Photochemical coatings for the prevention of bacterial colonization.

Photochemical Coatings for the Prevention of Bacterial Colonization S. G. DUNKIRK*

Department of Biochemistry North Dakota State University Fargo, ND 58105 S. L. GREGG, L. W. DURAN, J. D. MONFILS, J. E. HAAPALA, J. A. MARCY, D. L. CLAPPER, R. A. AMOS AND P. E. GUIRE Bio-Metric Systems, Inc. 9924 W. 74th St. Eden Prairie, MN 55344

ABSTRACT: Biomaterials are being used with increasing frequency for tissue substitution. Implantable, prosthetic devices are instrumental in the saving of patients’ lives and enhancing the quality of life for many others. However, the greatest barrier to expanding the use of biomedical devices is the high probability of bacterial adherence and proliferation, causing very difficult and often untreatable medical-device centered infections. The difficulty in treating such infections results in great danger to the patient, and usually retrieval of the device with considerable pain and suffering. Clearly, development of processes that make biomedical devices resistant to bacterial adherence and colonization would have widespread application in the field of biomedical technology. A photochemical surface modification process is being investigated as a generic means of applying antimicrobial coatings to biomedical devices. The photochemical process results in covalent immobilization of coatings to all classes of medical device polymers. A discussion of the photochemical surface modification process and preliminary results demonstrating the success of photochemical coatings in formulating microbial-resistant surfaces are presented in this paper. *Author to whom correspondence should be addressed. Current address is: of Chemistry, Moorhead State University, Moorhead, Minnesota 56563.

Department 131

Downloaded from jba.sagepub.com at UNIV OF MASSACHUSETTS on April 11, 2015

132 KEY WORDS: antimicrobial coatings, photochemistry, antibiotics, slow-release, antibiotic delivery.

hydrophilic coatings,

INTRODUCTION

_

The Problem-Medical Device-Centered Infections

mplantable medical devices are being used with increasing frequency in modern medical practice [1,2]. A wide variety of devices used for treatment of many diseases. Over 200,000 cardiovascular devices are implanted yearly in the U.S, devices such as vascular grafts and prostheses, arterial reconstructions, valve replacements, left ventricular assist devices and the total artificial heart [2]. Orthopedic implants for replacement or reconstruction of the joints, including the hip and knee, shoulder, wrists, ankles and elbows, result in over 485,000 implant procedures in the U.S., annually [2]. Additionally, intravascular catheters and urinary tract catheters are some of the most frequently used medical devices, with over 20 million patients requiring these devices, during their hospital stays [3,4]. Other biomedical devices include central nervous system shunts, endotracheal tubes, mammary prostheses and other soft tissue replacements, and intraocular lenses [1,2,5]. Devices which are in contact with physiological fluids and tissues, but not usually implanted, include hemodialysis and peritoneal dialysis membranes and tubing, hollow fiber blood oxygenators [6] and contact lenses (over 2 million wearers [2]). Just as varied as the number of biomedical device applications are the numerous materials from which these devices can be and are fabricated; polymeric materials ranging from smooth silicones, polyethylenes and polypropylene materials, to porous polyurethanes and dacron velours [1,2], with some devices being constructed from metals, ceramics and glasses [1,2]. The most frequent and serious complication of all prosthetic devices has been the development of infections [7-9]. are

Does the Environment of the Implanted 8Iedical Device Become Infected?

~Yhy

The presence of an indwelling foreign body increases the likelihood of infection and greatly complicates eradication of the problem [1,7,9]. The interactions of tissue and bacteria with the surface of the biomaterial is key in eliciting these problems [9]. Medical device polymers are relatively hydrophobic (low surface energies). Opportunistic bacteria, normally part of the skin flora, are transferred to the


133

.

.

.

wound and biomedical device in the surgical suite. The hydrophobic interactions between the device and the bacteria can result in tight adherence of these potential pathogens, which then become implanted along with the device [8,9]. These adherent bacteria then secrete a polysaccharide film, &dquo;slime&dquo; which protects them from the host’s systems for defense against such pathogens [1,8-15]. Foreign body infections are directly related to the ability of some bacteria to adhere to and grow on polymer surfaces and to produce the extracellular slime substance. Bacteria such as staphylococci, streptococci, Pseudomonas spp., and Acinetobacter spp., show this surface type of growth in vitro and in vivo [7-10]. Bacteria from retrieved, infected medical devices confirm that &dquo;slime&dquo; producers are the most common pathogens associated with these infections [7-10]. The most common organisms associated with medical device infections are the coagulase-negative Staphylococcus epidermidis and the pathogen Staphylococcus aureus [7--10]. Additional common organisms found in multi-microbial infections include Escherichia coli, Pseicdomonas aeruginosa, Proteus mirabilis, beta hemolytic Streptococcus spp. and Acinetobacter spp. [7,8,10]. In the presence of a device, normally non-virulent bacteria (e.g., Staphylococcus epidermidis) can cause serious infection [7]. Additionally, the minimum number of bacteria for virulence can be lowered as much as a thousand fold in the environment of the foreign body [7]. This increased virulence in the presence of the device is caused by interference with the host’s defense mechanisms against the establishment of infection in the device’s immediate environment [8]. How to Reduce Bacterial Adherence and Proliferation on Medical Devices?

