KONA Powder and Particle Journal No. 41 (2024) 265–279/https://doi.org/10.14356/kona.2024005

Original Research Paper

Lipooligosaccharide Ligands from Respiratory Bacterial
Pathogens Enhance Cellular Uptake of Nanoparticles †Mai H. Tu 1, Timothy M. Brenza 1, Margaret R. Ketterer 2, Morgan Timm 3, Benjamin M. King 4,
Michael A. Apicella 2 and Jennifer Fiegel 1,4*
1 Department of Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa, USA
2 Department of Microbiology and Immunology, The University of Iowa, USA

3 Department of Biochemistry and Molecular Biology, Gustavus Adolphus College, USA
4 Department of Chemical and Biochemical Engineering, The University of Iowa, USA% Cells associated with particles

Several bacterial pathogens contain membrane ligands that40
facilitate their binding and internalization into human tissues.
LOS-coated NPs
30
Cell
In this study, lipooligosaccharides (LOS) from the respiratory
pathogen non-typeable Haemophilus influenzae (NTHi) were20
isolated from the bacterial surface and evaluated as a nanoparticle
PAFR
LOS
coating material to facilitate uptake into the respiratory epithelium.
10
NTHi clinical isolates were screened to select a strain with high0binding potential due to their elevated phosphorylcholine content.
0
30
10
20
Incubation time (hours)
The association of particles with human bronchial epithelial
cells was investigated as a function of particle surface chemistry
and incubation time, and the uptake mechanism was evaluated
via chemical inhibitor and receptor activation studies. A morethan two-fold enhancement in particle uptake was achieved by coating the particles with LOS compared to uncoated or gelatin-coatedparticles, which was further increased by activating the platelet-activating factor receptor (PAFR). These findings demonstrate thatbacterial-derived LOS ligands can enhance the targeting and binding of nanoparticles to lung epithelial cells.
Keywords: endocytosis, platelet-activating factor receptor, lipooligosaccharides, non-typeable Haemophilus influenzae,
nanoparticle, bronchial epithelial cells

1. Introduction

Targeted drug delivery is a powerful strategy to treat
diseases that affect human tissues, offering high drugconcentrations at the target site with limited exposure ofother tissues. Efficient delivery can be achieved using drugloaded nanoparticles with chemical and physical properties
tuned to avoid host defense mechanisms and inhibit nonspecific distribution (Fenton et al., 2018; Patra et al., 2018).
Active targeting can further enhance delivery to specifictarget organs or cells within the body. With appropriate
surface functionality, such as through the attachment ofligands that recognize receptors on cellular surfaces, particulate carriers show improved efficiency in penetrating cells
in vitro. One challenge in achieving active targeting has
been the identification of cellular targets, which is limited
by our knowledge of disease-specific cell receptors andtheir ligands. A potentially useful class of ligands is produced and used by human pathogenic organisms to infectthe respiratory tract.
Received 28 May 2022; Accepted 29 August 2022
J-STAGE Advance published online 25 March 2023
* Corresponding author: Jennifer Fiegel;
1,4 Add: Iowa City, IA, 52242, USA
E-mail: jennifer-fiegel@uiowa.edu
TEL: +1-319-335-5162 FAX: +1-319-335-1415†

Haemophilus influenzae (H. influenzae), a Gramnegative bacterium that colonizes the nasopharynx of up
to 80 % of humans, is a leading cause for bacterial meningitis and other widespread bacteremic diseases (Cochi
and Broome, 1986; Foxwell et al., 1998). While a polysaccharide conjugate vaccine is available for and is effective
against H. influenzae serotype b, the vaccine is not effective
against the subtype that lacks a capsule, called non-typeable H. influenzae (NTHi) ( St. Geme, 1996). NTHi causes
localized opportunistic infections including otitis media,
sinusitis, conjunctivitis, bronchitis and pneumonia (Turk,
1984). It can infect cystic fibrosis (CF) patients early inchildhood and prime the lungs for later infection with thebacterium Pseudomonas aeruginosa, which is correlatedwith higher morbidity and mortality in CF patients (Starner
et al., 2006).
Diseases due to NTHi often start with colonization of therespiratory epithelium (Turk, 1984). The surface of NTHi isdecorated with lipooligosaccharides (LOS) which contain alipid structure that embeds in the outer leaf of the bacterial
outer membrane and an oligosaccharide structure thatfacilitates bacterial pathogenesis. By expressing specificcarbohydrates, such as phosphorylcholine (ChoP), as aterminal structure on the LOS, the bacteria are imbued with
two important defenses. First, ChoP expression reduces the

Copyright © 2024 The Authors. Published by Hosokawa Powder Technology Foundation. This is an open accessarticle under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

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ability of LOS-specific or bactericidal antibodies to bind tothe bacteria through molecular mimicry of host structures,
enabling the bacteria to evade the host immune system andlimit their pulmonary clearance (Clark et al., 2012; Pang etal., 2008; Weiser et al., 1998). Second, the presence of ChoPon the NTHi surface enhances bacterial adherence to hostcells by binding to the platelet-activating factor receptor(PAFR), effectively reducing mucociliary clearance of thebacteria and enhancing bacterial invasion of the respiratory
epithelium (Swords et al., 2000, 2001; Turk, 1984). The effectiveness of OS in inducing cellular uptake is dependent
on the specific LOS glycoform, with LOS containing higher
ChoP content resulting in enhanced NTHi colonizationand persistence in the lungs (Swords et al., 2000; Weiser
et al., 1998). These findings suggest that isolated ChoPcontaining LOS could be a potential targeting ligand for
drug delivery particles to mimic bacterial entry into lungcells and thus deliver drugs directly to infected lung cells.
We hypothesized that LOS ligands naturally expressedon bacterial cell surfaces from NTHi could be utilized
as targeting ligands to facilitate particle attachment anduptake into lung epithelial cells. The expression of ChoPon several clinical isolates of NTHi was measured andan NTHi strain with high ChoP content chosen for LOS
isolation. Nanoparticle uptake was evaluated in 16HBEobronchial epithelial cells in the absence and presence ofendocytosis pathway inhibitors and activators of PAFR.

2. Experimental

2.1 Culture of NTHi bacteria

NTHi clinical strains 2019 and 3198, isolated frompatients with chronic obstructive pulmonary disease(Campagnari et al., 1987), and strain 956 were reconstituted from frozen glycerol stocks and propagated on
chocolate agar (a non-selective, enriched growth medium)or brain heart infusion (BHI) agar (selective medium for
NTHi) supplemented with 10 μg/mL hemin and 1 μg/mL
nicotinamide adenine dinucleotide (NAD) at 37 °C and
5 % CO2. All culture chemicals were obtained from Sigma
Aldrich.

