Mucoadhesive Nanoparticles May Disrupt the Protective
Human Mucus Barrier by Altering Its Microstructure
Ying-Ying Wang1, Samuel K. Lai2¤, Conan So1, Craig Schneider2, Richard Cone3, Justin Hanes1,2,4,5,6,7*
1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2Department of Chemical and
Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America, 3Department of Biophysics, Johns Hopkins University, Baltimore,
Maryland, United States of America, 4Department of Ophthalmology, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United
States of America, 5Department of Environmental Health Sciences, Johns Hopkins School of Public Health, Baltimore, Maryland, United States of America, 6Center for
Cancer Nanotechnology Excellence, Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland, United States of America, 7Center for
Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Mucus secretions typically protect exposed surfaces of the eyes and respiratory, gastrointestinal and female reproductive
tracts from foreign entities, including pathogens and environmental ultrafine particles. We hypothesized that excess exposure
to some foreign particles, however, may cause disruption of the mucus barrier. Many synthetic nanoparticles are likely to be
mucoadhesive due to hydrophobic, electrostatic or hydrogen bonding interactions. We therefore sought to determine
whether mucoadhesive particles (MAP) could alter the mucus microstructure, thereby allowing other foreign particles to more
easily penetrate mucus. We engineered muco-inert probe particles 1 mm in diameter, whose diffusion in mucus is limited only
by steric obstruction from the mucus mesh, and used them to measure possible MAP-induced changes to the microstructure
of fresh human cervicovaginal mucus. We found that a 0.24% w/v concentration of 200 nm MAP in mucus induced a ,10-fold
increase in the average effective diffusivity of the probe particles, and a 2- to 3-fold increase in the fraction capable of
penetrating physiologically thick mucus layers. The same concentration of muco-inert particles, and a low concentration
(0.0006% w/v) of MAP, had no detectable effect on probe particle penetration rates. Using an obstruction-scaling model, we
determined that the higher MAP dose increased the average mesh spacing (‘‘pore’’ size) of mucus from 380 nm to 470 nm.
The bulk viscoelasticity of mucus was unaffected by MAP exposure, suggesting MAP may not directly impair mucus clearance
or its function as a lubricant, both of which depend critically on the bulk rheological properties of mucus. Our findings suggest
mucoadhesive nanoparticles can substantially alter the microstructure of mucus, highlighting the potential of mucoadhesive
environmental or engineered nanoparticles to disrupt mucus barriers and cause greater exposure to foreign particles,
including pathogens and other potentially toxic nanomaterials.
Citation: Wang Y-Y, Lai SK, So C, Schneider C, Cone R, et al. (2011) Mucoadhesive Nanoparticles May Disrupt the Protective Human Mucus Barrier by Altering Its
Microstructure. PLoS ONE 6(6): e21547. doi:10.1371/journal.pone.0021547
Editor: Wei-Chun Chin, University of California, Merced, United States of America
Received March 17, 2011; Accepted June 1, 2011; Published June 29, 2011
Copyright: ? 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the National Institutes of Health (http://www.nih.gov/) grants 5U01AI066726 (R.C.), P01HL51811 (J.H.),
R21HL089816 and R01HD062844 (J.H. and R.C.) and by a National Science Foundation (http://www.nsf.gov/) graduate research fellowship (Y.-Y.W.). The content is
solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute, the National Institute
of Biomedical Imaging and Bioengineering, the National Cancer Institute or the National Institutes of Health. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
Engineered or synthetic nanoparticles are increasingly used in
diverse applications, ranging from ultra-light high strength
materials to electronics, cosmetics and medicine. As the manufac-
turing and use of synthetic nanoparticles become more common,
the potential for unintended human exposure also increases.
Concerns over the safety of nanoparticles have centered primarily
on their small size and, therefore, potential for deeper tissue
penetration and higher reactivity (due to their high surface area to
volume ratio) [1–3]. Recent studies have shown that some non-
degradable environmental or engineered nanoparticles may
trigger significant pulmonary inflammation and/or immunological
responses in vivo [1–3]. Several investigators have also suggested
certain synthetic nanoparticles can be directly toxic to cells
through the generation of reactive oxygen species [2–3].