Changing the surface properties of biomaterials to reduce bacterial adherence initially and during the lifetime of the implant could reduce the incidence of device-centered infections [8]. Modifications of the biomaterial surfaces will allow control of the cell-to-surface events [8]. Infections could be diminished by enhancing tissue compatibility or integration, and by directly inhibiting bacterial adhesion. Changing the surface properties of biomaterials could be done by construction of devices with new polymer formulations, or by covering the devices with durable coatings which will impart improved properties to a surface. To have applicability to a wide range of medical devices, this coating should tenaciously bond to a variety of medical device materials [11-14]. A generic process which could be used to produce highly


134

durable (covalently coupled) antimicrobial coatings on a variety of materials would be very valuable to biomedical technology and the medical field. A coating process which fulfills this requirement is the use of photochemical coupling reagents [15,16]. This process is applicable to all polymeric biomedical materials, and preliminary results indicate that slight modifications of the reagents allow its successful use on some metals. We are currently testing the usefulness of photochemistry for prevention of bacterial attachment and colonization of medical devices. Several approaches are being developed for the use of photochemical coatings for reducing bacterial colonization; two of these approaches are presented in this paper. First, photochemically delivered hydrophilic polymers were applied to materials to increase hydrophilicity, thus reducing bacterial attachment and adhesion; second, slow release antibiotic systems were incorporated into the photochemically bonded hydrophilic coatings to prevent bacterial proliferation on device surfaces. A discussion of the photochemical surface-modification process, the choice of reagents for these purposes, and results of in aitro testing of the resistance of photochemically coated surfaces to microbial contamination

are

presented

in this paper

Mechanism of the Photochemical

(see Figure 1).

Coupling Process

The selection of the photogroup used to make the photochemical coupling reagents is dependent on the substrate surface material to be modified and the need for a highly reactive intermediate capable of forming covalent bonds with those surfaces. To conserve manpower and investment resources, it is advantageous to choose photogroups which can be used to couple to a wide variety of medical device polymers. The majority of our work has been conducted using the benzophenone type of photogroup. This was based on reports regarding its ability to form new carbon-carbon bonds when illuminated in the solution phase

[17]. Ultraviolet (UV) irradiation of aryl ketones, such as the benzophenones, results in initial formation of the singlet excited state. If the singlet has a sufficiently long lifetime, it can undergo an intersystem crossing to the triplet state. This highly reactive intermediate is then capable of insertion into carbon-hydrogen bonds by abstraction of a hydrogen atom from the polymer surface, followed by collapse of the resulting radical pair to form a new carbon-carbon bond (see Figure 2). The high energy of the triplet state of the benzophenone intermediate makes the photochemical covalent coupling process relatively


Figure 1. Schematic of photochemical coupling reagent. This is a representation of a heterobifunctional (therrno-photochemical) surface modification reagent used for the improvement of medical device surfaces A photogroup, capable of abstraction or insertion reactions with hydrocarbon-containing surfaces, is the foundation of this reagent. Then, a spacer (as short as a few carbons, or as long as hundreds of C’s) is linked to the photogroup. A chemical coupling group is added to the end of the spacer, which binds the desired coating reagent to the photochemical group. The photochemical surface modification reagent can then be applied to the medical device; production of a covalently im. mobilized coating occurs through illumination with the appropriate wavelength and intensity of light.

Figure 2. Mechanism of photoimmobilization.

135


136

independent of the chemical composition of the surface. This permits the modification of a wide variety of medical device materials. Photochemistry can be used to coat medical devices with a wide variety of surface modification reagents. The reagent choice is dependent upon the desired host response to the modified device surface. The make photochemical surface modification reagents, the benzophenone derivative must contain on one of the two aromatic rings a functionality capable of coupling to other molecules (the chemical coupling group, Figure 1). This is accomplished using standard synthesis techniques. It is preferred to have the site of substitution &dquo;para&dquo; to the carbonyl group of the benzophenone to minimize the opportunity for intramolecular coupling. Such a &dquo;para&dquo; positioning makes it more difficult for the substituent group on the benzophenone to lie in close proximity to the activated carbonyl group. Based on these criteria, the molecule chosen for preparation of the majority of our photoactivatible surface modification reagents is 4-benzoylbenzoic acid (BBA). The use of UV light for the illumination step may be undesirable for some materials. For example, UV light can induce unwanted crosslinking of biomaterials such as hydrogels, and can cause the degradation of polyurethanes. Also, some biomolecular surface reagents (e.g., natural carbohydrates, enzymes, immunoglobulins and other proteins) undergo a considerable amount of UV-induced denaturation. For applications where UV illumination is to be avoided, visible light activated photogroups, such as the aryl azides, have been used. For example, fluoro-2-nitro-4-azidobenzene (FNAB) is an example of a combined thermochemical-photochemical, heterobifunctional reagent used for immobilization of fragile biomolecules to medical device materials [16-18]. The dark stable aryl azide (ANP) group is activated by visible light (> 420 nm) to generate N2 and the aryl nitrene diradical, a shortlived species capable of insertion into carbon-carbon, carbon double bond carbon, and even carbon-hydrogen bonds with little dependence upon the temperature or pH of the reaction mixture [1fi,17]. Other photoactivatible reagents have the qualities to be used for preparation of photochemical surface modification reagents. Development of new photochemical coupling reagents and investigations as to their useful applications is an ongoing activity at Bio-Metric Systems, Inc. MATERIALS AND METHODS

Medical Device Substrate Materials. The substrates used for these studies were standard medical grade materials. Polystyrene (PS) was from bacterial grade petri dishes and 96-well microtiter plates (Corn-


137

ing Glass Ca, Corning, New York); polymethylmethacrylate (PMMA) was

obtained in sheet form from Precision Punch and Plastic (Minne-

tonka, MN). The silicone rubber (SR), polysulfone sheets and membranes (PSulf), Pellethane 55D (55D), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polycarbonate (PC), and latex catheters

proprietary client sources. Reagents and Chemicals. Benzoylbenzoic acid (BBA) and polyethylene glycol (PEG) were obtained in highest reagent purity from Aldrich Chem. Co. (Milwaukee, WI); acrylamide monomer, N-vinylpyrrolidone, N-(3-aminopropyl)methacrylamide and 2-aminoethylmethacrylate were from Polysciences (ivarrington, PA). All other reagents used in synthetic reactions were of highest reagent purities from repuwere

from

table commercial chemical sources. Bacteria. Bacterial adherence studies were conducted with several strains of bacteria. The bacteria used in the adhesion of proliferation studies were as follows: Pseudomonas aeruginosa (ATCC #27853);