2.2 Detection of ChoP on NTHi cells via ELISA
and dot immunoblot

The three NTHi strains were screened by enzyme-linked
immunosorbent assay (ELISA) to identify bacterial strains
and growth conditions that result in bacteria with high ChoP
activity. An NTHi cell suspension (8.0 × 107 cells/mL) was
added to each well of a 96-well plate (Corning Inc. product3590; clear, flat-bottom polystyrene, high-binding). The
plate was dried in an oven uncovered overnight at 40 °C.
The wells were then washed three times with 200 μL
ELISA wash solution (0.98 % w/v sodium acetate, 0.9 %w/v NaCl, 0.05 % v/v Tween 20 in purified water from
NanoPure® Infinity UV Barnstead system). The primary

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monoclonal antibody 12D9, which was raised directlyagainst ChoP, was diluted 1:25 and added to each well. The
plate was incubated overnight at room temperature, thenwashed. Alkaline phosphatase-conjugated goat anti-mouse
IgG AffiniPure F(ab’)2 (Jackson ImmunoResearch Inc.)
diluted 1:2000 in ELISA buffer was added to each well andincubated for 1 h, after which the wells were again washed.
The substrate, nitrophenyl phosphate bis (cyclohexylammonium) (pNPP), was prepared at 1 mg/mL in diethanolamine buffer (10 % w/v diethanolamine and 0.01 % w/v
MgCL2 in purified water, pH 9.8) immediately before use,
added to all wells, and incubated in the dark. The substrate
was then hydrolyzed by alkaline phosphatase to develop ayellow water-soluble product, p-nitrophenol. The relative
absorbance of p-nitrophenol at 405 nm was read using a
SpectraMax® M2 spectrophotometer (Molecular Devices)
after 2 h incubation. Wells with no cells, no primary antibody, or no secondary antibody served as negative controls(n = 4). Dot immunoblot confirmed the relative high ChoP
expression on NTHi 3198 cell surfaces.

2.3 Proteinase K/hot phenol method to isolate
LOS from NTHi bacterial cells

Due to its relatively high ChoP expression, LOS was
isolated from NTHi strain 3198 using the proteinase K
digest, phenol-water extraction method (Apicella, 2008;
Galanos et al., 1969). NTHi was cultured on supplemented
BHI agar overnight, harvested, digested in ELISA buffer
containing 1 % w/v sodium dodecyl sulfate (SDS, SigmaAldrich®) and 50 μg/mL proteinase K at 65 °C for 1 h,and incubated at 37 °C overnight to lyse the cells anddigest the proteins. The mixture was washed three times
via precipitation in 3 M sodium acetate and cold absoluteethanol overnight at –20 °C, then centrifuged at 3200 × g
at 4 °C (Eppendorf centrifuge I810R). The precipitate
was washed with ethanol to remove residual SDS. Afterthe final precipitation, the pellets were air-dried at room
temperature for 2 h and then suspended in 15 mL of DNase
I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2
in purified water, pH 7.6). Solutions of 10 μg/mL DNase(Roche) and 10 μg/mL RNase (Qiagen) were added andthe solution incubated at 37 °C in a water bath for 2 h todigest residual nucleic acids. Finally, a phenol extraction of
the LOS nuclease-treated mixture removed residual protein
contamination. The LOS mixture was isolated from phenol
soluble components using an equal volume of pre-heatedphenol and incubated at 65 °C for 20 min, then on ice for
1 h. Upon cooling, the suspension was separated into aphenol phase containing proteins, an interphase containing
cell-wall material, and an aqueous phase enriched in LOS.
The top aqueous layer was collected and washed threetimes before lyophilization and storage at 4 °C in a desiccant box. The activity of the isolated LOS was confirmed
using the colorimetric ELISA described above, except theMai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

primary antibody 12D9 was prepared at 1:50 dilution in
ELISA buffer, the secondary antibody was added at 1:2000
dilution in ELISA buffer, and the time for p-nitrophenol todevelop was 3 h.

2.4 Preparation of LOS-functionalized particles

Polystyrene particles (0.2 μm plain white Polybead®
or Fluoresbrite® polystyrene particles, Polysciences Inc.)
were prepared for functionalization by washing twice withcarbonate buffer (15.9 mg Na2CO3 and 25.9 mg NaHCO3
in 100 mL purified water, pH 9.6) an centrifuging at
12,000 × g for 30 min, followed by resuspension at a concentration of 2 × 109 particles/mL. Particles with a nominal
size of 0.2 μm were chosen to match the mean width of an
NTHi bacterium.
To achieve particle functionalization, polystyrene particles were added to a solution containing NTHi 3198 LOS ateither a low (5 ng/mL) or high (5 μg/mL) ligand concentration, mixed on a rotator for 2 h, washed twice with carbonate buffer, and resuspended in carbonate buffer. A subset
of uncoated and high ligand coated polystyrene particles
were exposed to a protein mixture of 0.1 % gelatin (Type B
from bovine skin derived from lime-cured tissue, average
MW 50,000–100,000 g/mol; Sigma G9382) solution incarbonate buffer. Particles were either stored in carbonate
buffer at a final concentration of 2 × 109 particles/mL
at 4 °C (cell association studies) or lyophilized overnightin a Labconco® FreeZone® 4.5-liter freeze dry system at achamber pressure <0.02 mbar and collector temperature ator below –50 °C.

2.5 Particle characterization

Lyophilized polystyrene particles were prepared for size
and zeta potential measurements by dispersion in water orserum-free medium (pH 7.4) via three cycles of vortexing
for 90 s and sonication for 10 min in cold water bath at
4 °C. Particle suspensions were placed into folded capillarycells (DTS 1060C cuvettes, Malvern Instruments) and theaverage particle hydrodynamic diameter and zeta potential(n = 3) measured using a Zetasizer® Nano ZS (Malvern
Panalytical LTD.). Particle size and morphology were confirmed via scanning electron microscopy using an S-4800
Field Emission Scanning Electron Microscope (FE-SEM).
Lyophilized particles were tapped onto SEM stubs coatedwith double-sided black carbon tape and sputter coatedwith Au-Pl at 10 mA and pressure above 7 × 10–2 mbar for
3 min (K550 Emitech® sputter coater).
To prepare particles for surface chemical analysis,
washed particles were dispersed in water, and 20 μL of
the suspension was added and dried on clean silicon wafers. Chemical analysis was carried out using a Kratos®
Axis Ultra X-ray photoelectron spectrometer (XPS) withconcentric hemispherical electron energy analyzers combined with an established delay-line detector. The incident

Original Research Paper

radiation from monochromatic Al Kα X-rays (1486.6 eV)
at 150 W (accelerating voltage 15 kV, emission current
10 mA) was projected 45° to the sample surface and thephotoelectron data was collected at a takeoff angle ofθ = 90°. The base pressure in the analysis chamber wasmaintained at 1.0 × 10–9 torr. Survey scans were taken ata pass energy of 160 eV and carried out over a 1200 eV ~–5 eV binding energy range with 1.0 eV steps and a dwell
time of 200 ms. High resolution scans of C 1s, O 1s, N
1s, P 2p, and S 2p were taken at a pass energy of 20 eVwith 0.1 eV steps and a dwell time of 2000 ms. Spectral
analysis was conducted using CasaXPS software (version
2.3.17dev6.4k). Spectra were calibrated using the adventitious carbon C 1s peak at 285.0 eV.

