In vivo and in vitro release of lysozyme from cross-linked gelatin hydrogels: a model system for the delivery of antibacterial proteins from prosthetic heart valves

Abstract

Prosthetic valve endocarditis may be reduced by the local delivery of antibacterial proteins from the Dacron sewing ring of a prosthetic heart valve. Dacron discs were treated with a carbon dioxide gas plasma to improve the hydrophilicity and thereby enabling homogeneous impregnation with gelatin type B. The gelatin samples were cross-linked to different degrees using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Lysozyme, a model protein for antibacterial proteins, was loaded into (non)-cross-linked gelatin gels incorporated in Dacron, or adsorbed onto non-treated and gas plasma-treated Dacron. The in vivo lysozyme release was measured after subcutaneous implantation of lysozyme-loaded samples in rats. The lysozyme content of the samples, and the lysozyme level of the surrounding tissue were determined at different explantation times (ranging from 6 h up to 1 week). For cross-linked gelatin gels, the lysozyme tissue level was elevated up to 2 days after implantation. In vitro release was measured using agarose medium or phosphate buffer. Lysozyme release in buffer solution under sink conditions was in good agreement with the in vivo lysozyme release profiles, and therefore considered a good model to describe in vivo release characteristics. The release was modelled with a solution of Fick’s second law of diffusion using the appropriate boundary conditions. In this way the lysozyme concentration in the gel and the surrounding tissue as a function of time and distance was obtained. The presence of cross-linked gelatin in Dacron did lead to an increased uptake of lysozyme and a delayed release during 30 h after implantation, whereas a burst release took place from Dacron, gas plasma-treated Dacron, or Dacron containing non-cross-linked gelatin.

Introduction

Prosthetic valve endocarditis is an infrequent but serious complication of cardiac valve replacement. The adherence of bacteria to the valve is considered to be the first step in the development of the infection. Infections giving rise to clinical signs within 60 days after surgery are designated as early onset infections. Early onset infections are generally caused by bacteria that have entered the body during surgery, e.g., Staphylococcus aureus and Staphylococcus epidermidis [1], [2], [3].

Thrombin-stimulated platelets secrete lysozyme as well as other bactericidal proteins, which are able to clear bacteria from vegetation and to kill pathogens [4], [5], [6]. These platelet-derived proteins belong to a class of antimicrobial proteins, that are small (3–10 kDa) and cationic [7], [8], [9]. Lysozyme, which is also small and cationic, was used as a model compound for antibacterial proteins in this study.

Considering the increasing resistance of bacteria against conventional antibiotics, the local application of these antibacterial proteins in a controlled release system in the Dacron sewing ring of a prosthetic heart valve provides a new and promising approach to reduce the incidence of early infective endocarditis. The requirements for such a release system are a good biocompatibility and biodegradability, and suitable release properties. A release time of 24–48 h after surgery (which is also the prophylaxis time for conventional antibiotics) is considered to be sufficiently long to kill all bacteria that have entered the implantation site at the time of surgery. The amount of antibacterial protein to be released during this period was estimated to be about 100 μg for purified protein [5], depending on the activity, the in vivo half-life, and the release profile. Gelatin was used as the matrix material for the controlled release system. Gelatin is a polymer of natural origin [10], it is biodegradable [11], biocompatible, and non-immunogenic [12], [13], [14]. These properties make gelatin a suitable compound for biomedical applications [15].

Dacron was treated with a carbon dioxide gas plasma to improve its hydrophilicity [16] thereby enabling homogeneous impregnation with gelatin. After impregnation, gelatin was cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). With this combination zero-length cross-links are introduced into the matrix without incorporating any foreign structural elements [11], [17]. The cross-link density of the matrices was varied, using different amounts of cross-linking agents.

In this study, the lysozyme release from Dacron impregnated with cross-linked gelatin was determined as a function of cross-link density of gelatin and compared to the lysozyme release from Dacron and Dacron impregnated with non-cross-linked gelatin.

In vivo release of lysozyme was measured after subcutaneous implantation of the samples in rats, which was used as a model for release from the sewing ring of a prosthetic heart valve. The lysozyme content of the samples and the surrounding tissue were measured at different release times. As mass loss of cross-linked gelatin samples was not observed up to 3 weeks of implantation, it is expected that lysozyme release is not substantially affected by degradation of the gel during the first week of implantation [15].

The in vitro lysozyme release profiles were measured using phosphate-buffered saline (PBS) solution or agarose medium, and compared to in vivo release profiles. Effective lysozyme diffusion coefficients were determined from the release profiles in PBS, and used to calculate the lysozyme concentration in the gel and the surrounding tissue during in vivo release, using a solution of Fick’s second law of diffusion applied for a semi-infinite composite medium. Using these mathematical equations, the concentration of antibacterial protein can be calculated as a function of time and distance from the implanted sample, depending on the initial loading of antibacterial protein and the cross-link density of the gelatin gel.