lewis 1994, Lewis et al. 2001), S. aureus has several MDR pumps that excrete a variety of toxic compounds from cells. For example, the NorA MDR pump protects cells from numerous amphiphilic cations, including the common fungicide benzalkonium chloride (Ng et al. 1994). Thus, mutants of S. aureus knocking out the nor A gene encoding the MDR pump are substantially more sensitive to such compounds (Hsieh et al. 1998). Because hexyl-PVP, like benzalkonium, is a hydrophobic quaternary ammonium cation, we decided to test the sensitivity of the norA mutant to immobilized hexyl-PVP. The result of this experiment is shown in FIG. 9A. The bactericidal efficiencies for the pumpless mutants were 99% ± 1% for airborne and waterborne organisms, somewhat higher than for their pump-competent parents-95 ± 2% and 97 ± 1%, respectively. It can be seen that
[0215]
An additional MDR pump (QacA) expressed from a natural transmissible plasmid⁺We performed a similar experiment with the S. aureus strain, which has (as designated as) compared to its parent. This strain is slightly more resistant to immobilized hexyl-PVP than its pump-deficient parent (FIG. 2B) and has 92 ± 3 of its parent in airborne and waterborne mutants, respectively. % And 94 ± 3%, respectively.
[0216]
Such very small differences in the sensitivity to hexyl-PVP between strains deficient / overexpressing MDR indicate that the preservative, which is in polymeric form, is not effectively pumped out by the pump Suggest that. Alternatively, N-hexylpyridinium may not be a substrate for MDR. To determine these possibilities, cells were treated with an aqueous solution of N-hexylpyridinium bromide to determine the minimum inhibitory concentration (MIC). This compound was found to be a very weak antimicrobial, with a MIC of 4 mg / mL against wild-type S. aureus (Table 4). This value exceeds 1,000 times that of the conventional preservative benzalkonium chloride (Table 4). The sensitivities of the strains to N-hexylpyridinium differed depending on their MDR status. Thus, the MIC of the norA strain is 0.5 mg / mL and QacA⁺That of the strain was above 4 mg / mL. This is quantitatively similar to the difference in sensitivity of these strains to benzalkonium chloride, indicating that N-hexyl-pyridinium is a weak antibacterial but a reasonable substrate for MDR. Is shown. When water-soluble poly (vinyl-N-methylpyridinium iodide) was tested instead (hexyl-PVP itself, which was previously used in its immobilized form in our studies, is not water-soluble and therefore used in MIC studies) Note that this was not possible), and the MIC value was found to be 75 μg / mL, much lower than that of the monomeric precursor N-hexylpyridinium bromide. There was no difference in the MIC value of this compound between the strains tested. Thus, polymerizing a preservative appears to have two consequences on its properties: increasing its potency and making it insensitive to the action of the MDR pump. is there.
[0217]
[Table 8]
[0218]
The development of resistance is a major concern when selling new antimicrobial agents. Excretion by MDR is the only known mechanism of resistance to hydrophobic cationic preservatives (Lewis 2001, Severina et al. 2002). Our findings indicate that resistance to hexyl-PVP attached to a surface is unlikely to occur through such a mechanism.
[0219]
References
Cunliffe D,
Smart CA, Alexander C, Vulfson EN (1999) Bacterial adhesion at synthetic
surfaces. Appl. Environ. Microbiol.
65: 4995-5002.
De Lencastre
H (2000) Archaic strains of methicillin-resistant Staphylococcus aureus:
molecular and microbiological properties of isolates from 1960s in Denmark.
Microb. Drug Resist. 6: 1-10.
Hiramatsu K
(1997) Dissemination in Japanese hospitals of strains of Staphylococcus aureus
heterogeneously resistant to vancomycin. Lancet 350: 1670-1673.
Hsieh P,
Siegel SA, Rogers B, Davis D, Lewis K
(1998) Bacteria lacking a multidrug pump: a sensitive tool for drug
discovery. Proc. Natl. Acad. Sci.
USA 95: 6602-6606.
Ingraham JL,
Maaloe O, Neidhardt FC (1983)
Growth of the Bacterial Cell, Chapter 5.
Sunderland (MA): Sinaner.