2.6 Isothermal titration calorimetry (ITC)

ITC measures the heat released or absorbed during a biomolecular binding event, enabling accurate determinationof the enthalpy of binding and the stoichiometry of eachbinding event at different concentrations of ligand, including proteins on nanoparticles. The GE MicroCal® iTC200(GE Healthcare) was used in this study. The accuracy of the
instrument was verified by titrating a 5 mM standard CaCl2
solution into a 0.4 mM standard EDTA solution, both provided by the manufacturer. LOS and nanoparticle solutions
were incubated in a 25 °C water bath for 15 min prior to
use to ensure temperature equilibration. LOS solution ata concentration of either 20, 30, 50, or 100 μg/mL wasloaded into the syringe and 625 μg/mL nanoparticle solution (the mass of nanoparticles in the original particle suspension was provided by the manufacturer) was loaded into
the sample cell, then the system was equilibrated to 25 °C.
Experimental parameters were set as follows: total numberof injections = 20 (one 0.4 or 0.5 μL injection followedby nineteen 2 μL injections); cell temperature = 25 °C;
initial delay = 60–200 s; syringe concentration = variable;
mixing speed = 700 rpm. Accounting for the dilution in the
ITC cell, the 30 and 50 μg/mL injections were equivalent toa final LOS concentration of about 4 and 7 μg/mL, respectively, interacting with the nanoparticles. Controls included
carbonate buffer titrated into 625 μg/mL of nanoparticles
and 50 μg/mL LOS titrated into carbonate buffer (to determine heat of dilution). Origin 7.0 software was used to convert all raw ITC data into DeltaH plots, and its regressionfunction was used to calculate the stoichiometry, enthalpy,
entropy and dissociation constant (Kd) of each titration.

2.7 Cultivation of immortalized bronchial
epithelial cells

The 16HBE14o- cell line, generated by transformationof normal bronchial cells obtained from a heart-lungtransplant patient, was generously provided by Professor
Gruenert, Department of Otolaryngology, University ofCalifornia, San Francisco (Gruenert et al., 1988). Cells

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were propagated in cell culture medium composed ofEagle’s minimum essential medium with Earle’s salts(Life Technologies Co.) supplemented with 10 % fetal
bovine serum (FBS, Atlanta Biologics Inc.), 2 mM Lglutamine (Life Technologies Co.), 100 μg/mL streptomycin and 100 units/mL penicillin G (Life Technologies Co.)in T-75 flasks (Greiner Bio-One). The cell culture medium
was replaced every other day. At 80 % cell coverage (4–6days), the cells were passaged by washing three times withbiological buffer (prepared as a 10-times concentrated
solution containing 3.795 g NaCl, 0.35 g Na2HPO4, 0.20 g
KCl, 0.99 g glucose, and 1.265 g HEPES in 1 L of purified
water, pH 7.4), detachment by treatment with Trypsin/
EDTA (Life Technologies Co.) for 5–10 min at 37 °C and
5 % CO2 with gentle agitation, and addition of the cellsuspension to new flasks with 12 mL of media per flask.
Passages 20 to 40 were used.

2.8 Immunofluorescent detection of PAFR on
16HBEo- cells

The expression of PAFR on the surface of 16HBEocells was determined using flow cytometry and confocal
microscopy. Cells were prepared for flow cytometry bysuspending cells in biological buffer containing 0.2 %Tween 20, centrifuging at 300 × g for 10 min, then incubating in cold methanol (–20 °C) for 10 min at –20 °C. The
fixed cells were washed with biological buffer containing
0.2 % Tween 20 (Sigma) and incubated with 10 % normal
goat serum in biological buffer containing 0.2 % Tween 20
at 4 °C overnight. Cells were then centrifuged, redispersed
in biological buffer at a concentration of 1 × 107 cells/mL.
To stain for PAFR expression, cells were incubated with
50 μL of 8 μg/mL solution of IgG2a PAFR (human)monoclonal antibody raised in mouse (product 160600,
Cayman Chemical) in biological buffer for 30 min at room
temperature. The controls were no stain, secondary antibody control, and isotype control using 8 μg/mL solutionof mouse IgG2ak clone eBM2a (eBiosciencesTM) in biological buffer. Cells were then washed six times with cold
biological buffer containing 0.2 % Tween 20 and incubatedwith 50 μL of either 6 % normal goat serum in biologicalbuffer (no stain control) or 0.1 μg/mL solution of Alexa
Fluor 647 Goat Anti-Mouse IgG (H+L) antibody (LifeTechnologies Co.) in biological buffer for 15 min. Cells
were again washed six times with cold biological buffer
containing 0.2 % Tween 20. After antibody treatment, cellsuspensions were analyzed immediately using an LSR II
flow cytometer (BDTM Biosciences).
To confirm and visualize PAFR expression, cells were
cultured onto collagen-coated cover slips in 24-well plates
and incubated overnight. The cells were washed threetimes with biological buffer containing 0.2 % Tween 20.
Immunostaining of the cell membranes was conducted as
described above, except 100 μL of each antibody solution

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was added to the cells and the secondary antibody Alexa
Fluor 568 Goat Anti-Mouse IgG (H+L) antibody (LifeTechnologies Co.) was added at a concentration of 4 μg/mL.
After cell washing, the glass coverslip containing the cell
membrane was mounted to a microscope slide using antifade reagent with the nuclear stain DAPI, then kept at 4 °C
until imaged. Cell layers were imaged using a Zeiss LSM
710 confocal microscope with 40× oil immersion objectivelens.

