(472c) Evaluation of a Nanoparticle Delivery Vehicle with Bacterial Targeting Ligand for Respiratory Treatment

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.