Kaatz GW, Seo
SM, Foste TJ (1999) Introduction of a norA promoter region mutation into the
chromosome of a fluoroquinolone-susceptible strain of Staphylococcus aureus
using plasmid integration. Antimicrob. Agents Chemother. 43: 2222-2224.
Kluytmans J,
van Belkum A, Verbrugh H (1997)
Nasal carriage of Staphylococcus aureus: epidemology, underlying mechanisms,
and associated risks. Clin. Microbiol. Rev. 10: 505-520.
Levy SB
(1998) The challenge of antibiotic resistance.Science 278: 46-53.
Lewis K
(2001) In search of natural substrates and inhibitors of MDR pumps.J. Mol.
Microbiol. Biotechnol. 3: 247-254.
Lewis K,
Lomovskaya O (2001) Drug efflux.In Bacterial Resistance to
Antimicrobials: Mechanisms,
Genetics, Medical Practice and Public Health.Lewis K, Salyers A, Taber H, Wax
R, eds. (2001) New York:
Marcel Dekker.
Lewis K
(1994) Multidrug resistance pumps in bacteria: variations on a theme.
Biochem. Sci. 19: 119-123.
Lewis K,
Salyers A, Taber H, Wax R, eds. (2001) Bacterial Resistance to
Antimicrobials: Mechanisms,
Genetics, Medical Practice and Public Health.New York: Marcel Dekker.
Ng EY,
Trucksis M, Hooper DC (1994)
Quinolone resistance mediated by norA: physiologic characterization and
relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus
chromosome. Antimicrob. Agents Chemother. 38: 1345-1355.
Rouch DA,
Cram DS, DiBerardino D, Littlejohn TG, Skurray RA (1990) Efflux-mediated
antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry
with tetracycline- and sugar-transport proteins. Mol. Microbiol. 4: 2051-2062.
Severina II,
Muntyan MS, Lewis K, Skulachev VP (2002) Transfer of cationic antibacterial
agents berberine, palmatine and benzalkonium through bimolecular planar
phospholipid film and Staphylococcus aureus membrane.Biochem.Internat. in
press.
Tiller JC,
Lee SB, Lewis K, Klibanov AM (2002)
Polymer surfaces derivatized with poly (vinyl -N-hexyl pyridinium) kill both
air- and water- borne bacteria. Biotechnol. Bioeng. 79: in press.
Tiller JC,
Liao C-J, Lewis K, Klibanov AM
(2001) Designing surfaces that kill bacteria on contact.Proc.Natl.Acad.Sci. USA 98:
5981-5985.
[0220]
Incorporation by citation
The entire text of all publications and patents referred to herein are hereby incorporated by reference in the same manner as specifically and individually indicating that each publication or patent is incorporated by reference. In case of conflict, the present application, including any definitions provided herein, will control.
[0221]
Equivalent
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.
[Brief description of the drawings]
[0222]
FIG. 1: Commercially available NH_Two-Aqueous suspension of Staphylococcus aureus cells (approximately 10 cells / mL of distilled water) on glass slides (left) and hexyl-PVP modified slides (right)⁶Are sprayed, air-dried for 2 minutes, and incubated at 37 ° C. overnight in 0.7% agar in bacterial growth medium.
FIG. 2: Commercial NH of Staphylococcus aureus colonies grown on infected surfaces of glass slides modified with PVP N-alkylated with various linear alkyl bromides_Two-Indicates the ratio to the number of colonies grown on a glass slide.
FIG. 3 shows compounds of the present invention prepared by Method A.
FIG. 4. SiO using combustion chemical vapor deposition_TwoCoating with a nanolayer, treating with 3-aminopropyltriethoxysilane, alkylating with 1,4-dibromobutane, and derivatizing with hexyl-PVP in the presence of 1-bromohexane 3 illustrates the derivatization of a polymer surface with poly (vinyl-N-hexylpyridinium bromide) (hexyl-PVP), comprising four steps:
FIG. 5: Commercially available HDPE slides (left) and hexyl-PVP derivatized slides (right) are loaded with aqueous suspensions of (a) S. aureus or (b) E. coli cells (approximately 10 cells / mL distilled water).⁶Are shown, sprayed, air-dried for 2 minutes, and incubated at 37 ° C. overnight in 0.7% agar in bacterial growth medium.