Staphylococcus aureus (ATCC #25923); Staphylococcus epidermidis (ATCC #12228); Escherichia coli (ATCC #25922). All organisms were cultured overnight in tryptic soy broth (Difco, Detroit, MI) at 37 °C prior to the adhesion studies. Selection and Preparation of Photochemical Surface Modifications Reagents Photochemical surface modification reagents were synthesized that expected to decrease the hydrophobicity of medical device polymers. For these studies, we synthesized and tested benzephenone derivatives of polyethylene glycol (MW 3350), (PEG), polyacrylamide (PAA) and polyvinylpyrrolidone (PVP). These reagents were also predicted to form adequately cross-linked matrices for slow release of antibiotics. Structures of several of these photochemical coating reagents were

are

given

in Table 1.

’

Synthesis of Photochemical Polyethylene Glycol (BBA-PEG) Polyethylene glycols (PEG’s) are polyethers possessing terminal hydroxyl groups. PEG’s are available in a wide range of molecular weights (200-20,000 D). The first steps in the synthesis of photochemical PEG derivatives is to prepare the acid chloride of BBA by reaction with thionyl chloride, to form BBA-C1. Thionyl chloride is two-fold molar excess of PEG in toluene solvent with an acid scavenger. For these applications, it is desirable to obtain a mono-functionalized (one photogroup/PEG) re-

reacted with

a

triethylamine

as


138 Table 7. Structural formulas of photoactivatible surface modification reagents.

agent. Thus, an excess of PEG is used to minimize the amount of disubstituted (2 photogroups/PEG) formed. This reaction produces a mixture of PEG, mono-functionalized PEG (BBA-PEG) and a small quantity of difunctionalized PEG CBBA-PEG-BBA). The monosubstituted product is isolated by differential solvent extraction using brinetoluene solvent mixtures. The reagent purity can be assessed by thin layer chromatography (TLC) and HPLC techniques, routinely exhibiting > 95% mono-substituted product.

Synthesis of Photoactive Polyacrylamide (BBA-PAA) In order to provide sites for attachment of photogroups on the polyacrylamide chain, it is necessary to prepare a special polyacrylamide derivative. This is done by copolymerizing acrylamide with a comonomer containing amine groups. Therefore, acrylamide is copolymerized with the chosen mole % of either N-(3-aminopropyl)methacrylamide or 2-aminoethylmethacrylate. The polymerization is run at 20 °C with aqueous solvent, using ammonium persulfate and N,N,N’,N’-tetramethylenediamine as catalysts. Control of molecular weight ifs accomplished by varying the % solids in the polymerization solution and the quantity of catalyst. The polymer is then purified from reactants by extensive dialysis against distilled water (DI H20), followed by lyophilization.


139

’

The BBA photogroup is then coupled to the aminated PAA by the reaction of BBA-CL using Schotten-Baumann conditions. Using excess acid chloride over primary amines, a chloroform-water reaction mixture is shaken vigorously to generate an emulsion with triethylamine or sodium hydroxide as acid scavenger. After centrifugation to remove suspended solids, the aqueous layer is then removed and dialyzed against DI H20 to remove non-coupled photogroups. The final product is then isolated by lyophilization. Using UV analysis of the aromatic content of the polymer, the quantity of incorporated photogroups is determined. Incorporation of photogroups of 0.04 itmole BBA/mg solids are routinely obtained with this procedure, Verification is obtained by comparison of amine values before and after derivatization.

Synthesis of Photo-polyvinylpyrrolidone (BBA-PVP) The synthesis of BBA-PVP is similar to the synthetic scheme used for the preparation of BBA-PAA. The first step is to prepare a PVP which contains reactive amine groups. This is done by copolymerization of N vinylpyrrolidone with either N (3-aminopropyl)methacrylamide or 2-aminoethylmethacrylate. The polymerization is run at 60 °C using azobisisobutyronitrile (AIBN) and TEMED as catalysts. Isolation of the product, photogroup incorporation and analysis of product are the same as those used for preparation of photo-PAA derivatives. Incorporation of photogroups to 0.047 pole BBA/mg monomer are routinely obtained with this procedure. Synthesis-of Radiolabeled BBA-PAA and BBA-PVP 1b quantify the loading of surface-modification reagents on medical device materials, we use radioisotopes to prepare radiolabeled derivatives of these coating reagents. This allows very sensitive measurements of surface-modification reagent loading per unit area of polymer material. For example, for PAA and PVP derivatives, the introduction of the BBA photogroup on the polymer chains leaves some residual unmodified primary amines. The quantity of the amine groups remaining can be partially controlled by adjusting the excess of the acid chloride over the amines in the original acylation reaction. A radiolabel can then be introduced into these polymers by an acetylation reaction of these remaining amines using [3H]acetic anhydride. The polymer is then extensively dialyzed against deionized water to remove non-bound radiolabel, lyophilized to dryness, and the specific radioactivity of the final product is determined as ~Cilmass of polymer using liquid scintillation counting. Typical specific radioactivities of these reagents were 1.7 ycjmg for [’H]BBA PAA and 5.7 pcihng for [3H]BBA PVP


140

Evaluation of Surfaces

Qualitative Evidence and Evaluation of Surface Coatings Photoimmobilization of hydrophilic reagents to hydrophobic medical polymers dramatically changes the surface energies of these materials. Qualitative evaluations of coated surfaces are done by measuring contact angles between a 3 ftl droplet of DI H20 and the surface of the pho-

tochemically treated device. Photoimmobilization of hydrophilic coatings results in decreases in contact angles (increased surface wettability). Routine screenings of photocoupling are conducted by this procedure. A more quantitative measurement of surface wettability is conducted by the Wilhelmy method of measuring advancing and receding contact angles [19]. This technique is used for characterizing the wetting behavior of fully hydrated polymeric materials. Measurements of both the advancing and receding contact angles can be made on a variety of sample shapes and with most wetting solutions [19]. Decreases in these angles indicate increased surface wettability. A Cahn Dynamic Contact Angle Analyzer (Cahn Instruments, Cerritos, CA) was

used for these measurements.