2.9 Particle association with bronchial epithelialcells

To prepare cells for particle binding and uptake studies,
16HBEo- cells were propagated on collagen-coated glass
coverslips. A volume of 100 μL of cells was dispensed, at
2 × 105 cells/well, onto dried collagen-coated coverslips
and incubated one day until reaching 80 % confluency, as
determined by visual inspection. Suspensions of fluorescent yellow particles with or without coating (0.22 μg/mL,
100 μL/well) were added to cells and incubated for 0.5, 2,
4, 18, or 24 h (n = 4 wells of cells per treatment) to quantify
uptake with time. In each case, after incubation the cells
were washed three times to remove unbound particles.
The number of cells associated with particles was quantified using an LSR II flow cytometer (BD Biosciences)with a 488 nm excitation source. Cells were processed forflow cytometry by incubating the cells with enzyme-freecell dissociation buffer (Life Technologies Co.) for 0.5–1 h
at room temperature, centrifuging at 300 × g for 10 min at
4 °C, then dispersing in cold biological buffer by gentle
pipetting to obtain a single cell suspension. Raw data wasprocessed to gate the live cell population(s) based on cellsize (forward scatter) and cell granularity (side scatter),effectively removing dead cells, cell debris and particle
agglomerates. Gating was confirmed with controls, i.e.
cells not treated with particles, particle alone, and cells andparticles mixed briefly together. The population of cells
containing particles were identified based on fluorescenceintensity using the Probability Binning (PB) Chi(T) value(Roederer et al., 2001). The percentage of cells in the population was quantified using the Enhanced Normalization
Subtraction (ENS) method in FlowJo® (Bagwell, 1996).
Results were collected for at least 10,000 cells (n = 4).
Confocal microscopy was used to assess the locationof particles within the cell cultures. After particle treatment, cell membranes were washed three times to remove
unbound particles and secretions produced during the
incubation period. Cell membranes were then stained with
1 μg/mL DiI D-282 (Life Technologies Co.), a lipophilic
indocarbocyanine fluorescent red dye (549 nm/565 nm),in cell culture medium for 30 min, washed with biological buffer three times to remove excess dye, fixed with
2 % w/v paraformaldehyde for 30 min, blocked with
50 mM ammonia chloride for 10 min, and washed with

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3. Results

3.1 Selection and characterization of NTHi
bacteria

Since ChoP content is vital for epithelial cell binding,

2.0

Chocolate agar
Brain heart infusion agar

*

1.5

*

1.0
0.5

31

98

0.0
6

Significance (p < 0.05) of differences was determined by
one-way ANOVA with Holm-Sidak’s multiple comparison
tests using GraphPad Prism software (version 6.05).

Electron microscopy images of the uncoated polystyrene
particles confirmed that particles were spherical, withsmooth surfaces (Fig. 2(a)). Upon particle exposure tocarbonate buffer, salt crystals were observed attached tothe particle surfaces (Fig. 2(b)). Coating the polystyrene
particles with 5 ng/mL LOS did not change their appearance compared to the carbonate buffer-washed particles(Fig. 2(c)), as salt crystals remained attached to the particle
surface; however, upon coating with the higher concentration of LOS (5 μg/mL), salt crystals were no longer95

2.11 Statistical analysis

3.2 Physical and chemical characterization of
uncoated and functionalized particles

19

The toxicity of inhibitors to 16HBEo- cells was first
determined using the MTS assay (CellTiter 96® Aqueous
One Solution Cell Proliferation Assay; Promega Co.).
Cells were seeded onto 96-well plates at 3 × 104 cells/
well, incubated for 24 h, then treated for 0–5 h with oneof the following inhibitors: 0.1 % NaN3 with 50 mM 2deoxyglucose, 5 μg/mL cytochalasin D, 80 μM dynasore,
200 μM genistein, 10 μg/mL chlorpromazine, or 5 mM
methyl β-cyclodextrin. Negative and positive control were
cells treated with biological buffer (absorbance normalized
to 100 % cell viability) and cells treated with 2 % w/v SDS,
respectively. Based on toxicity results, uptake studies in
the presence of inhibitors were limited to time points lessthan 3 h, and studies with inhibitors of clathrin-mediatedendocytosis were not conducted as cell viability with these
inhibitors was 40–95 % at all time points.
To determine the mechanisms by which particles weretaken up by cells, 16HBEo- cells were pre-treated with oneof the following inhibitors one hour prior to particle treatment: 0.1 % NaN3 with 50 mM 2-deoxyglucose, 5 μg/mL
cytochalasin D, 80 μM Dynasore, 200 μM genistein, or
5 mM methyl β-cyclodextrin. The receptor PAFR was
activated by pre-treating cells with PAF (10 μM) for 2 h.
The treatment solutions were removed then replaced withthe particle suspension containing fresh inhibitor or PAF
and incubated. After incubation, cells were washed threetimes to remove unbound particles and secretions produced
during the incubation period. Particle–cell association wasmeasured by flow cytometry.

20

2.10 Cell uptake inhibitor and activator studies

LOS with high ChoP content may enhance association withcells containing PAFR. Three NTHi strains were screened
to select for the strain with a high activity of ChoP. NTHi
strains 956 and 3198 expressed significantly higher ChoP
activity, as measured by absorbance of p-nitrophenol, thanthe 2019 strain (Fig. 1). The higher level of ChoP activity
on NTHi 3198 compared to NTHi 2019 was confirmedby dot immunoblot, with strain 3198 expressing more
ChoP on its surface at shorter exposure times (1 s) thanstrain 2019 (30 s), as observed in the higher intensity andpositive stained area of dark spots. This is in agreementwith published literature that reports 78 % of strain 3198
expresses ChoP with high reactivity, compared to 9 % ofstrain 2019 (Swords et al., 2000; Weiser et al., 1997). Further, the expression of ChoP on strains 2019 and 3198 was
higher when bacteria were cultured on BHI agar than on
chocolate agar; in contrast, no statistical difference in ChoP
expression was observed for strain 956 grown on the two
substrates (Fig. 1). Strain 3198 cultured on supplemented
BHI agar medium was chosen for LOS isolation and coating onto particles.

Absorbances of p-nitrophenol

biological buffer once to remove excess reagents. Triton
X-100 prepared at 0.05 % w/v in biological buffer wasused to permeabilize the cell membrane for 10 min. The
membranes were washed, the cover glass containing themembranes mounted to a microscope slide, and the cells
exposed to ProLong® Gold antifade reagent with theblue-fluorescent nuclear 4’,6-diamidino-2-phenylindole(DAPI 358 nm/461 nm; Life Technologies Co.) to stain
the cell nuclei. The cells were kept at 4 °C until imaged.
Z-stacks of cell layers were obtained using a LSM 710
upright confocal microscopy (Zeiss). A 40× oil immersion
objective lens was used to take images with step size of
200 nm. Images were rendered in MATLAB.

Original Research Paper

NTHi strain
Fig. 1 Relative absorbance of p-nitrophenol at 405 nm from colorimetric ELISA indicating the expression of ChoP on NTHi bacterial strains
2019, 256, and 3198 in either chocolate agar or brain heart infusion agar
growth medium (n = 3, mean ± SD). *indicates statistical difference
between NTHi grown in different agar media (p < 0.05).

