An innovative way of combating biofilms: delivery of quercetin and omega-3 fatty acids by nanostructured lipid carriers

Introduction: Biofilms are assemblies of microorganisms that are attached to biotic or abiotic surfaces or are associated with interfaces and are surrounded by a protective self-produced extracellular polymeric matrix1. Biofilms are usually associated with antimicrobial treatment failure and the development of chronic infections1. There are several options of treatment, however they are not all perfect. Therefore, it is important to develop new strategies. Nanocarriers can be engineered with precision, tailored to target specific bacterial strains, or disrupt biofilm architecture. Nanoparticles use their high surface area/volume ratio to deliver useful amounts of antimicrobial agents into the core of biofilms, where conventional formulations struggle to reach Their ability to penetrate the protective matrix of biofilms and directly interact with bacterial cells makes them promising agents in the fight against persistent infections2. Nanocarriers with encapsulated antibiofilm agents represent a promising frontier in antibiofilm therapy, offering targeted and potent solutions to combat the stubborn resilience of biofilm. Methodology: Nanostructured Lipid Carriers (NLCs) containing quercetin and omega-3 fatty acids were prepared by modified melt emulsification method3, and their size and potential zeta were measured. The antibiofilm activity of NLCs on S. aureus and E. coli biofilms was analyzed using colony-forming unit (CFU) counting. Biofilms were formed by adding 1 mL of each bacteria (1 × 106 CFU mL−1) to a 24-well tissue culture plates and incubating for 2 hours at 37°C and 100 rpm. After rinsing with 0.9% NaCl, fresh medium containing NLCs or the free compounds (quercetin and omega-3) was added for further incubation (24 and 48 h at 37°C and 100 rpm). The positive control was tryptic soy broth that did not contain any NLCs. Sessile populations were detached via 1-minute sonication. To assess the metabolic activity of the bacteria in the presence of NLCs, the rezazurin assay was also used. After the sonication, the sessile populations were incubated with resazurin for 3 h and then the fluorescence and/or absorbance was read. Finally, the toxicity of NLCs was tested on human dermal fibroblasts, previously cultured in α-MEM cell culture medium with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. The cells were kept in a 5% CO2 atmosphere at 37 °C. Once again, the resazurin assay was used to measure the toxicity. Results and conclusions: NLCs were homogeneous in terms of size and charge, with hydrodynamic size < 200 nm and negatively charged. Besides, NLCs seem to have an inhibitory effect on S. aureus and E. coli biofilms, especially the ones containing omega 3. The encapsulated compounds also demonstrated increased bioavailability and antibiofilm activity. In fact, in the CFU method, there was an inhibition of 6 logs or more in the case of NLCs containing omega-3 and 4 logs for NLCs with omega-3 and quercetin, a greater inhibition when compared to free quercetin (no inhibition) and free omega-3 (around 1 log). Finally, NLCs also show no cytotoxicity in fibroblasts and can be used on the skin. In conclusion, quercetin and omega-3-loaded NLCs may be a promising strategy as anti-biofilm treatment.