Quantitative

Measurements

of Photochemical Surface Modifications

Radiolabeled derivatives of photochemical reagents are used for quantitative evaluations of reagent loading on surfaces. First, surfacemodification reagents are applied to samples of medical device polymers, then illuminated. All non-covalently bound reagent is removed by extensive washing. Subsequently, liquid scintillation spectrometry is used to quantify the loading of reagent on the particular medical device polymer. Previous determination of the specific radioactivity of the photoreagent (dpm/ng or mCilttg) allows direct correlations between the dpmlunit area of polymer surface, and the loading of tenaciously bound reagents. Photoimmobilization of Coatings to Medical Devices

For’ in vitro experiments, we use disks or squares of polymer materials. The samples are first thoroughly cleaned by extracting residual leachables, followed by ultrasonic cleaning in non-ionic detergents and DI H20. All medical device materials, especially silicones and polysulfones, contain leachables. It is necessary to remove these materials to achieve efficient photochemical surface modification. The photochemical reagents will bind to the leachables, if they are of hydrocarbon nature, as well as the bulk polymeric backbone. Binding of


141

photohydrophilic polymers to these leachables makes them extremely water soluble, allowing leaching into surrounding fluids. Once the surface is clean and dry, it can be photochemically coated. Hydrophobic materials, such as silicone, polyethylene and polypropylene, are cleaned by corona discharge treatment Branson/IPC Model 4055 plasma generator, typically 250 m Thrr, 100 watts, 2 min. 0, atmosphere (Branson International Plasma Corporation, Hayward, CA.).

.

.&dquo;

immediately coated, while the surface is uniform coating. The photochemical coupling reagents are applied to substrate surfaces by spraying, painting, brushing or immersing in the coating reagents. Photoimmobilization of the coating reagent is then caused by illumination with a 320 nm high intensity UV light source (Model ELC 3000 Electro-Lite, Danbury, CT). Usually, the photobonding process is conducted at 20 ° C, for 1-3 minutes, 50 cm from the light source, in an air or nitrogen environment. After photoimmobilization, samples are usually washed to remove the non-covalently bound material. These materials

are

relatively hydrophilic,

then to

ensure

Microbial Attachment and Adherence Assays

.

Quantitative Bacterial Adherence Assays Several quantitative assays of microbial adherence have been developed for these studies. The first uses the method of Christensen et al. [20] with modifications for testing a variety of medical polymers and a number of pathogenic bacteria. These assays are conducted in 9fi-well microtiter plates. Polystyrene served as the standard material for initial testing of photoreagents; the bottoms of the wells were used at test surfaces. For evaluating other device materials using this assay procedure, disks are cut to fit snugly into the bottoms of flat-bottomed 96well plates. For the adherence assays, bacteria suspended in tryptic soy broth (105-108 colony forming units (CFU)/ml, depending on species) are added to the 96-well plates (control and surface-modified wells). Following incubation (37 ° C), the wells are washed with PBS to remove non-adherent bacteria. The adherent cells are then fixed with methanol and stained with 0.5% crystal violet in 40% methanol. After rinsing and drying, the absorption at 570 nm of each well is measured using a Biolek Instruments EL309 microtiter plate autoreader (Winooski, VT). The A5> is directly proportional to the number of attached bacteria in that well (determined from a standard curve that relates the AS70 absorbance to the number of bacteria). Alternate bacterial adherence assays have been developed for substrates not yielding to the shape of a microtiter plate well; an


142

is named the &dquo;colony count assay.&dquo; For this procedure, coated and control samples of medical device polymer are inoculated with 108 bacteria/ml, then incubated overnight in a thin layer of nutrient agar. Following incubation, photographs are taken with low magnification, and the bacterial colonies are counted from these photographs. Comparisons are then made between the number of colonies/unit area of the photochemically modified substrates and the control medical polymer substrate materials. A modification of an enzymatic assay for Escherichia coli adherence [21], which will be described in detail in a subsequent publication [Gregg et al., manuscript in preparation], has been developed and is referred to as the &dquo;¡3-glucuronidase assay:’ This assay measures j8-glucuronidase enzyme released from adherent bacteria to quantify numbers attached to medical device surfaces. For this assay, after sterilization of materials, the disks of control and modified medical device materials are grouped by coating method, then infected with E. coli (ATCC #25922). After incubation with bacteria, each group is separately and vigorously washed with sterile PBS. Next, each disk is placed in a separate well of a 96-well plate or in a culture tube. The enzyme substrate, ~-nitrophenyl-(3-n-glucuronide, is added to each sample. Following incubation with the substrate, a stopping reagent is added to terminate the enzyme reaction. The development of color generated from the enzyme-substrate reaction is then immediately monitored at 405 nm. Disks not infected with bacteria serve as negative controls for reagents. Presence of adherent E. coli is indicated by A40s at 2 X values of no bacteria controls.

example

Qualitative Bacterial Adherence Assays A qualitative assay of bacterial attachment was developed to visualize the effectiveness of the photochemical coatings for reducing bacterial adherence in vitro. To the medical device polymer material (e.g., polystyrene) which had previously been cleaned free of all extractables, peel-off letters are placed over part of the test sample surface. Then, the photochemical coating is applied to the entire surface of the material. Covalent bonding of the coating outside all of the masked areas is accomplished by illumination (3 min.) with UV light (320 nm}. The masking is removed, then bacteria in broth are added to and cultured on the sample plate The non-adherent bacteria are gently rinsed from the plate and the remaining bacteria fixed with methanol. Microscopic inspections of the surfaces are conducted (15 x and 100 x magnification) and photographs are taken of the control/coated surface interfaces. The percentage of bacterial coverage can be quantified with a digitizer.