Exposure to synthetic nanoparticles may occur via percutaneous
or trans-mucosal absorption. Nevertheless, the interactions
between nanoparticles and mucus in the airways, as well as other
mucosal organs, have yet to be fully characterized. Mucus is a
viscoelastic and adhesive gel that coats and protects nearly all
exposed surfaces of the human body not covered by skin, serving
as the first line of defense against foreign particles that impinge on
these surfaces. The dense mucin mesh network allows mucus to
efficiently trap most foreign particles through steric obstruction
and/or adhesion via hydrophobic, electrostatic or hydrogen
bonding interactions . Trapped particles are quickly eliminated
in as little as seconds to minutes (surface of the eye) to minutes to
hours (gastrointestinal, respiratory and female reproductive tracts)
by normal mucus clearance mechanisms [4–6]. Synthetic
nanoparticles typically feature hydrophobic, charged and/or
hydrogen bonding surfaces [7–10] and are, therefore, likely to
PLoS ONE | www.plosone.org1June 2011 | Volume 6 | Issue 6 | e21547
be strongly mucoadhesive due to interactions with periodic
exposed hydrophobic domains or negatively charged glycosylated
segments along mucin fibers . Although these and other
mucoadhesive particles are unlikely to perturb the microstructure
of mucus at low exposure levels, at high concentrations, they may
crosslink mucin fibers and cause them to bundle together,
enlarging pores in the mucus gel (Figure 1). Larger pores may
compromise the ability of mucus to trap foreign particles, and
increase the likelihood that other foreign particles or pathogens
will reach and potentially injure the underlying epithelia.
Here, we sought to investigate the ability of synthetic
mucoadhesive particles (MAP) to alter the microstructure of
human mucus, using 200 nm amine-modified polystyrene nano-
particles as model MAP. We performed our studies with freshly
obtained and minimally altered ex vivo human cervicovaginal
mucus (CVM), a physiological, highly viscoelastic gel that is
difficult to reproduce in cell culture. To detect changes in the
mucus microstructure, we use 1 mm muco-inert particles as probes
whose diffusional speeds can be related to effective pore sizes in the
mucus gel. This method involves minimal perturbation of the
mucus sample, in contrast to electron microscopy, which is known
to introduce artifacts due to dehydration and fixation steps during
sample preparation [11–13]. We have recently employed this
technique to characterize the pore size of native CVM  and
cystic fibrosis sputum . The speeds of the probe particles also
allow us to estimate the fraction of foreign particles that may
penetrate a physiologically thick mucus layer over time, providing
another assessment of how MAP may compromise the mucus
Materials and Methods
Research ethics approval for the collection of human CVM
samples used in this study was received from the Institutional
Review Board of the Johns Hopkins University. Written
informed consent was obtained from each participant enrolled
in the study.
Human cervicovaginal mucus collection
Mucus samples were obtained from women of reproductive age
(ranging from 18 to 27 years old) with healthy vaginal micro-flora.
Donors stated they had not used vaginal products nor participated
in unprotected intercourse within 3 days prior to donating.
Undiluted CVM, averaging 0.3 g per sample, was collected using
a self-sampling menstrual collection device following protocols
approved by the Institutional Review Board of the Johns Hopkins
University [8,16]. The device was inserted into the vagina for
approximately 30 s, removed, and placed into a 50 mL centrifuge
tube. Samples were centrifuged at 1000 rpm for 30 s to collect the
secretions. Collected mucus was stored at 4 uC until used for
microscopy the same day. The samples were collected at random
times throughout the menstrual cycle, but none were obtained
during ovulation based on the absence of spinnbarkeit by visual
inspection. Samples that were non-uniform in color or consistency
Nanoparticle preparation and characterization
For model MAP, we used amine-modified polystyrene nano-
particles sized 200 nm (Molecular Probes, Eugene, OR), which
were concentrated to 8% w/v (,1013particles/mL) by centrifu-
gation and resuspension in water. These particles feature a
hydrophobic core and a positively charged surface at the native
pH of CVM (pH,4), and may adhere to mucus through
hydrophobic interactions with oily domains along mucin fibers
or through electrostatic and/or hydrogen bonding interactions
with the negatively charged, glycosylated domains of mucins .