FIG. 6: Commercially available HDPE slides (left) and hexyl-PVP derivatized slides (right) on (a) Staphylococcus aureus or (b) E. coli cells in aqueous PBS, pH 7.0 suspension (per mL of PBS) About 10 cells⁶Shows a photograph of cells washed with PBS and incubated overnight at 37 ° C in 1.5% agar in bacterial growth medium.
FIG. 7 shows the killing efficiency of hexyl-PVP derivatized HDPE against aerial (filled circles) or water (open circles) Staphylococcus aureus cells deposited on the surface as a function of the surface density of pyridinium groups. Negative percentages correspond to a greater number of bacterial cells attached to the modified slide than on the unmodified slide.
FIG. 8 graphically depicts the bactericidal activity of hexyl-PVP covalently coated polyethylene slides against wild-type Staphylococcus aureus and various antimicrobial resistant strains. Bacterial suspensions, either in air (grey bars) or in water (shaded bars), were deposited on slide surfaces. The standard deviation from the average kill efficiency value is represented by an error bar.
FIG. 9 graphically illustrates the bactericidal activity of polyethylene slides covalently coated with hexyl-PVP against two sets of S. aureus parental / mutant strains. In the first set (A), the mutant lacks the MDR pump encoded by the norA gene (which has been knocked out), while the otherwise identical parent strain has it. In the second set (B), the parent strain lacks the MDR pump encoded by the qacA gene, and the strain having the plasmid has it.

Claims (35)

A composition comprising a substance and a compound fixed to said substance, wherein said fixed compound is a polymer and said polymer is not insoluble in water. 2. The composition of claim 1, wherein said immobilized compound is a polycation. The composition of claim 1, wherein the immobilized compound is a water-soluble lipophilic polycation. 2. The composition of claim 1, wherein said immobilized compound is covalently attached to said solid substance. The composition of claim 1, wherein said immobilized compound comprises poly (N-alkylvinylpyridine) or poly (N-alkylethyleneimine). A composition comprising a surface and a compound covalently bonded to said surface, wherein said compound is represented by Formula I:
(Formula I)
In the formula
R is each independently hydrogen, alkyl, alkenyl, alkynyl, acyl, aryl, carboxylate, alkoxycarbonyl, aryloxycarbonyl, carboxamide, alkylamino, acylamino, alkoxyl, acyloxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino ) Represents alkyl, thio, alkylthio, thioalkyl, (alkylthio) alkyl, carbamoyl, urea, thiourea, sulfonyl, sulfonate, sulfonamide, sulfonylamino, or sulfonyloxy;
R ′ each independently represents an alkyl, an alkylidene tether to the surface, or an acyl tether to the surface,
Z independently represents Cl, Br, or I,
n is an integer of about 1500 or less,
A composition represented by the formula: 7. The composition of claim 6, wherein said tether is an acyl tether. 7. The composition of claim 6, wherein said tether is an alkylidentate. 9. The composition according to claim 8, wherein said alkylidentether is -C4H8-tether. 7. The composition of claim 6, wherein the compound covalently bonded to the surface has a molecular weight of at least 10,000 g / mol. 7. The composition of claim 6, wherein the molecular weight of the compound covalently bonded to the surface is at least 150,000 g / mol. 7. The composition of claim 6, wherein the compound covalently bonded to the surface is poly (4-vinyl-N-alkylpyridinium bromide). 7. The composition of claim 6, wherein the compound covalently bonded to the surface is poly (4-vinyl-N-hexylpyridinium bromide). The composition of claim 6, wherein the surface comprises a layer of silicon oxide. 7. The composition of claim 6, wherein said surface is from the group consisting of low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), nylon, and poly (ethylene terephthalate) (PET). The composition selected. The composition of claim 6, wherein the surface is included in a medical device. 17. The composition of claim 16, wherein the medical device is a pin, screw, plate, ventricular atrial shunt, ventricular peritoneal shunt, dialysis shunt, heart valve, pacemaker, infusion pump, vascular prosthesis, stent, Suture, surgical mesh, replacement prosthesis, breast implant, tissue dilator, contact lens, fistula instrument, artificial larynx, endotracheal tube, tracheal tube, gastrostoma tube, bile drainage tube, biliary stent, catheter, bandage , An adhesive tape, and a transparent plastic adhesive sheet. A method of disinfecting a surface,