143 ANTIBIOTIC SLO~’-RELEASE ASSAYS

Slow Release Antibiotic Assay This assay is used. to assess the slow release of antibiotics from oddly shaped medical devices, such as catheters. First, sterilized control and surface-modified catheters are soaked for a period of time in an antibiotic solution, then washed thoroughly. Zb test the longevity of antibiotic effectiveness, a leaching procedure was used to measure the duration of effective antibiotic release from the photochemically applied coating. The details of this assay for slow release activity of antimicrobial coatings on medical devices will be described in another report (J. A. Marcy et al., manuscript in preparation). DATA AND RESULTS

The data and results from these studies predict the usefulness of the for reduction of bacterial colonization of medical device materials. First, the generic binding of photochemical reagents was demonstrated. Next, we compared bacterial attachment and adherence to control and photochemically modified device materials. Finally, preliminary data and results are presented showing the success of the use of photochemically bonded hydrogel coatings as vehicles for slow release of antibiotics from medical device surfaces.

photochemical coatings

Demonstration of Photo-derivatization of Medical Device Polymers

,

Photochemical coating reagents were applied to a range of materials, and the contact angles were measured; a reduction in contact angle from that of the control material indicated successful bonding of the photochemical reagents. Figure 3 shows that photochemical surface modification with a BBA-PEG-OH reagent resulted in significant reduction in contact angles of all tested sample materials. Some reagents were more successfully photochemically modified than others. Polymers such as polystyrene, polymethylmethacrylate and polysulfone were made more wettable (i.e., lower contact angles were produced) than were materials such as silicone, polyethylene and polypropylene. This order of photoimmobilization efficiency would be predicted by the photochemical coupling mechanism presented in Figure 1. However, recent work has shown that O2 plasma treatment of very hydrophobic surfaces, producing a transient wettability, improves the surface coverage with the photochemical coupling reagent. Im-


144

on polymer surfaces before and after modification with photopolyethylene glycol BEE-PEG, AWV 3350). Squares of medical device polymers were cleaned, then divided into two groups. BBE-PEG (MW3350,1 mglml) was photoirnmobilized to one set of samples. After illumination and washing, sessile drop contact angles were measured. A decreased contact angle after photoimmobilization of BBE-

Figure 3. Contact angles activatible

PEG demonstrated successful surface modification.

proved coverage results in greater photoimmobilization efficiencies (e.g., silicone rubber can be modified to contact angles in the 50’s with BBA-PEG, after plasma cleaning). This photochemical surface modification remains tenaciously bound to the materials after several rounds of manual &dquo;scrubbing&dquo;; the plasma treated surfaces, alone, cannot withstand these cleaning regimens. Different photochemical coupling reagents impart different degrees of wettability to a given medical device polymer. It was postulated that the most successful coatings for reduction of bacterial adherence would be those that caused the greatest reductions in medical device hydrophobicity (greatest increases in wettability). Several surface modification reagents were photochemically applied to various materials. Wettability data was obtained using the Wilhelmy method [19], with comparisons made to the non-coated controls. These studies showed that BBA-PEG, BBA-PAA and BBA-PVP all produced dramatic de-


145 in medical device polymer hydrophobicity. The results of these studies on polymethylmethacrylate are given in Table 2. The results of these studies showed that all of these reagents produced hydrophilic (wettable) coatings. Samples were also tested for lubricity by manual methods. The manual testing indicated that BBAPAA and BBA-PVP coated samples were both wettable and lubricious, but BBA-PEG coated samples were only wettable. Quantitative loading studies using radiolabeled photoreagents showed that changes in wettability were the result of photoimmobilization of surface modification reagent. Loadings of 1.5 pg/cm2 (application of 150 teg/cm2) and 2.1 ~,glcm~ (application of 100 Jlg/cm2) were obtained for [’H1BBA-PAA and (3H]BBA-PEG on PMMA. creases

B

Bacterial Adherence to Control and Photochemically Surface-Modified Medical Device Polymers

.

Quantitative and qualitative assays were conducted to test the ability of photochemistry to reduce bacterial colonization of medical device materials in vitro. First, reagents were tested on a representative polymer, polystyrene. Then, successful reagents were tested on other medical device substrates. Assays were done using Staphylococcus epidermidis, ~S aureus, Pseudomonas aeruginosa and Escherichia coli. Surface modification with BBA-PEG, BBA-PAA and BBA-PVP made polystyrene more wettable (sessile drop contact angle changes from 90° to 36°). These coatings also significantly reduced adherence of bacterial pathogens. The results of these in vitm studies are given in Figure 4. Photoimmobilization of BBA-PEG, BBA.PAA and BBA-PVP to °

Table 2. Wilhelmy analyses of photochemically coated PMMA.