For muco-inert probe particles, we covalently modified fluores-
cent, carboxylated polystyrene particles sized 1 mm (Molecular
Probes, Eugene, OR) with 2 kDa amine-modified poly(ethylene
glycol) (PEG; Nektar Therapeutics, San Carlos, CA) via a
carboxyl-amine reaction, as published previously . PEG is a
hydrophilic and uncharged polymer that, at high surface density
and low MW, can effectively shield the hydrophobic polystyrene
core from adhesive interactions with mucins, while also minimiz-
ing interpenetrating network effects (polymer chain entanglements)
and hydrogen bonding between PEG chains and mucins [8,17].
Particle size and j-potential (Table 1) were determined by
dynamic light scattering and laser Doppler anemometry, respec-
tively, using a Zetasizer Nano ZS90 (Malvern Instruments,
Southborough, MA). Size measurements were performed at
25uC at a scattering angle of 90u. Samples were diluted in
10 mM NaCl solution (pH 4 or pH 7) and measurements
performed according to instrument instructions. A near-neutral
j-potential at pH 7 was used to confirm PEG conjugation onto the
1 mm probe particles . Based on our previous findings, muco-
inert particles 1 mm or larger in diameter are highly hindered in
native human mucus by steric obstruction, whereas 500 nm muco-
inert particles are nearly freely diffusive due to the availability of
Figure 1. Schematic illustrating the potential effects of
synthetic mucoadhesive particles (MAP) on the microstructure
of mucus. MAP may increase mucus pore sizes by bundling mucin
fibers through polyvalent adhesive interactions. This would allow larger
non-mucoadhesive particles to penetrate mucus more readily.
Table 1. Characterization of mucoadhesive (MAP) and muco-
inert (PEG-coated) probe particles.
pH 4pH 7
aProvided by the manufacturer.
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org2June 2011 | Volume 6 | Issue 6 | e21547
sufficiently large spaces or pores in the mucus mesh [14,18]. As
such, 1 mm muco-inert particles are sensitive probes for detecting
increases in mucus pore size.
Multiple particle tracking
Each mucus sample was separated into aliquots and treated with
8% w/v MAP or, as controls, (i) saline, (ii) a diluted solution of
MAP (0.02% w/v) or (iii) 8% w/v of 200 nm muco-inert particles
(prepared and characterized as described above for 1 mm probe
particles). Solutions were added to mucus at 3% v/v, with gentle
stirring to achieve visually uniform particle distribution, yielding
final particle concentrations of 0.24% w/v or 0.0006% w/v. The
concentration of 0.24% w/v falls within dose limits (0.17–0.35%
w/v) estimated based on the Occupational Safety and Health
Administration’s Permissible Exposure Limits for airborne partic-
ulates, such as metal oxide nanoparticles (,10 mg/m3, 8 hr time-
weighted average ); an average breathing rate of 3.2 m3/hr for
heavily active adults ; a deposition fraction of ,5–10% for
0.1–1 mm particles in the tracheobronchial (TB) region of the
lungs ; a TB surface area of 2741 cm2; and an average
TB mucus layer thickness of 30 mm . Treated samples were
incubated at least 15 min prior to addition of diluted probe
particle solutions (,1010particles/mL) at 5% v/v. Samples were
transferred to custom-made chambers holding 20–30 mL of mucus
each and incubated 2 hr before microscopy. Samples incubated at
room temperature or at 37uC yielded similar results. The
trajectories of the fluorescent probe particles in CVM were
recorded using a silicon-intensified target camera (VE-1000, Dage-
MTI, Michigan, IN) mounted on an inverted epifluorescence
microscope equipped with 100X oil-immersion objective (numer-
ical aperture 1.3). Movies were captured with Metamorph
software (Universal Imaging Corp., Downingtown, PA) at a
temporal resolution of 66.7 ms for 20 s. The tracking resolution
was 10 nm, as determined by tracking the displacements of
particles immobilized with a strong adhesive . Trajectories of
n.100 probe particles were analyzed for each experiment, and six
experiments were performed in independent CVM samples from
different donors. The coordinates of particle centroids were
transformed into time-averaged mean squared displacements
(MSD), calculated as ,Dr2(t).= [x(t + t) – x(t)]2+ [y(t + t) –
y(t)]2(where t = time scale or time lag), from which individual
particle and ensemble average MSD and effective diffusivities (Deff
= MSD/(4t) for 2D particle tracking) were calculated, as
previously demonstrated [8,24]. Immobile particles are defined
as those with an average MSD below the 10 nm tracking
resolution at a time scale of 1 s.
