(681g) 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.