Selective mechanical removal of bacterial biofilms by conjugated magnetic nanoparticles

Biofilms are microbial communities protected by a self-synthesized layer of extracellular polymeric substance that form on surfaces. Biofilms protect the bacteria from the host immune system and enable them to persist in adverse environmental conditions. They are an important clinical and public health problem, as up to 80% of all bacterial infections in humans are associated with biofilms. Healthcare-associated biofilm infections affect 3.8 million people in the EU each year and are estimated to cause 90,000 deaths annually. Furthermore, biofilms facilitate gene transfer and the spread of antimicrobial resistance, and are up to 1000-fold more resistant to antibiotics than the planktonic form of bacteria. Antibiotics in high doses are toxic to humans; they also lack specificity for pathogens, affecting beneficial commensals as well. The lack of effective and specific antimicrobial agent for pathogenic biofilm removal creates the need for new effective and specific anti-biofilm approaches. To address this problem, we propose the conjugation of protein and glycan binders specific for bacteria in biofilms with recently-developed anisotropic magnetic nanoparticles. These nanoparticles (termed “nanomowers”) will specifically bind to a selected biofilm and mediate its mechanical removal upon exposure to the external rotating magnetic field. We hypothesize that this will decrease the concentration of antibiotic needed for complete biofilm elimination. In this proposal, we will demonstrate the proof-of-principle on bacteria with high pathological relevance, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. The main aim of the present proposal is to develop and verify a new strategy for selective mechanical biofilm removal by coupling protein or glycan biofilm binders with anisotropic magnetic nanoparticles. This will be achieved by the interdisciplinary collaboration of research groups from Jožef Stefan Institute (IJS), Faculty of Pharmacy (FFA) and Biotechnical Faculty (BF). The group of Prof Aleš Berlec (Dept. of Biotechnology, IJS) will use biotechnological approach to select and produce different types of protein binders specific for biofilm-forming bacteria, such as lectins and alternative scaffolds, and assess safety of nanoparticles using cell biology approach. The group of Prof Marko Anderluh (FFA) will use a medicinal chemistry approach in a complementary effort to develop glycan binders selective for these bacteria, with a particular focus on FimH and LecA antagonists with a functional group to enable attachment to magnetic nanoparticles. Both types of bacterial binders will be used to functionalize magnetic nanoparticles prepared by the group of Prof Slavko Kralj (Dept. for Materials Synthesis, IJS) by assembling nanochains with or without sharp edges. These nanoparticles will be coated with a thin layer of silica, enabling conjugation with bacterial binders. Prepared nanoparticles will be investigated for their ability to bind and remove biofilms by the groups of Dr Jerica Sabotič (Dept. of Biotechnology, IJS) and Dr Anja Klančnik (BF) by using microbiology approach. The selectivity and efficacy of nanoparticles in biofilm removal will be assessed using crystal violet staining, CFU counting, bioluminescence assay and fluorescence microscopy, while safety and efficacy of the nanoparticles will be evaluated in Caco-2 and HT-29 epithelial cell models. The highly interdisciplinary project consortium will be coordinated by Prof. Aleš Berlec and will involve experienced researchers, as well as PhD and MSc students. The project is justified by preliminary results on binder development, nanoparticle synthesis and biofilm assays, as well as by previous collaboration of project partners. The proposal has a realistic timeframe, risk analysis and intellectual property management strategy and aims to deliver high quality publications by providing novel disruptive approach to biofilm control.