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observed, suggesting LOS displaced the salt on the particle
surface (Fig. 2(d)). Similarly, exposing uncoated particles
with a protein solution (gelatin) resulted in visible residue
on the particle surface (Fig. 2(f)); however, this residue
was not observed on particles that were pre-coated with
5 μg/mL LOS prior to gelatin coating (Fig. 2(e)).
When suspended in water, uncoated particles exhibited
low polydispersity with a hydrodynamic diameter of~255 nm, and a zeta potential of about –50 mV (Table 1).
When placed in carbonate buffer, the particles agglomerated
slightly. Agglomeration was maintained with LOS coating
and was intensified with gelatin coating. When placed inserum-free media for cell studies, particles generally deagglomerated, except for the 5 μg/mL LOS coated particles
which exhibited slightly higher agglomeration. In addition,
particles suspended in media exhibited more neutral zeta
potentials than the same particles in water. Serum-free

Original Research Paper

media contains various salts that likely compressed theelectrical double layer surrounding the particles, resulting
in a reduction in the zeta potential. Particles coated with
5 μg/mL LOS exhibited the highest zeta potential in media
at about –25 mV, suggesting significant surface coveragewith the negatively charged LOS. LOS molecules have
COOH groups on their carbohydrate structures which arenegatively charged at physiologic pH, in addition to the
ChoP moieties which are zwitterionic at physiologic pH.
The surface chemistry of uncoated and functionalized
particles was investigated by XPS (Table 2). To confirm
the existence of a coating on the functionalized particle
surfaces, the raw coating materials were also analyzed.
LOS contained mostly carbon (49.6 %) and oxygen(35.2 %), with the remaining composed of 9.3 % nitrogen,
5.6 % phosphate, and 0.3 % sulphur. Gelatin contained nophosphate, but was comprised of 65.8 % carbon, 18.7 %

a

b

c

d

e

f

Fig. 2 Scanning electron microscopy images of (a) uncoated polystyrene particles, (b) particles washed with carbonate buffer, (c) particles coatedwith 5 ng/mL LOS, (d) particles coated with 5 μg/mL LOS, (e) particles coated with 5 μg/mL LOS and exposed to gelatin, (f) uncoated particles
exposed to gelatin. Scale bar = 500 nm (a) or 1 μm (b–f).
Table 1 Characterization of geometric size and zeta potential of polystyrene particles (200 nm nominal diameter) with various surface coatings in waterand serum-free media. Values are reported as mean ± SD (n = 3).

Particle coating

Suspension in water

Suspension in serum-free media

Particle diameter (nm)

Zeta potential (mV)

Particle diameter (nm)

Zeta potential (mV)

Uncoated

255.15 ± 6.6–50.0 ± 5.3——

Carbonate buffer

381.0 ± 14.2

–40.9 ± 0.7

157.7 ± 9.9–10.4 ± 1.2

5 ng/mL LOS

295.2 ± 6.4–46.8 ± 2.2

183.7 ± 7.4–8.8 ± 0.55 μg/mL LOS

351.6 ± 14.8

–47.8 ± 2.1

461.1 ± 7.8–24.5 ± 2.2

5 μg/mL LOS and gelatin

555.4 ± 254.6–39.5 ± 1.1

187.7 ± 13.8

–7.6 ± 0.9

Gelatin1053.8 ± 372.5–36.4 ± 4.9

165.7 ± 3.2–7.3 ± 0.9270Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

Original Research Paper

Table 2 Surface elemental analysis of LOS, gelatin, and polystyrene particles with various surface coatings via X-ray photoelectron spectroscopy.

% Atomic concentration

Material

C/O ratio

C 1s

O 1s

N 1s

P 2p

S 2p

LOS

49.6

35.2

9.3

5.6

0.3

1.4

Gelatin

65.8

18.7

15.3

0.0

0.2

3.5

Uncoated

19.5

54.3

0.0

6.7

19.5

0.36

5 ng/mL LOS

38.6

40.8

1.0

5.0

14.6

0.9

5 μg/mL LOS

57.7

29.2

0.5

3.1

9.6

2.0

5 μg/mL LOS and gelatin

62.7

24.9

4.1

2.4

5.9

2.5

Gelatin

75.9

16.7

4.5

0.6

2.3

4.5

Particles

oxygen, 15 % nitrogen and 0.2 % sulfur. Gelatin also
had a higher C/O ratio at 3.5, compared to LOS at 1.4. In
contrast, the uncoated particle surface contained carbon,
oxygen, and sulfur since the polystyrene particles used inthis study had a polystyrene backbone composed of carbon
and oxygen, and sulfate function groups. The polystyrene
particles had the smallest C/O ratio at 0.36. Some residual
phosphate (7 %) was also observed on the uncoated particle surface, likely due to contamination from the water
washes. Given these differences among the raw materials,
the phosphate, nitrogen, and C/O ratio were used to confirm functionalization of the particles.
Particles coated with a 5 ng/mL LOS solution exhibited
an elemental surface indicative of a mostly uncoatedpolystyrene surface with a small amount of LOS adsorbed(C/O ratio of 0.9, between that of LOS and polystyrene;
with existence of N indicating some LOS adsorption).
This suggests that LOS was on the particle surface but did
not completely coat the entire surface. With a higher LOS
coating solution concentration (5 μg/mL), the C/O ratio
increased to 2.0, suggesting a higher concentration of LOS
on the surface. In addition, the phosphorus and sulphur
concentrations at the surface decreased to 3.1 % and 9.6 %,respectively, suggesting that these moieties were buried
beneath the carbon and oxygen backbone of the LOS.
When uncoated particles or LOS-coated particles were
coated with gelatin, the nitrogen concentration and C/Oratio increased significantly compared to their uncoatedcounterparts, to values closer to that of pure gelatin. Thissuggests that gelatin partially masked the surface of theparticles, even in the presence of LOS.
The LOS coating process was further analyzed by ITC to
determine the stoichiometry of LOS binding to the polystyrene particles. ITC experiments were carried out using four
different concentrations of LOS (Fig. 3). The regression
lines for the 20 and 100 μg/mL titrations exhibited negligible
slope and therefore not analyzed. For the 30 and 50 μg/mL
titrations, the amount of LOS bound to the particle surface
increased with an increase in LOS concentration resulting

in an estimated 17.7 LOS binding sites per particle (titration with 30 μg/mL LOS) and 33.9 LOS binding sites per
particle (titration with 50 μg/mL LOS). Enthalpies of binding were large and negative for all titrations (–2 kcal/mol
to –10 kcal/mol), indicating strong binding of LOS to theparticle surface.