146

polystyrene, respectively, decreased adherence of Pseudomonas aeruginosa by 97%, 99% and 99%; Staphylococcus epidermidis by 92%, 85% and 91%; S aureus by 86%, 85% and 96%. Preliminary studies using the ¡3-glucuronidase assay demonstrated that these same coatings were successful in reducing the adherence of Escherichia coli. These results are given in Figure 5. E. coli adherences to BBA-PEG, BBA-PAA and BBA-PVP coated polystyrene were reduced 66%, 68% and 72%, respectively. The qualitative bacterial adherence assay was used to venerate Figures 6 and 7, which pictorially demonstrate the dramatic numerical results of Figure 4. Figure 6 results demonstrate nearly complete reduction in attachment af ~Pseudomonas to BBA-PVP modified polystyrene, using the qualitative adhesion assay. Bacteria adhered only to the areas which had been masked from the photoimmobilization procedure (the areas covered by the peel-off letters). The same dramatic results were demonstrated using a modified colony count assay for demonstration of the reduction of Staphylococcus epidermidis attachment and adherence (Figure 7). Photochemical coating of the polystyrene with BBA-PAA

Figure 4. Bacterial attachment to control and photochemically coated polystyrene. Bacterial adherence to control and photochemically coated polystyrene samples was studied using the assay of Christensen et al. [20] with some modifications. Surface modification with BBE-PEG, BBA-PAA and BBA-PVP, resulted in 97%, 99% and >99% reduction in adherence of Pseudomonas aeruginosa, 92%, 85% and 91% reduction in adherence of Staphylococcus epidermidis, and 86%, 85% and 96% reduction in adherence of S. aureus,

respectively.


147

Figure 5. E. coli adherence to control and photochemically coated polystyrene. The (3-glucuronidase assay system was used to study the attachment of E. coli to control and surface-modified polystyrene. Samples which were dry or had been fully hydrated in PBS were incubated with 3.5 x 10& CFUhnl for one hour, 37°. After extensive washing, the substrate was added, and color formation was measured. Photoimmobilization of BBEPEG, BBA-PVP and BBA-PAA resulted in 66%, 68% and 72% reduced adherence to hydrated samples.

and BBA-PVP reduced S. epidermidis adherence by an average of 90% and 94%, respectively. Assays of other substrate materials show that these same photoreagents decrease bacterial attachment to a number of medical device polymers. The degree of reduction varied for each material, but reduction in attachment and adherence was generally demonstrated by photochemical coating with the BBA-PEG, BBA-PAA and BBA-PVP

reagents. The results of the bacterial adherence assays demonstrated that modification reagents significantly reduce bacterial attachment to medical device surfaces in vitro. Reduction in bacterial adherence during the surgical implant procedure, and after implant, is predicted to be achieved with these coatings. However, though bacterial adherence can be reduced as much as 99% by photoimmobilization of hydrophilic polymers, even these small numbers of bacteria might result in development of infections. Therefore, to make the photochemical coatings even more effective, we tested the use of the photoimmobilized hydrogel coatings to be used, not only as hydrophilic

photochemical surface


Figure 6. Pseudomonas adherence to control and photochemically modified polystyrene. Vinyl letters spelling the work INFECTED were placed on a sterile polystyrene petri dish, followed by photoimmobilization of BBA-PVP to the entire surface of the dish. The letters were then peeled away, exposing the raw polystyrene. Pseudomonas aeruginosa was cultured in broth for 24 hours on the plate surface. The non-adherent bacteria were rinsed from the surface; the remaining bacterial were fixed with methanol. Figure (a) shows the D of INFECTED at a 15 x magnification (scratches on the picture are on the opposite side of the plate). In (b) an edge of the D is shown at 100 x magnification. Photographs were taken using dark field microscopy.


Figure 7. Staphylococcus epidermidis attachment to control and surface-modified polystyrene. Control and photochemically coated polystyrene microtiter plate wells were exposed to Staphylococcus epidermidis in PBS at a concentration of 10’ CFU/ml. Bacteria were

in contact with the surfaces for

one

minute. l~Ton-adherent bacteria

were

then

care-

fully washed from the wells with PBS. A solution of 0.7% agar in growth media was then poured into each well and allowed to solidify. Colonies were counted following overnight incubation at 37 °C. Representative photographs are shown in (a), with numerical results given in the graph, (b).

149


150

surfaces to reduce initial bacterial adherence, but as vehicles for the slow release of antibiotics to prevent bacterial colonization. Antibiotics were incorporated into the BBA-PAA or BBA-PVP hydrogel coatings by soaking the photochemically coated samples in a concentrated antibiotic solution. The degree of photogroup intra-matrix cross-linking of the photochemical surface modification reagent was controlled to produce relatively thick coatings ( > 300 nm) with maximal retention of antibiotic. Early investigations showed that colonization of Staphylococcus epidermidis and Staphylococcus aureus could be completely controlled in vitro, for more than 24 hours by BBAPAA + antibiotic coatings on silicone rubber. These results were en. couraging and would be predicted to be effective, since most devicecentered infections are established shortly after surgery [1,9]. Further work to optimize the synthetic organic chemistries and the antibiotic incorporation steps have resulted in coatings which serve to release effective doses of antibiotic for many days [J. A. Marcy et al., manuscript in preparation]. Preliminary results using the improved coatings on latex catheter materials are given in Figure 8. These results show that effective antibiotic release sufficient to completely inhibit proliferation of E. coli, in vitro, for at least 12 days, was produced by photochemical coating with BBA-PAA and slow release of antibiotic. DISCUSSION In this paper, we describe a photochemical coating technology for reducing bacterial colonization of biomedical implants. The photochemistry technology is highly effective in modifying a wide variety of implants, imparting beneficial properties to preformed medical devices. The application of such a coating significantly reduces the adherence of a variety of bacterial strains, resulting in as much as 99% reduction in bacterial adherence after photochemical surface modification of medical device polymers. The incorporation of a slow release antibiotic within this matrix is effective in preventing the proliferation of bacteria which adhere to the devices for at least 12 ’days. Such a technology is expected to significantly reduce the incidence of device-centered infections on a wide number of medical devices, thus alleviating the most severe problem associated with the use of artificial medical devices, device-centered infection [1,8]. Current studies are being conducted to manipulate the synthesis of photochemical reagents to increase the photochemical coupling efficiencies, further reduce the bacterial adherence to these surfaces, and