A step of coating the surface with SiO _2,
Hydroxylating a portion of the SiO ₂ on the surface to form a plurality of Si-OH groups;
Converting at least a portion, tri (alkoxy) Si-O-(alkyl) more Si-O-(alkyl) NH2 group by treatment with -NH ₂ reagent plurality of Si-OH groups of said on said surface Steps to
1 a portion of a plurality of Si-O-(alkyl) -NH ₂ group of the on the surface, and alkylated with n- dihaloalkane, a plurality of Si-O-(alkyl) -NH- (alkyl) - halogen Generating a halide group;
Treating the plurality of Si-O- (alkyl) -NH- (alkyl) -halide groups with an amine-containing polymer and an alkyl halide to covalently bond the amine-containing polymer to the surface;
Including, methods. 19. The method of claim 18, wherein said surface is a polymer surface. 20. The method of claim 19, wherein the polymer surface is selected from the group consisting of LDPE, HDPE, PP, nylon, and PET. The method of claim 18, wherein the tri (alkoxy) Si-O-(alkyl) -NH ₂ reagent is 3-aminopropyl triethoxysilane, method. 19. The method according to claim 18, wherein said 1, n-dihaloalkane is 1,4-dibromobutane. 19. The method according to claim 18, wherein the amine-containing polymer is poly (4-vinylpyridine). 19. The method according to claim 18, wherein the alkyl halide is 1-bromohexane. The method of claim 18, wherein the surface is LDPE, HDPE, PP, nylon, and is selected from the group consisting of PET, the tri (alkoxy) Si-O-(alkyl) -NH ₂ reagent 3- An aminopropyltriethoxysilane, wherein the 1, n-dihaloalkane is 1,4-dibromobutane, the amine-containing polymer is poly (4-vinylpyridine), and the alkyl halide is 1- The method, which is bromohexane. A method of disinfecting a surface,
A step of coating the surface with SiO _2,
Hydroxylating a portion of the SiO ₂ on the surface to form a plurality of Si-OH groups;
At least a portion of the plurality of Si-OH groups of said on said surface, tri (alkoxy) Si-O-(alkyl) of the plurality by treatment with -NH ₂ reagent Si-O-(alkyl) -NH ₂ group Converting to
Acrylating the Si-O- (alkyl) NH ₂ groups with activated acrylic acid to produce a plurality of Si-O- (alkyl) -NH acrylate groups;
Polymerizing the Si-O- (alkyl) -NH acrylic acid group with an amine-containing monomer to form an amine-containing polymer covalently linked to the surface;
Treating the covalently linked amine-containing polymer with an alkyl halide;
A method that includes 27. The method of claim 26, wherein said surface is a polymer surface. 27. The method according to claim 26, wherein the polymer surface is selected from the group consisting of LDPE, HDPE, PP, nylon, and PET. The method of claim 26, wherein the tri (alkoxy) Si-O-(alkyl) -NH ₂ reagent is 3-aminopropyl triethoxysilane, method. 27. The method of claim 26, wherein said activated acrylic acid is acryloyl chloride. 27. The method according to claim 26, wherein the amine containing monomer is 4-vinylpyridine. 27. The method according to claim 26, wherein the alkyl halide is 1-bromohexane. 27. The method of claim 26, wherein the surface is a polymer surface selected from the group consisting of LDPE, HDPE, PP, nylon, and PET, wherein the tri (alkoxy) Si-O- (alkyl) a -NH ₂ reagent is 3-aminopropyl triethoxysilane, the activated acrylic acid a acryloyl chloride, the amine-containing monomer is a vinylpyridine, wherein the alkyl halide is 1-bromo-hexane Is the way. 27. The method of claim 18 or 26, wherein the surface is bactericidal against Gram-positive bacteria. 27. The method according to claim 18 or 26, wherein the surface is bactericidal against Gram-negative bacteria.