3.3 Uptake of particles in 16HBEo- lung epithelial
cells

PAFR expression in 16HBE14o- cells was confirmed
via immunostaining (Fig. 4). The secondary and isotype
controls exhibited low levels of staining due to non-specificreceptor interactions (Figs. 4(b) and 4(c), respectively),
which was quantified by flow cytometry via fluorescenceintensity (Fig. 4(e)). PAFR expression on the cell surface
was observed as diffuse staining across the entire cell surface (Fig. 4(d)). Flow cytometry confirmed that the entire
population of 16HBEo- cells stained for PAFR exhibited
higher intensity of fluorescence than controls, confirming
the cells expressed PAFR (Fig. 4(e)).
Next, lung epithelial cells were exposed to uncoated,gelatin-coated, or functionalized particles for up to 24 hand the percentage of cells associated with particles quantified by flow cytometry. Uncoated particles and particles
coated with 5 ng/mL LOS behaved similarly, with little
uptake from 0–19 h (<10 % of cells were associated withparticles) followed by a small increase at 24 h to 10 %(Fig. 5(a)). Gelatin coated particles were associated with at
most 12 % of cells at 4 h, which was not statistically different than uncoated particles. However, particles coated with
5 μg/mL LOS were associated with about 12 % of cells attime 0 and this rose steadily to about 35 % at 24 h. Confocal microscopy confirmed that the 5 μg/mL LOS-coatedparticles were internalized into the 16HBEo- cells and notjust attached to the cell surface (Fig. 5(b)).
The energy dependence of particle uptake in 16HBEocells was quantified after cell incubation with NaN3/
2-deoxyglucose, which inhibits all energy-dependent
pathways (Fig. 6). Incubation of cells with NaN3/2-

271

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aLOS:nanoparticle molar ratio
0

0

5

10

15

20

25

b

ΔH (kcal/mol)

ΔH (kcal/mol)-6

Trial 1
Trial 2

-10

0

20

40

60

40

-4
-6

d

Trial 1
Trial 2

LOS:nanoparticle molar ratio
0

0

25

50

75

100

125

-2

-4
-6
Trial 1
Trial 2
Trial 3

ΔH (kcal/mol)-2
ΔH (kcal/mol)

30

-12

LOS:nanoparticle molar ratio

-12

20

-10

-12

-10

10

-8

-8

-8

0

-2

-4

0

LOS:nanoparticle molar ratio
0

-2

c

Original Research Paper

-4
-6
-8
-10

Trial 1
Trial 2

-12

Fig. 3 Isothermal titration calorimetry (ITC) plots of ΔH versus LOS–nanoparticle molar ratio with single injections of (a) 20 μg/mL, (b) 30 μg/mL,(c) 50 μg/mL, or (d) 100 μg/mL of LOS. Colored symbols represent unique replicates. Lines represent regression curves originated by Origin software.
A buffer–nanoparticle titration was used as a reference and subtracted from all data sets.

deoxyglucose resulted in complete loss of uptake of the
5 μg/mL LOS-coated particles, suggesting these particles
are taken up by endocytosis. No other particle type experienced inhibited cell uptake after cell exposure to NaN3/
2-deoxyglucose and thus may be taken up by both energydependent and energy-independent pathways.
Further studies were conducted with specific pathway
inhibitors to help elucidate specific uptake pathway for different particle types. Inhibitors of micropinocytosis (cytochalasin D) and caveolin-mediated endocytosis (genistein)
did not inhibit the uptake of any particle type. Instead, particle uptake was sometimes enhanced with cell exposure tothese inhibitors, suggesting that particles were taken up by
multiple pathways. The inhibitor of cholesterol- dependent
pathways (methyl β-cyclodextrin), which inhibits both
caveolin-mediated and clathrin-mediated endocytosis, significantly reduced cell uptake of the 5 μg/mL LOS-coatedparticles. This suggest that the LOS-coated particles wereprimarily taken up by receptor-mediated endocytosis.
However, due to the significant cell toxicity exhibited by
the inhibitors of clathrin-mediated endocytosis, this could
not be confirmed via chemical pathway inhibitors.
To prove that receptor-mediated endocytosis was theroute by which LOS-coated particles were taken up into
cells, we investigated whether PAFR-mediated endocytosis could be altered by the cognate ligand of the receptor(PAF). Upon cell incubation with PAF, the 5 μg/mL
LOS-coated particles exhibited a 5- to 10-fold increase in272

uptake (Fig. 7). No significant change in the associationof particles with lung cells was observed with the otherparticle types.

4. Discussion

Lipopolysaccharides (LPS), also known as endotoxins,
are large lipid- and polysaccharide-containing molecules
constituting the major component of the outer membraneof Gram-negative bacteria. They induce a strong immune
response in animals and have been implicated in various
pathogenic functions (Alexander and Rietschel, 2001;
Simpson and Trent, 2019). Key studies have shown thatisolating LPS from the bacterial surface and attaching it toa particle surface may not alter its functionality, enabling
the particles to mimic some bacterial properties (Piazza
et al., 2011). For example, titanium dioxide particles
coated with LPS from E-coli induced pro-inflammatory
signaling in primary human mononuclear phagocytes(Ashwood et al., 2007). LPS associated with polymericand metallic components of orthopedic implants stimulate
toll-like receptor (TLR) activation, which contributed to an
inflammatory response (Greenfield et al., 2010; Hold andBryant, 2011). However, the activation of the inflammatory
response by LPS-coated particles was not desired for thecurrent application.
Lipooligosaccharides (LOS) share a similar lipid A
structure to LPS, but are lower in molecular weight,
lack the O-antigens that allow serotyping, and have

Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279cba% Cells associated with particlesaOriginal Research Paperd

Uncoated 0.2 µm particles
LOS-coated 0.005 µg/ml 0.2 µm particles (no gelatin)
LOS-coated 5 µg/ml 0.2 µm particles (no gelatin)
Gelatin-coated 0.2 µm particles
LOS-coated 5 µg/ml 0.2 µm particles (with gelatin)

40
30
20
10
0

0

10
20
Incubation time (hours)

30

b

20 µm

e

% Cells

PAFR
Isotype control
Secondary control
No stainFluorescence intensity
Fig. 4 (a–d) Confocal micrographs of 16HBE14o- cells with nuclei
stained using DAPI (blue) and incubated with the (b) secondary antibody (secondary control, red stain), (c) fluorescent antibody IgG2ak to
confirm the specificity of primary antibody binding (isotype control, red
stain), or (d) the primary and secondary antibodies (red stain for PAFR).
Scale bar = 20 μm. (e) Percent fluorescent cells as a function of fluorescence intensity for cells not stained (far left curve) or incubated with thesecondary antibody (secondary control, second curve from left), IgG2ak(isotype control, third curve from left), or the primary and secondary
antibodies (PAFR staining, far right curve).

significantly lower toxicity profiles (Preston et al., 1996).
LOS is the major glycolipid expressed on mucosal Gramnegative bacteria, including members of the genera
Neisseria, Haemophilus, Bordetella, and Branhamella.
It is a heat stable, amphipathic glycolipid complex thatcovers up to 75 % of the outer membrane (Silipo et al.,2011), and is crucial for the viability and survival of Gramnegative bacteria as it helps maintain proper outer membrane structure. It also contributes to host-bacterium interactions related to adherence, colonization, internalization,and ultimately survival in host cells.
The oligosaccharide region of LOS is highly heterogeneous within and between bacterial strains, containing bothFig. 5 (a) Percentage of 16HBE14o- cells associated with uncoatedand functionalized particles as a function of incubation time at 37 °C(n = 4, mean ± SD). (b) Confocal microscopy image of 16HBEo- cells
exposed to 5 μg/mL LOS-coated particles. Cell membranes were labeled by DiI (red fluorescence) and cellular nuclei were labeled by DAPI
(blue fluorescence), and particles fluoresce green. Two representative
cross-sections of the cell layer are displayed at top and right side of the
micrograph, with green fluorescing particles denoted by the arrows.