Mucus pore size analysis
The sizes of pores between mucin fibers of saline- and MAP-
exposed CVM were estimated based on an obstruction-scaling
model originally developed by Amsden and coworkers for
covalently cross-linked hydrogels, but equally applicable to gels
with physical entanglement cross-links, such as mucus [25–28].
The model is valid when there is no chemical interaction between
probe particles and the gel mesh, and particles traveling through
the pores of the gel experience the viscous drag of water. We have
shown previously that particles well coated with low MW PEG
exhibit minimal adhesive interactions with mucus constituents
[8,14,17], as well as other synthetic particles (unpublished
observations). The model describes the ratio of diffusion in a gel
and diffusion in water as Dg/Do= exp((2p/4)((rs+ rf)/(rg+ rf))2),
where Dgis the diffusion coefficient of the probe particle in the
polymer gel, Dois its diffusion coefficient in water, rsis the particle
radius, rfis the gel fiber radius, and rgis the effective radius of the
pore. An rfof 3.5 nm was used as the current best estimate for the
radius of individual mucin fibers from biochemical, electron
microscopy and atomic force microscopy observations [27–28].
Simulation of particle penetration
To estimate particle penetration across mucus, we performed a
Monte Carlo simulation of one-dimensional particle transport
across a mucus slab of uniform thickness. The simulation consisted
of ,20,000 particles with effective diffusivities equal to those
measured experimentally for the 1 mm PEG-coated probe particles
in saline- or MAP-exposed CVM at a time scale of 1 s (30 particles
simulated per measured diffusivity). Particles were initially located
at one surface of the slab, and then allowed to undergo Brownian
motion. Particle positions over time (in steps of 1 s) were recorded,
and the fraction of penetrable particles was calculated as the
fraction that reaches the opposite surface of the mucus slab.
Bulk rheological characterization of MAP-exposed CVM
Bulk rheological characterization of CVM was performed with
a strain-controlled cone and plate rheometer (ARES-100, Rheo-
metrics, Piscataway, NJ). CVM from 4–5 donor samples was
pooled (total volume ,1.3 mL) and stored at 4uC until use. The
temperature of specimens was maintained at 37uC during
measurements. Oscillatory deformations of small amplitude (1%
strain) and controlled frequency were applied to extract the
frequency-dependent viscoelastic properties with minimal shearing
damage to the CVM samples. We report the frequency-dependent
elastic and viscous moduli, G9(v) and G0(v), which are the in-
phase and out-of-phase components, respectively, of the stress
induced in the CVM samples divided by the maximum amplitude
of the applied deformation. Phase angle is defined as arctan(G0/
G9); a phase angle of 90u corresponds to a viscous liquid, while 0u
corresponds to an elastic solid. The rheology of control and MAP-
exposed CVM was evaluated in sequential order. As with particle
tracking experiments, MAP (8% w/v) were added at 3% v/v,
gently stirred to ensure exposure of the entire sample, and
incubated at least 15 min prior to subsequent measurements.
