Polymersome-Mediated Antibiotic Delivery to Treat Porphyromonas gingivalis Infected Keratinocytes

Porphyromonas gingivalis is a Gram-negative bacterium associated with periodontal disease. P. gingivalis can invade oral keratinocytes and thus evade local host defences and killing by antibiotics that cannot cross the plasma membrane. These intracellular bacteria may then repopulate the gingival crevice to cause of re-infection. Polymersomes are nano-sized delivery vehicles composed of amphiphilic co-polymers that can encapsulate hydrophilic drugs like metronidazole within their core. Polymersomes are rapidly taken into cells by endocytosis before dissociating, at the low pH that occurs in the lysosomal compartment of cells, to release the drug intracellularly. Objective: To deliver polymersome-encapsulated metronidazole into the cytoplasm of P. gingivalis-infected keratinocytes to kill the intracellular dwelling organisms. Methods: Human oral keratinocytes or the keratinocyte cell line H357 were cultured in 24-well plates and then infected with strains of P. gingivalis (W50, NCTC11834, ATCC33277, A245br, IO43C2) at an MOI of 100. P. gingivalis invasion into keratinocytes was determined using an antibiotic protection assay and the number of intracellular bacteria measured by colony counts. Confocal microscopy was used to visualize intracellular bacteria. Metronidazole was encapsulated into polymersomes using sonication. Infected keratinocytes were treated with polymersome-encapsulated metronidazole or free metronidazole for 90 mins and the number of intracellular P. gingivalis measured. Results: P. gingivalis invaded keratinocytes in a dose-dependent manner and clinical isolates (A245br, IO43C2) showed higher levels of invasion (3.03% and 2.82% respectively) than laboratory strains (W50 0.05%, NCTC11834 0.3%, ATCC33277 2.13%). Highest levels of invasion were seen with bacteria in early log phase (0.26%) compared to late log (0.08%) or stationary phase (0.055%) bacteria. Polymersome-encapsulated metronidazole significantly reduced the levels of intracellular bacteria compared to free metronidazole 33% (p=0.016, One way ANOVA). Conclusion: Polymersomes are effective at delivering metronidazole into keratinocytes and killing intracellular P. gingivalis. Polymersome-mediated antibiotic therapy could potentially prevent re-current episodes of periodontitis.