the recognition structures for host cells and componentsof the immune system, as well as carbohydrate moietiesthat may camouflage the bacterial surface from the hostthrough molecular mimicry of host structures (Mandrelland Apicella, 1993; Masoud et al., 1997; Moran et al.,
1996; Murphy and Apicella, 1987; Risberg et al., 1999a,
1999b; Silipo et al., 2011). In some bacterial species,
these carbohydrate structures further facilitate bacterial
adherence to the respiratory epithelium (Clark et al., 2012;
Jalalvand and Riesbeck, 2014; Swords et al., 2000, 2001).
Phosphorylcholine (ChoP) expressed as a terminal structure of the OS region facilitates adherence of multiple bacterial pathogens, including NTHi, to the platelet-activating
factor receptor (PAFR) on bronchial cells by molecularly
mimicking the structure of the cognate ligand plateletactivating factor (PAF) (Cundell et al., 1995; Gillespie etal., 1996; Harvey et al., 2001; Lysenko et al., 2000; Rose-

273

Normalized % cells associated with particles

Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

1000

Original Research Paper

Uncoated 0.2 µm polystyrene particles
LOS-coated 0.005 µg/ml 0.2 µm polystyrene particles (no gelatin)
LOS-coated 5 µg/ml 0.2 µm polystyrene particles
Gelatin-coated 0.2 µm polystyrene particles
LOS-coated 5 µg/ml 0.2 µm polystyrene particles (with gelatin)

800
600

*
*

*
*

400

*

*

200
0

Control

NaN3 /2-deoxyglucose

Cytochalasin D

Genistein*
Methyl β-cyclodextrin

1000

*

800
600
400
200

PA
F

0
C
on
tr
ol

Normalized % cells associated with particles

Fig. 6 Percentage of cells, as determined by flow cytometry, associated with 16HBEo- cells before (control) and after exposure to various inhibitorsfor 2 h. Data is reported as mean ± SD (n = 4). * indicates statistically significant differences compared to the absorbance of untreated cells.

Uncoated 0.2 µm particles
LOS-coated 0.005 µg/ml 0.2 µm particles (no gelatin)
LOS-coated 5 µg/ml 0.2 µm particles (no gelatin)
Gelatin-coated 0.2 µm particles
LOS-coated 5 µg/ml 0.2 µm particles (with gelatin)

Fig. 7 Normalized percentage of 16HBE14o- cells associated with
uncoated and functionalized polystyrene particles before (control) or
after activation with PAF (n = 8, mean ± SD).

now et al., 1997; Schweda et al., 2000; Weiser et al., 1997,1998). As a potential targeting ligand for particles, our previous studies indicated that coupling ChoP-containing LOS
from NTHi (strain 2019) to the surface of 1 μm particles facilitated significantly more binding to the human bronchial
epithelial surface compared to particles coated with gelatinor coated with truncated LOS lacking the oligosaccharide
chains that contain ChoP (Swords et al., 2000). Here, we
aimed to evaluate the ability of ChoP-containing LOS tofacilitate the uptake of smaller nanoparticles into respira-274

tory epithelial cells.
Given the heterogeneity of the OS expression between
bacterial strains, a screening of clinically isolated NTHi
strains was performed to select a strain with high ChoP
content to facilitate particle adherence and uptake into lungcells. Two of the three NTHi clinical isolates tested (956
and 3198) exhibited high ChoP activity. While the ChoP
content of strain 956 had not been previously reported, theobserved differences between strains 3198 and 2019 agreed
with published literature, which has reported that 78 % ofstrain 3198 expressed ChoP with high reactivity, compared
to 9 % of strain 2019 (Swords et al., 2000; Weiser et al.,1997). Based on these results and the known ability of
NTHi 3198 to associate with and invade primary lung epithelial cells (Ketterer et al., 1999), LOS from NTHi 3198
was chosen as the ligand for further studies.
LOS is a large molecule containing a lipid structurethat embeds in the bacterial outer membrane and an
oligosaccharide structure that is exposed on the surface.
The polystyrene particles used in this study exhibit a hydrophobic, negatively charged surface containing sulfate
ester functional groups. Due to the hydrophobicity of theparticles and the lipophilicity of the LOS lipid tails, we
anticipated that the lipid tails of the LOS molecules would
readily adsorb to the hydrophobic particle surface. In thiscase, the carbohydrate heads on the LOS structure containing ChoP could be available for cell targeting. Basedon analysis of the zeta potential and XPS data, LOS wasindeed coated onto the surface of polystyrene particles,
particularly with the more highly concentrated LOS
coating solution. However, the XPS data also suggested
that the phosphate groups were buried within a mostly
carbon-oxygen LOS backbone. This would be undesirable
if the ChoP functional groups were not available to function
as a targeting ligand on the particle surface. As XPS studies

Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

were conducted on dry particles, when the carbohydrate
chains become hydrated, the LOS structure could open andgive access to the ChoP moieties.
ITC further confirmed the ability of LOS to strongly
adsorb to the nanoparticle surface. Typical enthalpies ofbinding for proteins to polystyrene nanoparticle surfaces
has been reported to be on the order of 10 s to 100 s ofkcal/mol (Porzeller et al., 2019), similar to the values
obtained in this study. Using titration concentrations of 30
and 50 μg/mL, we estimated that there are about 34 LOS
binding sites per particle. The titration concentration of
50 μg/mL (~7 μg/mL final concentration in the cell after titration), resulted in slightly more adsorbed LOS molecules
than the 30 μg/mL titration concentration (~4 μg/mL final
concentration in the cell after titration). This suggests thathigher LOS concentrations enhance LOS adsorption to theparticle surface. Previous work by Swords et al. reported
a dose-response in which increasing the concentration of
LOS coating resulted in more particle adherence to respiratory epithelium (Swords et al., 2000). LOS concentration
is limited, though, as it readily forms micelles in aqueous
solution at low concentrations due to its amphiphilic nature(Steimle et al., 2016). Thus, a higher LOS coating might beachieved by using a co-solvent to better solubilize the LOS.
The ability of LOS to form micelles, as well as the complex
structure of LOS, are potential reasons for the relatively
high variability in the ITC data.
If the ChoP moieties on the LOS molecules remained
accessible on the particle surface, we would expect theparticles to exhibit better binding to and internalization into
the respiratory epithelium. PAFR is expressed on humanlung tissue membranes (Hwang et al., 1985; Shukla et al.,2016) and various lung epithelial cell lines (Chen et al.,2015). In the current study we confirmed PAFR expressionin 16HBEo- bronchial epithelial cell monolayers via immuno-fluorescent staining and used these cells to evaluate
the uptake of uncoated and coated particles. The 5 μg/mL
LOS-coated particles exhibited the highest cell uptake ofnanoparticles, with about 35 % uptake at the 24 h timepoint. All other particle types were taken up by less than
12 % of cells at any time. Confocal microscopy confirmedthat particles were not just bound to the surface, but wereinternalized into the cell layers. While the ability of PAFR
to mediate particle attachment to the respiratory epithelium
had been previously established (Swords et al., 2000), thisis the first time to our knowledge that bacterial-derived
LOS has been used as a targeting ligand to facilitate particle uptake into cells.
We further investigated the uptake mechanism by blocking specific endocytic pathways using chemical inhibitors.
NTHi bacteria have been shown to internalize into humanlung epithelial cells by macropinocytosis and receptormediated endocytosis, so these pathways were likely candidates for LOS-coated nanoparticle uptake (Clementi and

