Application of Novel 3,4-Dihydroxyphenylalanine-Containing Antimicrobial Polymers for the Prevention of Uropathogen Attachment to Urinary Biomaterials.

Introduction and Objective: Urinary catheters and stents are frequently prone to catheter-associated urinary tract infections (CAUTI) through biofilm formation. Several strategies have been evaluated in search of a stent coating to reliably prevent adherence of bacteria and biofilm. Previous in vivo and in vitro research with methoxylated polyethylene glycol 3,4-dihydroxyphenylalanine (DOPA) copolymer as a candidate coating showed promising results to reduce the bacterial attachment. We aimed to further enhance this antimicrobial activity by adding antimicrobial agents to newly synthesized DOPA-based copolymers. Materials and Methods: Building on our previous experience, novel copolymers were engineered based on DOPA. Quaternary ammonium groups and silver particles were added by cross-linking to increase the antimicrobial activity through both kill-by-contact and planktonic killing. After coating polyurethane sheets and measuring contact angles, all candidate coatings were challenged in vitro with an Escherichia coli culture. The most promising coatings were then further evaluated against a panel of seven clinically relevant uropathogens and planktonic killing, and microbial attachment was determined. Results: Initially, seven coatings were developed, referred to as Surphys 093-099. The most significant increase in contact angle was identified in Surphys-095 and -098. Surphys coatings S-094, S-095, and S-098 were cross-linked with silver and exhibited profound antimicrobial properties when challenged with E. coli. Further testing demonstrated S-095 to have antimicrobial efficacy against gram-positive and gram-negative bacteria at different silver-loading concentrations. The final coating, consisting of a 2 mg/mL solution of S-095 cross-linked with 0.25 mg/mL AgNO3, appeared to be highly bactericidal showing a ≥99.9% bacterial killing effect while remaining below cytotoxicity levels. Conclusions: We were able to engineer DOPA-based copolymers and add quaternary ammonium and silver particles, thus increasing the bactericidal properties of the coating. These coatings have exhibited a biologically significant ability to prevent uropathogens from attaching to biomaterials and represent a realistic opportunity to combat CAUTI.

Anti-adhesive coating and clearance of device associated uropathogenic Escherichia coli cystitis.

PURPOSE: A previous study showed decreased uropathogen adherence using a novel anti-fouling coating consisting of mussel adhesive protein mimics conjugated to poly(ethylene glycol). We assessed the ability of methoxy polyethylene glycol-dihydroxyphenylalanine (Nerites Corp. Ltd., Madison, Wisconsin) coated ureteral stents to resist bacterial adherence, infection development and encrustation in a rabbit model of uropathogenic Escherichia coli cystitis. MATERIALS AND METHODS: Sof-Flex stent curls that were uncoated and coated with 3 coatings, including Surphys 002, 008 and 009, respectively, and uncoated Percuflex Plus stents were inserted transurethrally into the bladder of 50 male New Zealand White rabbits (Charles River Laboratories, Montreal, Quebec, Canada), followed by instillation of uropathogenic E. coli strain GR12 (10(7) cfu). Urine was examined for bacteria on days 0, 1, 3 and 7, and for cytokine levels on day 7. On day 7 the animals were sacrificed. Stent curls and bladders were harvested for analysis. In a parallel experiment stents were challenged in vitro for 7 days with GR12 in human urine. RESULTS: Surphys 009 coated devices showed decreased urine and stent bacterial counts compared to those in controls. Eight of 10 rabbits in the Surphys 009 group had sterile urine by day 3 vs 1 in each control group (p = 0.013), while stent adherent organisms were decreased by more than 75%. While no statistical differences were found in encrustation and bladder inflammation across the groups, immune scoring was lowest in the uncoated Sof-Flex control and Surphys 009 groups (p = 0.030). CONCLUSIONS: Surphys 009 strongly resisted bacterial attachment, resulting in improved infection clearance over that of uncoated devices. However, this did not translate to decreased encrustation, which appeared to be independent of infection in this model.

A novel low-friction surface for biomedical applications: modification of poly(dimethylsiloxane) (PDMS) with polyethylene glycol(PEG)-DOPA-lysine.

Aqueous biocompatible tribosystems are desirable for a variety of tissue-contacting medical devices. L-3,4-dihydroxyphenylalanine (DOPA) and lysine (K) peptide mimics of mussel adhesive proteins strongly interact with surfaces and may be useful for surface attachment of lubricating polymers in tribosystems. Here, we describe a significant improvement in lubrication properties of poly (dimethylsiloxane) (PDMS) surfaces when modified with PEG-DOPA-K. Surfaces were characterized by optical and atomic force microscopy, contact angle, PM-IRRAS, and X-ray photoelectron spectroscopy. Such surfaces, tested over the course of 200 rotations ( approximately 8 m in length), maintained an extremely low friction coefficient (mu) (0.03 +/- 0.00) compared to bare PDMS (0.98 +/- 0.02). These results indicate the potential applications of PEG-DOPA-K for the modification of device surfaces. Extremely low mu values were maintained over relatively long length scales and a range of sliding speeds without the need for substrate pre-activation and in the absence of excess polymer in aqueous solution. These results were only obtained when DOPA was bound to lysine (modification with PEG-DOPA did not have an effect on mu) suggesting the critical role of lysine in obtaining a lowered friction coefficient.