All data are presented as a mean with standard error of the
mean (mean 6 SEM) indicated. Statistical significance between
saline and MAP conditions was determined by a one-tailed, non-
parametric Wilcoxon signed-rank test, since the response of mucus
samples to MAP depends on a number of factors (see Discussion)
and cannot be assumed to be normally distributed. p values less
than 0.05 were considered statistically significant.
Results and Discussion
Effect of MAP on mucus microstructure
In agreement with our previous work [14,18], the diffusion of
1 mm muco-inert (PEG-coated) probe particles was strongly
hindered in control mucus samples treated with 3% v/v saline,
as evident by their constrained and non-Brownian trajectories
(Figure 2A and Video S1). In contrast, the probes exhibited more
diffusive trajectories in aliquots of the same native mucus samples
treated with 3% v/v MAP at a toxicologically relevant dose of
0.24% w/v final concentration (see Methods for details; Video S2).
As we have previously observed , MAP were nearly completely
immobilized in mucus (data not shown), whereas similarly sized
and larger (up to at least 500 nm) muco-inert particles diffuse
freely through mucus, suggesting MAP must be immobilized by
adhesive interactions to the mucin mesh network. Using multiple
particle tracking [8,24], we quantified probe particle motions in
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org3 June 2011 | Volume 6 | Issue 6 | e21547
terms of ensemble average mean square displacements (,MSD.;
Figure 2B). Five out of six independent samples showed an
increase in probe particle ,MSD. upon exposure of mucus to
MAP, while one showed no significant change. On average, MAP
exposure increased probe particle effective diffusivity by ,10-fold
for each sample, at a time scale of 1 s. This improvement was also
reflected by the slope a of the log-log ,MSD. vs. time scale plot
(a=1 represents unconstrained Brownian transport, while de-
creasing a corresponds to increased obstruction to particle
movement). The average a was 0.68 for probe particles in treated
samples, compared to only 0.36 in control samples. The increase
in particle transport rates was due in part to a significant drop in
the fraction of immobile probes (from 33% to 20%; Figure 3).
Since well-PEGylated particles have minimal affinity to mucin
fibers [8,17], the increase in probe particle speeds in MAP-
exposed mucus suggests MAP enlarged the pores in the mucus
mesh. To quantify this change in the mucus microstructure, we
fitted the effective diffusivities of the probe particles to an
obstruction-scaling model, which relates the obstruction experi-
enced by non-interacting solute particles to the pore size of the
surrounding mesh network [25–26]. The average pore size of
mucus samples increased significantly from 380630 nm in the
saline control to 470680 nm in the MAP-exposed condition
(p,0.05). The fraction of pores with spacings $500 nm increased
from 11% to 19%, and the fraction of pores $1 mm increased
from 3% to 9%.
In contrast to our observations with a relatively high MAP dose
(0.24% w/v), the same amount of 200 nm muco-inert nanoparticles
affect the mucus microstructure (data not shown), suggesting the
effects of synthetic nanoparticles on mucus depend on both particle
concentration and surface chemistry. Synthetic nanoparticles may
adhere to mucins through hydrophobic or electrostatic interactions
with hydrophobic and glycosylated domains along mucin fibers,
respectively. At low concentrations, the number of MAP is likely
insufficient to form the bonds with mucins (i.e., avidity) necessary to
crosslink or bundle mucins, which are normally held apart by
entanglements or terminal disulfide bridges. Likewise, particles that
interact minimally with mucins would have little effecton the mucus
microstructure, even at high concentrations. However, the
collective binding interactions of large numbers of MAP with
multiple mucin fibers may generate sufficient avidity to cause
mucins to crosslink and bundle together, thus yielding larger pores
by Olmsted and coworkers of high concentrations of fluorescent
polystyrene nanoparticles agglomerating mid-cycle cervical mucins
into thick cables . In that work, polystyrene nanoparticles
caused a striking collapse of the mucus gel, to an extent greater than
what we observed here. A key difference that may explain this
discrepancy is the use of ovulatory cervical mucus (OCM) by
Olmsted et al. compared to non-ovulatory cervicovaginal mucus
here. OCM is more dilute , so there are fewer entanglements
and crosslinks to resist the displacement of mucin fibers during
bundling by MAP. In addition, electron microscopy studies have
suggested ovulatory mucus contains long strands of mucin fibers
arranged in parallel with minimal entanglements, which may
promote bundling by MAP compared to non-ovulatory mucus that
consists of a random mesh of highly entangled mucin fibers .