Original Research Paper

Murphy, 2011; Ketterer et al., 1999; Swords et al., 2000, 2001).
Pre-incubation of the lung cells with NaN3-2-deoxyglucose
completely knocked out their ability to take up the 5 μg/mL
LOS-coated particles, suggesting an energy-dependent
pathway for these particles, but had no impact on the otherparticle types. We further saw no evidence of the LOScoated particles being taken up by macropinocytosis. Due
to significant cell toxicity upon cell incubation with inhibitors of the clathrin-mediated pathway, we were unable toconfirm this pathway using chemical inhibitors of endocytic pathways. Thus, a different approach was chosen inwhich the impact of a PAFR activator on the cellular uptakeof uncoated and functionalized particles was investigated.
A549 cells were pre-treated with PAF, the natural substratefor PAFR (Chao and Olson, 1993). Prior studies haveshown that PAFR stimulation is involved in bacterial entry
and could assist the delivery of siRNA to airway epithelia(Krishnamurthy et al., 2014; Zhang et al., 2000). In the current study, PAF pre-treatment significantly enhanced cell
uptake of the 5 μg/mL LOS-coated particles, suggestingthat PAF activation aided their uptake. Activation of PAFR
via PAF exhibited no significant change on the associationof any other particle type, demonstrating receptor targetingwith the LOS coating.
In summary, the functionalization of polystyrene nanoparticles with LOS bacterial ligands resulted in better cellular uptake in PAFR-expressing lung cells. This strategy
could be extended to drug-containing nanoparticles toenhance the therapeutic efficacy of drug compounds that
require cellular internalization. Further, receptor-ligand
interactions could be enhanced by activating the cell
receptor, suggesting that enhanced cellular uptake could
be achieved in disease states where the receptor is alreadyactivated, such as in the instance of infection, COPD,
asthma, and cancer.

Acknowledgments

Funding for this work was provided by the National
Institutes of Health (R21HL113876 and P30 DK054759).
M. Timm was supported through an NSF-REU programin Nanoscience and Nanotechnology (NSF 1359063). We
thank the University of Iowa Central Microscopy Research
Facility, a core resource supported by the Vice President
for Research & Economic Development, the Holden
Comprehensive Cancer Center, and the Carver College ofMedicine, for access to and training on the confocal andelectron microscopes. Flow cytometry data presented wereobtained at the Flow Cytometry Facility, which is a Carver
College of Medicine / Holden Comprehensive Cancer
Center core research facility at the University of Iowa. The
Facility is funded through user fees and the generous financial support of the Carver College of Medicine, Holden
Comprehensive Cancer Center, and Iowa City Veteran’s
Administration Medical Center. Part of the study has been275

Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

published in the PhD thesis of author Mai Tu (Tu, 2015).

Nomenclature
BHI

Brain heart infusion

BSA

Bovine serum albumin

ChoP

Phosphorylcholine

ELISA

Enzyme-linked immunosorbent assay

ITC

Isothermal titration calorimetry

LOS

Lipooligosaccharide

LPS

Lipopolysaccharide

NAD

Nicotinamide adenine dinucleotide

NTHi

Non-typeable Haemophilus influenzae

PAF

Platelet-activating factor

PAFR

Platelet-activating factor receptor

pNPP

Nitrophenyl phosphate bis (cyclohexyl ammonium)

SDS

Sodium dodecyl sulfate

SEM

Scanning electron microscope

XPS

X-ray photoelectron spectrometer

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Original Research Paper

Authors’ Short Biographies
Mai H. Tu
Mai H. Tu earned a Ph.D. in pharmaceutics from the University of Iowa, USA under the guidanceof Dr. Jennifer Fiegel. Her doctoral work focused on developing lipooligosaccharide-modified polymeric particles for targeted pulmonary drug delivery.

Timothy M. Brenza
Timothy M. Brenza completed a postdoctoral fellowship under the guidance of Dr. Jennifer Fiegel
from 2008–2010. He is currently an Assistant Professor of chemical and biological engineering at
South Dakota School of Mines and Technology.

Margaret R. Ketterer
Margaret R. Ketterer has worked at the University of Iowa Department of Microbiology and
Immunology, in the laboratory of Dr. Michael Apicella, since 1993. Her research efforts have concentrated on the role of bacterial lipo-oligosaccharides in infections of human cells by Neisseria species
and by Haemophilus influenzae.

Morgan Timm
Morgan Timm received a B.S. degree in biochemistry and molecular biology from Gustavus
Adolphus College, USA and is currently an MD/PhD student at Washington University, USA. She
conducted a summer research project in the laboratory of Dr. Jennifer Fiegel as part of the Nano NSF
REU program at the University of Iowa.

Benjamin M. King
Benjamin M. King earned a Ph.D. in chemical and biochemical engineering from the Universityof Iowa, USA in 2018 under the guidance of Dr. Jennifer Fiegel. His doctoral work focused on interactions of environmental and therapeutics particles with the airway microenvironment.

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Mai H. Tu et al. / KONA Powder and Particle Journal No. 41 (2024) 265–279

Original Research Paper

Authors’ Short Biographies
Michael A. Apicella
Michael A. Apicella is a Professor Emeritus of microbiology and immunology at the Universityof Iowa. His research focused on understanding the factors involved in human infections caused byGram-negative bacterial pathogens.

Jennifer Fiegel
Jennifer Fiegel is a Professor of chemical and biochemical engineering at the University of Iowa
(Iowa City, Iowa), with a secondary appointment in pharmaceutical sciences and experimental therapeutics. Her research expertise is in the design of improved therapeutic drug delivery strategies forthe treatment and prevention of infections.

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