Biocompatibility and biofilm inhibition of N,N-hexyl,methyl-polyethylenimine bonded to Boston Keratoprosthesis materials
Introduction
Indwelling prosthetic devices have been critical in saving numerous lives and they have enhanced the quality of life for even more patients [1]. Yet the presence of an indwelling foreign body both predisposes to, and complicates the eradication of, infections, often with biofilm formation [2]. Device-related biofilm infections account for some 60% of all hospital-associated infections [3] and add about $5 billion per year to U.S. hospital costs [4]. Biofilms, heterogenous collections of bacteria often encased by a secreted matrix called extracellular polymeric substance (EPS) [5], adhere strongly to surfaces, are resistant to antibiotics and other biocides, and are protected from host immune defenses [6]. The risk of a biofilm-associated medical device infection depends on its location, with any implantable device that translocates from the surface of the body (skin, cornea) into a sterile body site (blood, urine, intraocular) being at greater risk [2], [7].
The Boston Keratoprosthesis (B-KPro) is an artificial cornea that is a treatment option for corneal disorders not amenable to standard penetrating keratoplasty (corneal transplantation) [8]. The B-KPro has the shape of a collar button and consists of a transparent medical-grade poly(methyl methylacrylate) (PMMA) front plate with a stem, that houses the optical portion of the device, and a back plate with holes composed of either PMMA or medical-grade titanium. During implantation, the device is assembled with a donor corneal graft positioned between the front and back plates with extension of the optic stem into the anterior chamber of the eye; a titanium C-ring locks the assembly (Fig. 1A–C) [9]. Its postoperative management includes the continuous wearing of a soft contact lens to prevent ocular surface dehydration and tissue melt [10]. Daily low-dose topical antibiotic prophylaxis is required [11], as well as low-dose topical steroids in many patients [9]. While the use of life-long daily antibiotic prophylaxis to prevent infection has been effective, long-term medication adherence and emergence of resistant organisms continue to be concerns, especially in the developing world [12].
Thus there is a great need for keratoprostheses and other implantable medical devices that inherently resist bacterial infection long-term. Most biocidal products depend on a timed release of antibiotics, heavy metal ions (notably silver), or other biocides. Once all biocide has been released, the antimicrobial activity ends, leaving foreign material in situ; consequently, the overall long-term benefit of antibiotic-impregnated materials remains unclear [13]. Given the large health care costs of medical-device and hospital-acquired infections [4], non-toxic materials that effectively and permanently limit microbial adherence and viability are very much needed.
It would be most desirable to permanently attach coatings onto PMMA and other currently used prosthetic materials that either prevent adherence of bacteria or kill them on contact, thereby inhibiting the formation of biofilms. Recently, non-leaching, long-chained hydrophobic polycations that can be attached covalently to the materials’ surfaces and render them strongly antimicrobial have been developed [14], [15]. Specifically, immobilized N,N-hexyl,methyl-polyethylenimine (HMPEI) (Fig. 1D) has broad antibacterial, antifungal, and antiviral properties [16], [17], [18], [19].
Herein we assess HMPEI covalently attached to PMMA and titanium materials used in the construction of the B-KPro. Antimicrobial efficacy against hyperbiofilm-forming clinical isolates of Staphylococcus aureus and in vitro cell cytotoxicity using immortalized human corneal epithelial cells were investigated. In vivo biocompatibility studies with new ocular models in the rabbit comparing HMPEI-derivatized and parent PMMA and titanium, as well as the B-KPro with applicability to other implantable medical devices, were examined.
Section snippets
Materials
Laboratory chemicals, branched 750-kDa PEI [19], 500-kDa poly(2-ethyl-2-oxazoline), and organic solvents were from Sigma–Aldrich Chemical Co. (St. Louis, MO). Linear PEI (217 kDa) was prepared by deacylation of poly(2-ethyl-2-oxazoline) as previously described [19].
The Boston Keratoprosthesis (B-KPro) parent materials were medical-grade poly(methyl methacrylate) (PMMA) (Spartech Townsend, Pleasant Hill, IA) and titanium (Ti) 6-4 ELI (Dynamet, Washington, PA) processed by J.G. Machine Co
In vitro studies
Since there is suggestive evidence that ocular infection may be caused by a subset of organisms that possess virulence traits [20], [21], [22] and because media composition affects in vitro biofilm production [33], we evaluated S. aureus laboratory strains and clinical isolates for biofilm production under various media and growth conditions (Table 1). As others [34], we found that S. aureus ocular isolates formed biofilms variably under different conditions. We selected those ocular-associated
Discussion
Immobilized HMPEI exerts its antimicrobial effects by long hydrophobic polycationic chains that are able to penetrate and lyse the cell membranes/walls of microorganisms [14], [15]. This contact-dependent killing has been demonstrated for planktonic bacteria, as well as viruses and fungi [17], [18], [19], but heretofore not for bacterial biofilms that are the major cause of device-associated infections [3] and naturally resistant to antimicrobial agents [5]. Using large inocula (>107) of S.
Conclusions
HMPEI covalently attached to B-KPro materials inhibits biofilm formation by S. aureus. The immobilized hydrophobic polycation confers no additional toxicity or reactivity compared to the control in vitro or in vivo in enucleated eyeballs in the adjacent corneal stroma, anterior chamber, or iris. Because it is easily observable and the prosthetic material can be easily removed or exchanged, the B-KPro may represent an ideal format for testing these antimicrobial coatings in humans.
Acknowledgments
We thank Therese F. Labrecque, M. Susan Parker, Rick P. Boody, David W. Sheridan, and Nancy Sutcliffe (MEEI Henry Whittier Porter Bacteriology Laboratory); Donald Pottle (SERI Confocal Microscopy facility); Sandra J. Spurr-Michaud (SERI - tissue culture); Norman A. Michaud and Valdemar Araujo (MEEI Core Morphology); John M. Graney (JG Machine, Woburn, MA); Kathryn V. Martin (MEEI animal research assistance); Marcus Rauch, Susan R. Heimer, James Chodosh, Roberto Pineda II, and the MEEI B-KPro
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