151

Figure 8. Effectiveness of antibiotic release from catheters coated with a photoimmobilized hydrogel matrix. Antibiotic (proprietary source) was incorporated into the BBAPPA coating of photoderivatized latex catheters by soaking them in a 10 mg/ml solution, followed by rinsing and drying. Control samples were not soaked in the antibiotic solution. The samples were then placed in the E. coli inoculated broth and incubated overnight. Changes to fresh samples of inoculated broth were made daily. At each time point, the proliferation of bacteria was monitored by OD6~ measurements. Comparisons were made to a &dquo;No Bacteria&dquo; broth control. A value of ~00 nm) which do not flake or crack off of surfaces, and do not interfere with the performance of the medical devices. The coatings are tenaciously bound to polymeric materials, withstanding many rounds of durability testing. The first in vivo testing of these coatings indicates this same

oped

durability. 1bxicity and pathology data predict that this technology will result in coatings which are safe for implantation. Cytopathology studies (agar onlay with MRC 5 fibroblasts or L929 cells) have been unable to demonstrate cytotoxicity at concentrations 1000-fold higher than those required for effective coatings on medical devices. Ames mutagenicity testing indicates to mutagenic properties at concentrations 10,000-fold higher than needed for applications. Subcutaneous injections in mice


153 and rabbits, contact lens studies in rabbits, and intraocular implantations in rabbits and cats all predict that these coatings are safe. Currently, this coating is being used for investigational coating of a human ocular device, with no indications of adverse effects (in fact, greater than 80% improvement of conditions with coated devices). The data presented in this review centers on the antimicrobial properties of these materials. However, this technology can be used to impart a variety of properties to preformed medical devices. In addition to their antimicrobial properties, the coating reagents used in the presented studies also reduce protein and lipid deposition, highly increase wettability and lubricity, and improve hemocompatibility, in vitro. Photochemical derivatives of cell adhesion proteins and peptides increase the rate of epithelial and endothelial cell attachment and proliferation on the surfaces, thus resulting in greater tissue integration of the medical device (increased biocompatibility of the artificial devices). This widely flexible technology is expected to have a great impact on a number of medical device applications. ACKNOWLEDGEMENT

Bio-Metric Systems, Inc., wishes to acknowledge generous support over several years from a number of federal agencies, for the development and demonstration of our photochemical surface modification chemistry, including especially the National Science Foundation and the National Institutes of Health. Partial support for our work on control of microbial colonization has been provided by NIH grant and contract projects: 2-R44-AI28125, 1-R43-AI30410, 2-R44-HL40280, 2-R44DK38271, 1-R43-DK42763, N44-DK9-2299. REFERENCES 1.

2. 3.

Bisno, A. L. and F. A. Waldvogel. 1989. Infections Associated with Indwell-

ing Medical Devices. Washington, D.C.: American Society for Microbiology. Helmus, M. N. 1990. "Outlook for Biomedical Materials," in Decision Resources, DR Reports, Burlington, MA. Maki, D. G. 1989. "Pathogenesis, Prevention, and Management of Infec-

tions Due to Intravascular Devices Used for Infusion Therapy," Chapter 8 of Infections Associated with Indwelling Medical Devices. Washington, D.C.: American Society for Microbiology, pp. 161-177. 4. Hessen, M. T. and D. Kaye. "Infections Associated with Foreign Bodies in the Urinary Tract," Chapter 10 of Infections Associated with Indwelling Medical Devices. Washington, D.C.: American Society of Microbiology, pp. 199-213.


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Stark, W. J., D. E. Whitney, J. W. Chandler and D. M. Worthen. 1986. "Trends in Intraocular Lens Implantations in the United States," Arch. Ophthalmol., 104:1769-1770. 6. Herman, C. L. 1987. "Contact Lens Safety: Interim Status Report on FDA Efforts," CLAO File, 2(2):2. 7. Christensen, G. D., L. M. Baddour, D. L. Hasty, J. H. Lowrance and W. A. Simpson. 1989. "Microbial and Foreign Body Factors in the Pathogenesis of Medical Device Infections," Chapter 2 of Infections Associated with Indwelling Medical Devices, A. L. Bisno and F. A. Waldvogel, eds., Washington, D.C.: American Society for Microbiology, pp. 27-59. 8. Gristina, A. G. 1987. "Biomaterial-Centered Infection: Microbial Adhesion Versus Tissue Integration," Science, 237:1588-1595. 9. Dankert, J., A. H. Hogt and J. Feigen. 1986. "Biomedical Polymers: Bacterial Adhesion, Colonization and Infection," CRC Crit. Rev. Biocompat., 2:219-301. 10. Peters,G., E. D. Gray and G. M. Johnson. 1989. "Immunomodulating Properties of Extracellular Slime Substance," Chapter 3 of Infections Associated with Indwelling Medical Devices, A. L. Bisno and F. A. Waldvogel, eds., Washington, D.C.: American Society for Microbiology. 11. Christensen, G. D., W. A. Simpson, A. L. Bisno and E. H. Beachey. 1982. "Adherence of Slime-Producing Strains of Staphylococcus epidermidis to Smooth Surfaces, Infect. Immun., 37:318-326. 12. Franson, T. R., N. K. Sheth, H. D. Rose and P. G. Sohnle. 1984. "Scanning Electron Microscopy of Bacteria Adherent to Intravascular Catheters," J . Clin. Microbiol., 20:500-505. 13. Marrie, T. J. and J. W. Costerton. 1984. "Scanning and Transmission Electron Microscopy of in situ Bacterial Colonization of Intravenous Catheters," J. Clin. Microbiol., 19:687-693. 14. Peters, G., R. Locci and G. Pulverer. 1982. "Adherence and Growth of Staphylococci on Surfaces of Intravenous Catheters," J. Infect. Dis., 146:479-482. 15. Guire, P. E., S. G. Dunkirk, M. W. Josephson and M. J. Swanson. 1991. "Preparation of Polymeric Surfaces via Covalently Attaching Polymers," U.S. Patent 5002582. 16. Guire, P. E. 1976. "Photochemical Immobilization of Enzymes and Other Biochemicals," in Methods in Enzymology, Vol. 44, K. Mosbach, ed., New York, NY: Academic Press. 17. Galardy, R. E., L. C. Craig, J. D. Jamieson and M. P. Printz. 1974. "Photoaffinity Labeling of Peptide Hormone Binding Sites," J. Biol. Chem., 249:3510. 18. Nash, P., S. G. Dunkirk, M. M. Golubowicz, R. J. Drexler and P. E. Guire. 1986. "Coupling Cell Factors on Anatomical Substitutes with Proclivity for Cell Linking," in Proceedings of the 8th Annual Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 1, G. V. Kondraske and C. J. Rinson, eds., pp. 157-159. 19. Smith, L. M., L. Bowman and J. D. Andrade. 1985. "Contact Angle Analysis of Hydrated Contact Lenses," in Proceedings of the Durham, England Conference on Biomedical Polymers, (July):12-15. 5.

