3D cell assemblies for controlled evaluation of response to dual agent delivery nanofibers targeting bacterial...

Inflammatory response to Gardnerella during bacterial vaginosis (BV) is linked to complications including preterm birth and infection susceptibility. Platforms for local antibiotic and probiotic co-delivery have recently emerged to promote user adherence and avoid recurrence. This study designed a novel 3D cell assembly for controlled response evaluation to co-delivery platforms. Vaginal bilayer model was configured with 3.0 μm pore inserts in 24-well plates, with confluent vaginal epithelial cells (VK2 E6/E7) in apical chamber and peripheral mononuclear cells (PBMCs) in basal chamber. PEO (rapid-dissolving) and PEO:PLGA (sustained-release) nanofibers were loaded with 7.5 mg metronidazole and 5x10⁷CFU L.crispatus, and added to apical chamber. After 24-h Gardnerella treatment followed by 72-h nanofiber treatment, IL-8 and TNF-α levels were measured. Probiotic-loaded nanofibers elicited weak inflammatory response compared to Gardnerella or L. crispatus exposure. TNF-α was lower with sustained L. crispatus recovery compared to burst release (without metronidazole). IL-8 was comparable between rapid vs. sustained release. Metronidazole elicited transient Gardnerella decrease while L. crispatus inhibited longer term. Burst release yielded greater Gardnerella inhibition than sustained release, while L. crispatus recovery was relatively unaffected. Nanofibers had no cytotoxicity on VK2 cells or PBMCs. A 3D cell assembly model was established for controlled response evaluation to dual-agent nanofibers targeting BV, furthering the goal of more refined in vitro new approach methodologies (NAMs) for FRT research.