Optimization of a Nanoparticle Delivery Vehicle to Mimic Bacterial Invasion of Lung Epithelium

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 to mimic the invasion characteristics of NTHi, 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.  However, 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.  Therefore, the attachment of polyethylene glycol (PEG) was investigated to minimize particle interactions with the respiratory fluid barrier.  The goal of our research is to minimize nanoparticle interactions with respiratory fluids while retaining the improved cellular adherence and penetration afforded by the LOS coating. 

Commercially available fluorescent nanoparticles were modified with various NTHi LOS glycoforms (2019 and 3198), PEG, and combinations thereof.  Upon coating, the modified nanoparticles exhibited an increase in size proportional to the molecular weight of the coating material and more neutral surface charge.  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 was used to quantify the subpopulation of the cell monolayer with particles present on the cell membrane or internalized within the cell at different time periods. 

The presence of secreted apical fluid results in a reduction of the cell population with particles at 2 hr for both the NTHi LOS glycoforms and PEG.  The maximum populations were achieved for the particles directly applied to the apical surface at 6 hr for all particles (36-49 percent).  Whereas, the maximum populations were achieved at 12 hr for the particles applied to the apical side of the cells with apical secretions for all populations (19-47 percent).  The presence of the natural secretions on the apical surface is seen to modify the amount of cells in the monolayer interacting with the particles and the rate at which these interactions take place.  The particles containing both PEG and LOS showed enhanced cellular uptake (20, 22, 39, 32 percent) compared to the single modified LOS particle (11, 10, 18, 18 percent) for each time period (2, 6, 12, 24 hr).  However, the amount for the bifunctional particle still fall below that of the single modified PEG particles (27, 28, 47, 40 percent).  This suggests that further optimization of the bifunctional particle is possible with better control of attachment for both the PEG and LOS glycoform.