Identification of Specific Target Ligands to Overcome Periodontal Disease Through Phototherapy

Abstract

Background: Conventional antibacterial treatment fails to eradicate biofilms associated with dental caries and periodontal disease. Biofilm targeted-photodynamic therapy (PDT) could be a solution to these problems. Objective: to identify broad-spectrum bacterial binding ligands from one-bead one-compound (OBOC) libraries to develop targeting ligands against biofilm thorough phototherapy. Material & Methods: Biofilm bacteria of S. mutans and E.coli were created on tissue flasks. After thorough washing to remove the free bacteria, the biofilm bacteria were scraped with a sterile blade. The scraped bacteria were ultra-sonicated and were incubated with OBOC libraries. The beads with S. mutans binding were sorted. After removing the bacteria from beads, the S. mutans binding beads were then re-incubated with E.coli. The sequences were determined by edman chemistry. The focused library was generated according to the mortif. The bacteria of S. mutans, P.gingivitis, E.coli, P. aeruginosa, Klebsiella sp, E. faecalis, citrabacter sp., and S. aureus were screened with focused library one by one. The compound beads with the property of binding to all of above bacteria were isolated and then incubated with human oral epithelial cells as well as human endothelial cells. The beads that bind to bacteria, but not to human oral epithelial cells and endothelial cells were finally identified. The chemical structures of identified compounds were determined by using peptide sequencing. The bacterial binding compounds were then conjugated with biotin through lysine and challenged with biofilm to testing their binding efficacy through streptavidin 488. The “all in one” targeting nanoparticles with hydrophobic core of photosensitizers were developed by decorating these bacterial binding ligands with porphyrins through click chemistry. The phototoxic efficacy of targeting nanoparticles on biofilm was evaluated by using viable account. Results: four bacterial compounds were identified for possessing the property of specifically binding to all of the above bacteria, but not binding to normal human oral cells and endothelial cells. MTT assays indicated that these four compounds have no cytotoxicity to human epithelial cells and endothelial cells at up to 500 uM. Zonal inhibition assay indicated these four ligands do not have bactericidal effects. Compared with the porphyrin nanoparticles control, targeting nanoparticles caused a 100-fold reduction in the number of mix E. coli and S.mutans in the biofilm, whereas control group phototherapy had no effect on the number of E. coli. Conclusion: A high throughput bacterial binding compound screening method was established and four novel bacterial binding compounds were identified. The use of a compounds photosensitizer conjugate is a more selective method of delivery thermal and dynamic therapy to biofilm. Work is currently under way to evaluate the phototoxic efficacy of these targeted nanoparticles on S. mutans, P. gingivitis, E. coli, P. aeruginosa, Klebsiella sp, E. faecalis, citrabacter sp., and S.aureus biofilm to identify the optimal parameters to fight biofilm.