Other experimental differences include the volume of particles
added (20% v/v by Olmsted vs. 3% v/v here), the total amount of
particles added (final concentration 2% w/v vs. 0.24% w/v here)
and the degree of particle mixing into mucus (relatively rigorous vs.
gentle stirring here).
We observed substantial heterogeneity in MAP-induced chang-
es to the microstructure, which may be attributed to compositional
differences between CVM samples collected at random points
throughout the menstrual cycle (excluding the ovulatory phase)
and from different donors. Due to hormonal changes, the contents
of mucins (both total mucin concentration and different MUC
types) [29,31], lipids  and other mucus constituents differ
markedly throughout the menstrual cycle. As discussed above,
mucin concentration may determine the extent of mucin bundling
possible. Lipids, which naturally coat the hydrophobic domains of
Figure 2. Transport of probe particles in saline- (Control) or MAP-exposed CVM. (A) Sample trajectories of probe particles with effective
diffusivities within one S.E.M. of the mean at a time scale of 1 s. (B) Ensemble-averaged geometric mean square displacements (,MSD.) as a function
of time scale for probe particles. Data represent six independent experiments with samples from different donors (n$100 particles per experiment). a
represents the slope of the log-log ,MSD. vs. time scale plot. * indicates statistical significance at p,0.05.
Figure 3. Fraction of immobile probe particles in saline-
(Control) or MAP-exposed CVM. * indicates statistical signifi-
cance at p, ,0.05.
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org4 June 2011 | Volume 6 | Issue 6 | e21547
mucins , may also interfere with adhesive interactions be-
tween mucins and MAP. It is likely that the effect of a
higher particle concentration in improving the mucus-altering
ability of MAP may eventually saturate, since the number of
adhesive interactions mucins can form with particles is finite. In
addition, physical entanglements and disulfide crosslinks within
the mucin mesh network may restrict the extent of mucin bundling
Predicted particle penetration into MAP-exposed mucus
The increased mobility of the probe particles in MAP-exposed
mucus suggests a greater fraction may penetrate physiologically
thick mucus layers. To model the long range diffusion of the
particles, we performed a Monte Carlo simulation of particles
undergoing Brownian diffusion across a mucus slab, using the
diffusivities of hundreds of probe particles measured above
(Figure 4). The thickness of physiological mucus layers varies
from tens to several hundred microns depending on anatomical
location [4,34–36] here, we assumed thicknesses ranging from 10
to 55 mm, which correspond to values reported for the large
airways . For a thickness of 10 mm, about 2-fold more of 1 mm
probe particles were predicted to penetrate the mucus layer within
1 hr in MAP-exposed vs. saline-treated mucus (11% vs. 6%,
respectively), and, for a thickness of 30 mm, 3-fold more probe
particles were predicted to penetrate (3% vs. 1%, respectively). For
comparison, if the particles were diffusing through pure water,
64% and 17% of probe particles would theoretically be able to
penetrate 10 mm and 30 mm, respectively, over the same duration.
The increase in probe particle penetration suggests exposure of
mucus to MAP may similarly enhance penetration by pathogens
or other foreign particles with muco-inert surfaces (e.g., Norwalk
virus and human papilloma virus ), which may significantly
increase risk of infection or toxicity. Nevertheless, the window of
opportunity for harmful particles to penetrate the mucus barrier is
likely limited, since MAP-exposed mucus is likely to be cleared by
natural mucus clearance mechanisms as the mucus blanket is
continuously renewed, on the order of minutes to hours depending
on mucosal site .