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155 20. Christensen, G. D., W. A. Simpson, J. J. Younger, L. M. Baddour, F. F. Barrett, D. M. Melton and E. H. Beachey. 1985. "Adherence of CoagulaseNegative Staphylococci to Plastic Tissue Culture Plates: A Quantitative Model for the Adherence of Staphylococci to Medical Devices," J. Clin.

Microb., 22(6):996-1006. 21. Adams, M. R., S. M. Grubb, A. Hamer and M. M. Clifford. 1990. "Colorimetric Enumeration of Escherichia coli Based on β-Glucuronidase Activity," Appl. Environ. Microbiol., 56:2021-2024. 22. Zasloff, M. 1987. "Magainins, a Class of Antimicrobial Peptides from Xenopus Skin: Isolation, Characterization of Two Active Forms, and Partial cDNA Sequence of a Precursor," Proc. Natl. Acad. Sci. USA, 84:5449-5453. 23. Christensen, G. D., A. Simpson, A. L. Bisno and E. H. Beachey. 1983. "Experimental Foreign Body Infections in Mice Challenged with SlimeProducing Staphylococcus epidermidis," Infection and Immunity, 40: 407-410.

ABOUT THE AUTHOR

S. G. Dunkirk Dr. Dunkirk has been

a

senior researcher/consultant at Bio-Metric

Systems, Inc., (BSI), Minneapolis, MN. Since joining the company in 1984, her work has focused on application of the photochemistry technology to the improvement of a wide variety of medical devices. She has been a prolific winner of NIH grants through the Small Business Innovative Research granting program, servicing as Principal Investigator on a number of projects allowing basic and applied research projects with a wide range of medical devices, including cardiac, ophthalmic, catheter and intravascular and peritoneal devices. Currently, Dr. Dunkirk holds an academic position in the Department of Chemistry, Moorhead State University, Moorhead, MN. Dr. Dunkirk received a B.S in Chemistry and a Ph.D. in Chemistry/ Biochemistry from North Dakota State University prior to a PostDoctoral fellowship at the University of Minnesota. Such Biochemistry/ Chemistry experience provided the background for development of testing protocols for the effective properties of the photochemical surface modification process. Currently, she is a member of the society for Biomaterials and the American Chemical Society, and teaches Biochemistry Courses at the university level. Bio-Metric Systems, Inc., is a small biotechnology company located in a western suburb of the twin cities of Minneapolis-St. Paul, MN. The company was founded in 1979, and has several principal investigators who have been prolific winners of research and commercial grants and contracts. BSI is one of the top companies who have received SBIR


156

funding from NIH, NSF, USDA, DOA and other agencies. Currently, BSI holds patents to the photochemical technologies for improvement of medical devices, as well as surface properties of other materials. The

technology is applicable to a wide range of medical devices and other products, and is being licensed to individual manufacturers for each particular application.


The primary photochemical reaction to bacterial photosynthesis.

The quest for a unified view of bacterial land colonization.

Prevention of bacterial colonization on polyurethane in vitro by incorporated antibacterial agent.

Physical stress and bacterial colonization.

Antibiotic-loaded synthetic calcium sulfate beads for prevention of bacterial colonization and biofilm formation in periprosthetic infections.

Bacterial Colonization of Thrombosed Dialysis Arteriovenous Grafts.

Mechanisms of Bacterial Colonization of the Respiratory Tract.

Electrically conductive catheter inhibits bacterial colonization.

The effect of bacterial colonization on venous ulcer healing.

Prospects for the prevention of bacterial meningitis with polysaccharide vaccines.

Prevention of bacterial endocarditis.

Prevention of capsular contracture with photochemical tissue passivation.

Reply: prevention of capsular contracture with photochemical tissue passivation.

Bacterial Abilities and Adaptation Toward the Rhizosphere Colonization.

A novel electrical method for the prevention of microbial colonization of intravascular cannulae.

The prevention and treatment of bacterial endocarditis.

Electrolyzed Saline Irrigation for Elimination of Bacterial Colonization in the Empyema Space.

The relationships between environmental bacterial exposure, airway bacterial colonization, and asthma.

Prophylactic miconazole oral gel for the prevention of neonatal fungal rectal colonization and systemic infection.

Bacterial colonization of psoriasis plaques. Is it relevant?

Pattern of bacterial colonization of atopic dermatitis in saudi children.

Bacterial colonization of double J stents and bacteriuria frequency.

Self-decontaminating photocatalytic zinc oxide nanorod coatings for prevention of marine microfouling: a mesocosm study.

Abstract 108: photochemical tissue passivation for prevention of vein graft intimal hyperplasia.