A variety of phylogenetically distinct bacterial pathogens, such as non-typeable Haemophilus influenzae (NTHi), invade host cells in the upper airways by binding the platelet-activating factor (PAF) receptor. Lipooligosaccharide (LOS) glycoforms naturally expressed on the bacterial cell surface facilitate bacteria-epithelial interactions. We are utilizing LOS ligands isolated from the bacteria surface to coat the surface of nanosized delivery vehicles enabling targeting of drug molecules to the respiratory epithelium. We have observed that modification of the surface of nanoparticles with absorbed LOS glycoforms from the 2019 NTHi strain increased cellular adherence and penetration when applied to bronchial epithelial cell cultures in vitro.
Commercially available fluorescent nanoparticles were modified with various NTHi LOS glycoforms (2019 and 3198). The efficacy of the targeted nanoparticles to attach and be internalized by the respiratory epithelium was evaluating using a cell line to model the upper respiratory tract. Calu-3 bronchial epithelial cells were grown to confluent monolayers under air-interfaced culture conditions on semi-permeable Transwell membranes. The adherence and uptake of nanoparticles into the epithelial cells was determined under two apical fluid conditions: removed apical fluid and natural secretions produced by the cells. Flow cytometry and confocal microscopy were used to evaluate the subpopulation of the cell monolayer with particles present on the cell membrane or internalized within the cell at different time periods. We observe that when LOS modified nanoparticles were applied to a more physiologically relevant model of the respiratory system their diffusion through the respiratory fluid barrier is hindered.
Cellular toxicity of the was measured through the use of a MTS (tetrazolium salt) assay to quantify metabolically active cells, NR (neutral red) assay to examine membrane permeability and lysosomal integrity, and a Live/Dead Fixable dye from Invitrogen to evaluate cell membrane integrity. The MTS and NR assays are designed for 96 well plates so the seeding density, cell incubation time, amount of MTS and NR dyes, and dye incubation time were optimized for the 96 well plates. The Live/Dead Fixable dye with the Calu-3 cells grown to confluence under air-interface conditions on semi-permeable Transwell membranes was utilized to evaluate cellular health in a more relevant model of the respiratory tract. For the Calu-3 cell line the presence of the natural secretions on the apical surface reduced the toxicity observed in the Live/Dead stain when compared to particles directly applied to a washed surface of Calu-3 cell monolayer. This suggests that for the Calu-3 cell line the MTS and NR assays in the 96 well plates overestimated the amount of cell toxicity for particles when compared to the more physiologically relevant confluent monolayer grown under air-interface culture conditions.
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