Bulk rheology of MAP-exposed mucus
Rapid mucus clearance is critical to the effectiveness of the
mucus barrier. Since proper mucus clearance depends strongly on
the bulk rheological properties of the mucus gel , we also
investigated whether MAP may alter the bulk viscoelasticity of
mucus. We pooled mucus from several donors and performed
strain-controlled cone and plate rheometric measurements on
native and MAP-exposed samples sequentially. The bulk viscous
and elastic moduli were quantified by applying a small, fixed-
amplitude oscillatory stress at specified frequencies. Both the
viscous and elastic moduli changed minimally upon MAP
exposure (Figure 5), decreasing on average by ,4% and 2%,
respectively. The phase angle (d) for both native and MAP-
exposed samples was ,14u, indicating that mucus remained a
viscoelastic solid (d=0u indicates a Hookean solid, d=90u
indicates a purely viscous fluid and 0u,d,90u indicates a
viscoelastic material). These values are comparable to those we
have measured before for fresh mucus . Thus, although MAP
may alter mucus microstructure substantially, the bulk rheology of
mucus appeared unperturbed.
Figure 4. Monte Carlo simulation of particle penetration across a layer of saline- (Control) or MAP-exposed CVM over time. The
simulation consists of ,20,000 particles undergoing random diffusion with diffusivities equal to the experimentally measured diffusivities of probe
particles in either condition. The % of particles able to reach the opposite side of the mucus layer is reported.
Figure 5. Bulk elastic (G9) and viscous (G0) moduli of unexposed
(Control) and MAP-exposed pooled CVM.
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e21547
Classical polymer physics models [18,38–40] would predict a
35–65% decrease in the bulk elastic modulus as pore size increases
from 380 nm to 470 nm, in contrast to the ,2% decrease
observed here. However, the essentially unchanged bulk rheology
of mucus upon MAP exposure is in agreement with our previous
finding that the microstructure of mucus can be selectively altered
without perturbing bulk rheology . In that work, we showed
that treatment of mucus with a non-ionic detergent disrupted
adhesive interactions between mucins, reducing natural mucin
bundling and, hence, pore sizes. We postulated that the resulting
increase in the number of entanglements between mucins
(expected to increase bulk viscoelasticity) likely offset the decrease
in adhesive interactions (expected to decrease bulk viscoelasticity),
resulting in no detectable change in the bulk rheology. Similarly,
an increase in mucin fiber bundling upon MAP exposure here may
result in a decrease in the number of entanglements under shear,
as well as a simultaneous increase in adhesive interactions between
mucins. These two opposing effects may balance each other to the
extent that no change in bulk rheology was detected even with a
sensitive strain-controlled rheometer. Based on visual observation
of the fluorescently-labeled particles, MAP appeared to mix
uniformly into the entire mucus sample; thus, it is unlikely that
non-uniformly distributed MAP caused only local changes to
mucus microstructure that were not detectable by bulk rheological
measurements. Our results suggest proper mucus clearance, which
depends critically on the bulk rheological properties of mucus, may
not be directly impaired by exposure to MAP, even at relatively
high exposure levels. However, it should be noted that toxic
nanoparticles that bypass the mucus barrier may cause toxicity to
underlying epithelial cells, which may lead to mucus hypersecre-
tion, altered mucus bulk rheology and poor mucus clearance,
similar to the pathogenesis of inflammatory lung diseases [41–44].
Implications for drug delivery applications
Finally, our results may offer a unique approach for potentially
enhancing drug or nucleic acid delivery to mucosal surfaces. Large
drug carriers are preferred for improved drug loading and release
kinetics, but even those engineered to have muco-inert surfaces are
markedly slowed by steric obstruction from the mucus mesh,
particularly in thicker secretions like cystic fibrosis sputum .