First prize: Novel uropathogen-resistant coatings inspired by marine mussels.

BACKGROUND AND PURPOSE: Success in the prevention of urinary device infections has been elusive, largely due to multiple bacterial attachment strategies and the development of urinary conditioning films. We investigated a novel anti-fouling coating consisting of mussel adhesive protein mimics conjugated to polyethylene glycol (mPEG-DOPA(3)) for its potential to resist conditioning film formation and uropathogen attachment in human urine. METHODS: Model TiO(2) -coated silicon disks ( approximately 75 mm(2)) were either coated with mPEG-DOPA(3) or left uncoated and sterilized using ethylene oxide gas. For bacterial attachment experiments, coated and uncoated surfaces were separately challenged with bacterial strains comprising six major uropathogenic species for 24 hours at 37 degrees C in human pooled urine. Starting inoculum for each strain was 10(5) CFU/mL and 0.5 mL was used per disk. Following incubation, the disks were thoroughly rinsed in phosphate buffered saline to remove non-adherent and weakly-adherent organisms and cell scrapers were employed to dislodge those that were firmly attached. Adherent bacteria were quantitated using dilution plating. Representative disks were also examined using scanning electron microscopy, energy dispersive x-ray analysis, and live/dead viability staining. RESULTS: The mPEG-DOPA(3) coating significantly resisted the attachment of all uropathogens tested, with a maximum >231-fold reduction in adherence for Escherichia coli GR-12, Enterococcus faecalis 23241, and Proteus mirabilis 296 compared to uncoated TiO(2) disks. Scanning electron microscopy and viability staining analyses also reflected these results and demonstrated the ability of the coating to resist urinary constituent adherence as well. CONCLUSION: Model surfaces coated with mPEG-DOPA(3) strongly resisted both urinary film formation and bacterial attachment in vitro. Future in vitro and in vivo studies will be conducted to assess whether similar findings can be demonstrated when these polymer coatings are applied to urologic devices.

Algal antifouling and fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels.

The marine antifouling and fouling-release performance of titanium surfaces coated with a bio-inspired polymer was investigated. The polymer consisted of methoxy-terminated poly(ethylene glycol) (mPEG) conjugated to the adhesive amino acid l-3,4-dihydroxyphenylalanine (DOPA) and was chosen based on its successful resistance to protein and mammalian cell fouling. Biofouling assays for the settlement and release of the diatom Navicula perminuta and settlement, growth and release of zoospores and sporelings (young plants) of the green alga Ulva linza were carried out. Results were compared to glass, a poly(dimethylsiloxane) elastomer (Silastic T2) and uncoated Ti. The mPEG-DOPA3 modified Ti surfaces exhibited a substantial decrease in attachment of both cells of N. perminuta and zoospores of U. linza as well as the highest detachment of attached cells under flow compared to control surfaces. The superior performance of this polymer over a standard silicone fouling-release coating in diatom assays and approximately equivalent performance in zoospore assays suggests that this bio-inspired polymer may be effective in marine antifouling and fouling-release applications.

Biomimetic anchor for surface-initiated polymerization from metal substrates.

In this paper, we demonstrate the first use of a catecholic initiator for surface-initiated polymerization (SIP) from metal surfaces to create antifouling polymer coatings. A new bifunctional initiator inspired by mussel adhesive proteins was synthesized, which strongly adsorbs to Ti and 316L stainless steel (SS) substrates, providing an anchor for surface immobilization of grafted polymers. Surface-initiated atom transfer radical polymerization (SI-ATRP) was performed through the adsorbed biomimetic initiator to polymerize methyl methacrylate macromonomers with oligo(ethylene glycol) (OEG) side chains. X-ray photoelectron spectroscopy, surface FT-IR, and contact angle analysis confirmed the sequential grafting of initiator and polymer, and ellipsometry indicated the formation of polymer coatings of up to 100 nm thickness. Cell adhesion experiments performed with 3T3-Swiss albino fibroblasts showed substantially reduced cell adhesion onto polymer grafted Ti and 316L SS substrates as compared to the unmodified metals. Moreover, micropatterning of grafted polymer coatings on Ti surfaces was demonstrated by combining SI-ATRP and molecular assembly patterning by lift-off (MAPL), creating cell-adhesive and cell-resistant regions for potential use as cell arrays. Due to the ability of catechols to bind to a large variety of inorganic surfaces, this biomimetic anchoring strategy is expected to be a highly versatile tool for polymer thin film surface modification for biomedical and other applications.

Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA.

In the present study, we have utilized X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry (ELM), and optical waveguide lightmode spectroscopy (OWLS) to examine the surface adsorption and protein resistance behavior of bio-inspired polymers consisting of poly(ethylene glycol) (PEG) conjugated to peptide mimics of mussel adhesive proteins. Peptides containing up to three residues of 3,4-dihydroxyphenylalanine (DOPA), a key component of mussel adhesive proteins, were conjugated to monomethoxy-terminated PEG polymers. These mPEG-DOPA polymers were found to be highly adhesive to TiO2 surfaces, with quantitative XPS analysis providing useful insight into the binding mechanism. Additionally, the antifouling properties of immobilized PEG were reflected in the excellent resistance of mPEG-DOPA-modified TiO2 surfaces to protein adsorption. Measurements of mPEG-DOPA and human serum adsorption were related in terms of ethylene glycol (EG) surface density and serum mass adsorbed and demonstrated a threshold of approximately 15-20 EG/nm2, above which substantially little protein adsorbs. With respect to surface density of adsorbed PEG and the associated nonfouling behavior of the adlayers, strong parallels exist between the nonfouling properties of the surface-bound mPEG-DOPA polymers and PEG polymers immobilized to surfaces using other approaches. Peptide anchors containing three DOPA residues resulted in PEG surface densities higher than those achieved using several existing PEG immobilization strategies, suggesting that peptide mimics of mussel adhesive proteins may be useful for achieving high densities of protein-resistant polymers on surfaces.

Mussel adhesive protein mimetic polymers for the preparation of nonfouling surfaces.

A new biomimetic strategy for modification of biomaterial surfaces with poly(ethylene glycol) (PEG) was developed. The strategy exploits the adhesive characteristics of 3,4-dihydroxyphenylalanine (DOPA), an important component of mussel adhesive proteins, to anchor PEG onto surfaces, rendering the surfaces resistant to cell attachment. Linear monomethoxy-terminated PEGs were conjugated either to a single DOPA residue (mPEG-DOPA) or to the N-terminus of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (mPEG-MAPD), a decapeptide analogue of a protein found in Mytilus edulis adhesive plaques. Gold and titanium surfaces were modified by adsorption of mPEG-DOPA and mPEG-MAPD from solution, after which surface analysis by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectroscopy confirmed the presence of immobilized PEG on the surface. The ability of modified surfaces to resist cell attachment was examined by culturing 3T3 fibroblasts on the surfaces for up to 14 days. Quantitative image analysis revealed that cell adhesion to mPEG-DOPA and mPEG-MAPD modified surfaces decreased by as much as 98% compared to control surfaces. Modified Ti surfaces exhibited low cell adhesion for up to 2 weeks in culture, indicating that the nonfouling properties of mPEG-DOPA and mPEG-MAPD treated surfaces persist for extended periods of time. This strategy paradoxically exploits the strong fouling characteristics of MAP analogues for antifouling purposes and may be broadly applied to medical implants and diagnostics, as well as numerous nonmedical applications in which the minimization of surface fouling is desired.

Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels.

3,4-Dihydroxyphenylalanine (DOPA) residues are known for their ability to impart adhesive and curing properties to mussel adhesive proteins. In this paper, we report the preparation of linear and branched DOPA-modified poly(ethylene glycol)s (PEG-DOPAs) containing one to four DOPA endgroups. Gel permeation chromatography-multiple-angle laser light scattering analysis of methoxy-PEG-DOPA in the presence of oxidizing reagents (sodium periodate, horseradish peroxidase, and mushroom tyrosinase) revealed the formation of oligomers of methoxy-PEG-DOPA, presumably resulting from oxidative polymerization of DOPA endgroups. In the case of PEG-DOPAs containing two or more DOPA endgroups, oxidative polymerization resulted in polymer network formation and rapid gelation. The amount of time required for gelation of aqueous PEG-DOPA solutions was found to be as little as 1 min and was dependent on the polymer architecture as well as the type and concentration of oxidizing reagent used. Analysis of reaction mixtures by UV-vis spectroscopy allowed the identification of reaction intermediates and the elucidation of reaction pathways. On the basis of the observed reaction intermediates, oxidation of the catechol side chain of DOPA resulted in the formation of highly reactive DOPA-quinone, which further reacted to form cross-linked products via one of several pathways, depending on the presence or absence of N-terminal protecting groups on the PEG-DOPA. N-Boc protected PEG-DOPA cross-linked via phenol coupling and quinone methide tanning pathways, whereas PEG-DOPA containing a free amino group cross-linked via a pathway that resembled melanogenesis. Similar differences were observed for the rate of gel formation as well as the molecular weight between cross-links ((-)M(c)), calculated using equilibrium swelling and the Flory-Rehner equation.