(208g) Biocompatible Nanoparticles to Probe and Treat Adverse Pregnancy Outcomes
AIChE Annual Meeting
2023
2023 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Faculty Candidate Session: Food, Pharmaceuticals, and Bioengineering II
Tuesday, November 7, 2023 - 9:48am to 10:06am
Methods: For the first study, EVs were isolated by sequential ultracentrifugation from the supernatants of Lactobacillus crispatus (LC, ATCC 33197), Gardnerella vaginalis (GV, ATCC 14019) and Mobiluncus mulieris (MM, ATCC 43064) bacterial cultures and NYC media as a negative control. EV concentration was quantified by nanoparticle tracking analysis (NTA) and imaged by transmission electron microscopy (TEM). Human ectocervical (Ecto), endocervical (Endo) and vaginal (VK2) epithelial cells were co-cultured with EVs from LC, GV and MM (1x109 EVs/well) and NYC control for 24hrs. For fluorescence imaging, EVs were labeled with rhodamine B isothiocyanate before incubation and cells were fixed and stained for E-cadherin after 4 and 24h. For immune characterization, cell media was collected and cytokines were quantified by ELISA (n=6). Cell death was assessed by cell cytotoxicity assays measuring lactate dehydrogenase (LDH). For the second study, catalase-loaded nanoparticles were formulated and characterized by dynamic light scattering and catalase activity assays. We conducted an in vivo model of HIE using the Vannucci method in 7-day-old rat pups. Pups received saline (n=23) or catalase-loaded nanoparticles (n=19) 30 minutes, 24 hours, and 48 hours after injury and were sacrificed at 72 hours. Injury severity was determined by extent of 2,3,5-triphenyltetrazolium chloride (TTC) staining in freshly extracted brains.
Results: EVs were successfully isolated from LC, GV and MM supernatants and confirmed with TEM images. EVs ranged in diameter from 50-300 nm (avg. 170) across all three bacterial strains. No particles were present in isolates from NYC. After 24h of incubation, EVs appear internalized within vaginal and cervical cells as both punctate structures and diffuse signal, potentially indicating EV uptake and digestion by host cells, are observed. No cytotoxicity was observed after any EV treatment. In all three cell types, GV and MM EVs increased IL-8 production (p<0.0001 compared to NT) while LC EVs had no effect on epithelial immune response. NYC control media had no effect on cytotoxicity or IL-8 levels. In the second study, catalase-loaded nanoparticles were 70.2 nm in diameter with a polydispersity index of 0.22, zeta potential of -6.3 mV, and 7.2% encapsulation efficiency, which is a ratio of catalase activity in the nanoparticles to catalase activity in the loading solution. Results from the in vivo experiment demonstrated a significant (p=0.047) neuroprotective effect of the catalase-loaded nanoparticle treatment (median injury=4.9% tissue loss) compared to the saline control (median injury=13% tissue loss).
Conclusion: Due to their small size and physicochemical properties, nanocarriers can traffic through biological barriers, interact with target cells, and induce various functional responses. Nanoparticles can drive disease: in the context of PTB, EVs from vaginal microbes associated with adverse reproductive outcomes (GV and MM) activate a cervical epithelial immune response while EVs from a known healthy microbe (LC) have no immunogenic effect. In other contexts, nanoparticles are a promising therapeutic platform: catalase can be successfully loaded into polymeric nanoparticles and provide significant neuroprotection after HIE. Nanotechnology must continue to be used to further understand and treat adverse pregnancy and neonatal outcomes.