Evaluación de un recubrimiento sol-gel biodegradable cargado con antifúngicos para la prevención y el tratamiento de infecciones protésicas producidas por especies del género Candida

Abstract

Joint replacement is a procedure that improves the life of millions of people every year, providing pain relief, independence and restoring joint function. It is expected that the number of arthroplasties will increase by more than 150% in the next twenty years, which consequently will lead to an increase in the associated complications, such as loosening, vascular injuries, instability, poor surgical wound healing or infections. Prosthetic joint infections, despite being infrequent, represent a devastating condition for the patient and entail elevated costs for the healthcare system. The majority of these infections are caused by bacteria and 1-2% are caused by fungi, being Candida species the most frequently isolated. There currently exist large amounts of literature describing bacterial prosthetic joint infections and therapeutic guidelines are available for patient management and treatment. In contrast, standardized therapeutic guidelines are not available for fungal prosthetic joint infections mainly due to their low frequency, the different clinical characteristics of the patients and the high variability of presentation, and the treatment often depends on the experience of each center. These infections are characterized by the development of a fungal biofilm on the implant surface that protects the fungus from the treatment and the immune system reaction, damages the periprosthetic tissue and can cause a systemic infection. Fungal biofilms are especially resistant and resilient to treatment, which often leads to reinfections and therapeutic failure, so prolonged antifungal therapy and repeated interventions are often necessary to disinfect the area and perform a joint replacement. To deal with the challenge of these infections, three complementary strategies have been addressed in the literature. The first focuses on promoting osseointegration of the implant, which minimizes the possibility of developing an infection. Second is based on surface modification of the biomaterial to reduce the adhesion of microorganisms, which is the previous step for biofilm development. Third strategy is focused on the development of efficient local drug delivery systems to prevent and treat these infections. The main objective of this thesis is the in vitro evaluation of a hybrid organo-inorganic sol-gel coating, biofunctionalized with organophosphite and loaded with fluconazole or anidulafungin to prevent and treat fungal prosthetic infections caused by Candida albicans and Candida parapsilosis, the most frequently isolated species. This objective will be achieved through two phases: the previous characterization of the strains and the evaluation of the coatings. In the first phase, the results from these species are compared to those obtained from the characterization of few Candida auris strains since we had access to them within the first months of this thesis. The clinical importance of C. auris has increased rapidly since it was discovered in 2009 due to its high colonization and transmission capacities in and between healthcare centers all around the world and the antifungal resistance displayed by clinical isolates. These characteristics have positioned this pathogen as one of the main fungal etiological agents causing nosocomial systemic infections. First, biofilm production was characterized in all strains using confocal microscopy and fluorescence spectroscopy. Characterization of C. auris strains included an in vivo infection model in Galleria mellonella, the evaluation of their planktonic and biofilm antifungal susceptibility and the evaluation of the antifungal treatment of biofilms using confocal microscopy. Results obtained with C. auris strains were compared with those obtained with C. albicans and C. parapsilosis strains. In the second phase, the adherence of C. albicans and C. parapsilosis strains to the coatings without addition of antifungals was evaluated. Then, prevention of biofilm formation and biofilm treatment were studied using the antifungal-loaded coatings. Finally, a cytotoxicity assay using mammalian cells was performed. Results obtained in all the experiments revealed the different behavior of clinical strains compared to reference strains in the three species: they produced more abundant biofilms, were more pathogenic and presented different susceptibility patterns. Characterization studies showed that all strains had the ability to form a mature biofilm, varying their characteristics depending on the species and growth conditions. As example, C. albicans produced the most abundant and complex biofilms, composed of yeasts and filaments embedded in large amounts of extracellular matrix, and C. auris strains produced the least abundant biofilms, composed mainly of densely packed yeasts. Results of the Galleria mellonella in vivo model showed that C. albicans was more pathogenic than C. auris, and the phenotype of C. auris strains induced different outcomes in the model. Susceptibility studies revealed that C. auris strains displayed greater tolerance to antifungals in comparison to C. albicans and C. parapsilosis strains. In addition, biofilms produced by all strains were much more resistant than planktonic yeasts, and biofilms produced by C. auris clinical isolates were resistant to all tested antifungals. Finally, antifungal treatment of C. auris biofilms significantly reduced its viability and covered surface, being anidulafungin the most effective drug. Characterization of sol-gel coatings revealed that the presence of the coating without antifungal load decreased the adherence of C. albicans, but increased up to 65 times the adherence of C. parapsilosis. Anidulafungin-loaded coatings were more effective in preventing biofilm formation by C. albicans, while fluconazole-loaded coatings prevented more effectively biofilm formation by C. parapsilosis. Treatment with antifungal-loaded coatings showed that the presence of the coating without antifungals was sufficient to reduce biofilms produced by C. albicans, and the load with fluconazole or anidulafungin slightly enhanced this effect. In C. parapsilosis, presence of the coating stimulated biofilm formation, and the load with fluconazole drastically reduced them by up to 99%. Finally, no cytotoxicity events were observed due to the presence of antifungal-loaded coatings. A successful biomaterial with real applications in medicine would address these characteristics: being non-cytotoxic and minimally invasive, promoting osseointegration and effectively prevent and/or combat microbial infections. In this work a novel antifungal-loaded hybrid organo-inorganic sol-gel coating has been characterized. It can be easily manufactured and directly applied to the implant surface. The sol-gel is biodegraded within the first 24-48 hours post-intervention, the critical period for the development of an infection. In addition, biodegradation allows the release of phosphorous compounds and antifungal drugs to the surrounding environment, promoting osseointegration and preventing fungal biofilm formation, presenting this coating as a promising tool for reducing fungal prosthetic joint infections incidence.