Flavonoids and omega-3 fatty acids-loaded lipid nanocarriers enriched hydrogels as promising antimicrobial biofilm strategies

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 matrix. Biofilms are usually associated with antimicrobial treatment failure and the development of chronic infections. There are several treatment options available, but not all of them are 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. Nanocarriers can 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 delivery systems of antimicrobial agents in the fight against persistent infections. Nanocarriers with encapsulated antibiofilm agents represent a promising frontier in antibiofilm therapy, offering potent solutions to combat the stubborn resilience of biofilm. Therefore, the present work aimed to develop quercetin (Q) and/or omega-3 fatty acids (ω3) - loaded Nanostructured Lipid Carriers (NLCs) enriched hydrogels that may act as an innovative strategy against biofilms, preventing their formation on wounds. NLCs were prepared by modified melt emulsification method, and their size, polydispersity index and zeta potential were measured. NLCs were homogeneous in terms of size and charge, with mean hydrodynamic size less than 200 nm and a negative charge. First, the susceptibility of Staphylococcus aureus and Escherichia coli facing Q and ω3, both free and loaded in NLCs, was tested using the diffusion method. Only free quercetin showed an inhibition halo in some of the strains, contrary to free ω3 and NLCs formulations. The antibiofilm activity of Q and ω3, both free and loaded on NLCs, on S. aureus and E. coli biofilms was analyzed using colony-forming unit (CFUs) counting. To assess the metabolic activity of the bacteria in the presence of NLCs, the resazurin assay was also used. NLCs seem to have an inhibitory effect on S. aureus and E. coli biofilms, especially those containing ω3. The encapsulated compounds also demonstrated increased antibiofilm activity compared to free compounds. In fact, in the CFUs method, there was an inhibition until 8 Logs CFUs/mL in the case of NLCs containing Q and ω3, a greater inhibition when compared to free Q (no inhibition) and free ω3 (around 1 Log CFUs/mL). The results were similar when using the resazurin assay. Next, the cytotoxicity of Q and ω3, both free and loaded on NLCs was tested on human dermal fibroblasts and on the fibroblasts line L929. The resazurin assay was used again to assess toxicity. Neither the free compounds nor the NLCs showed cytotoxicity on fibroblasts, and after 7 days, NLCs, particularly those containing ω3, had a positive effect on cell proliferation. The NLCs containing Q and ω3 were then incorporated into hydrogels, and the rheological properties and release profile were assessed. In terms of rheology, the hydrogels with the NLCs showed pseudoplastic behaviour and the absence of thixotropy. Regarding the release assay, it was obtained a cumulative release of around 42 % of quercetin at 24 hours. In conclusion, Q and ω3-loaded NLCs enriched hydrogels may be a promising antibiofilm treatment strategy, allowing for proper delivery of these antimicrobial agents while preventing biofilm formation on wounds.