Dynamic 3-hydroxyflavone-modified D-alanine nanoparticles for metabolic-incorporation in anaerobic, pathogenic biofilms generating CO in infected periodontal pockets

Light-activatable CO-generating molecules such as 3-hydroxyflavone (3-HF) derivates, are water-insoluble, which limits their applicability for infectious biofilm control. Here, a 3-HF-modified type of D-alanine (3-HFD) was coupled with hexadecyltrimethylammonium-bromide (CTAB) to self-assemble into water-suspensible, hydrophilic nanoparticles. Self-assembled 3-HFD nanoparticles did not generate CO upon visible-light irradiation, but dynamically disassembled upon metabolic-incorporation in bacterial cell-wall peptidoglycan of Gram-positive and Gram-negative periodontal pathogens, leaving molecular 3-HFD inside the cell-wall that generated CO upon visible-light irradiation. In vitro , visible-light irradiation of aerobic Staphylococcus aureus and anaerobic Porphyromonas gingivalis biofilms in presence of 3-HFD nanoparticles yielded CO-generation with strong antibiofilm activity under atmospheric oxygen conditions and in a nitrogen atmosphere. 3-HFD nanoparticles were subsequently applied for treatment of ligature-induced periodontitis in specific-pathogen-free rats with P. gingivalis inoculated, periodontal pockets. Irrigation with nanoparticle suspensions and visible-light irradiation of P. gingivalis infected periodontal pockets was demonstrated to yield in-pocket CO-generation. CO-generation improved bone-regeneration, modulated macrophages to attenuate immune-inflammation and reduced P. gingivalis colony-forming-unit counts, while hardly affecting total colony-forming-unit counts. This points to selective elimination of CO-susceptible pathogens and restoration of a healthy periodontal microbiome, as confirmed using 16S rRNA microbiome analyses. Herewith, an effective, non-surgical and non-antibiotic based three-in-one CO-based treatment of periodontitis is proposed.