The use of biodegradable and biocompatible MAP that transiently
enlarge mucus pores may significantly enhance the penetration of
large drug carriers through mucus. These ‘sacrificial’ mucoadhe-
sive nanoparticles may be employed prior to administration of the
drug carriers. As mucus is continuously secreted, MAP will be
displaced over time; however, mucus clearance in the vagina and
other mucosal surfaces occurs on the order of several hours ,
providing sufficient time for drug carriers to penetrate MAP-
altered mucus. In pilot studies, exposure of mucus to a high
concentration of MAP did not cause existing MAP that were
already immobilized to become mobile. This suggests that, in the
absence of exposure to exogenous pathogens, the use of ‘sacrificial’
nanoparticles would not increase the risk of infection by already-
present pathogens while facilitating sustained and/or targeted
drug delivery to mucosal surfaces.
In practice, the amount of mixing of MAP into mucus that
occurs during particle administration may be limited, depending
on the mucosal surface of interest. For example, little to no mixing
is expected in the respiratory tract, where aerosolized MAP would
deposit and remain trapped on the surface of the mucus layer. In
contrast, some mixing may occur in the cervicovaginal tract,
where intra-abdominal pressure generates squeezing flows that can
cause mixing of MAP into the mucus layer. Nevertheless, even if
MAP are distributed only in the outermost, luminal mucus layer,
they may still provide therapeutic benefits by sufficiently enlarging
pores to enhance the penetration of drug carriers into deeper
mucus layers. This is of particular significance at mucosal surfaces
like the vagina, where secreted mucins form two distinct layers, a
quickly cleared luminal mucus layer and a more slowly cleared
adherent mucus layer adjacent to the epithelium. By reaching the
slowly cleared adherent mucus layer, drug carriers may be
retained at the mucosal surface in close proximity to target cells
for prolonged periods of time, leading to enhanced efficacy.
In summary, we have characterized the effect of synthetic
particles on the microstructure of native mucus gel, and
demonstrated that both particle concentration and surface
chemistry play a role in whether the particles disrupt the mucus
barrier. High concentrations of MAP can increase pore sizes in
mucus through the crosslinking or bundling of mucin fibers,
allowing greater penetration of foreign particles across mucus.
While we used CVM here, lung and other human mucus
secretions have generally similar bulk rheological properties and
compositions as CVM, with 2–5% w/w mucins (predominantly of
the MUC5B mucin type ) and 90–98% w/w water [4,33].
Thus, our findings may also extend to these other types of human
mucus. These results highlight the importance of understanding
nanoparticle-mucus interactions and their implications for nano-
toxicology, as well as for biomedical applications.
particles in human cervicovaginal mucus samples treated with 3%
v/v saline, over the course of 20 s. The trajectories of the probe
particles are strongly hindered.
Transport of 1 mm muco-inert (PEG-coated) probe
particles in human cervicovaginal mucus samples treated with 3%
v/v mucoadhesive particles (MAP; 0.24% w/v final concentra-
tion), over the course of 20 s. The trajectories of the probe
particles are much more diffusive than in the same mucus samples
treated with saline.
Transport of 1 mm muco-inert (PEG-coated) probe
We thank the Integrated Imaging Center at the Johns Hopkins University.
Conceived and designed the experiments: YYW SL RC JH. Performed the
experiments: YYW. Analyzed the data: YYW SL C. So C. Schneider.
Wrote the paper: YYW SL RC JH.
1. Donaldson K, Stone V, Clouter A, Renwick L, MacNee W (2001) Ultrafine
particles. Occup Environ Med 58: 211–216.
2. Nel A (2005) Atmosphere. Air pollution-related illness: effects of particles.
Science 308: 804–806.
3. Nel A, Xia T, Madler L, Li N (2006) Toxic potential of materials at the
nanolevel. Science 311: 622–627.
4. Lai SK, Wang YY, Hanes J (2009) Mucus-penetrating nanoparticles for drug
and gene delivery to mucosal tissues. Adv Drug Deliv Rev 61: 158–171.
5. Ali MS, Pearson JP (2007) Upper airway mucin gene expression: a review.
Laryngoscope 117: 932–938.
6. Greaves JL, Wilson CG (1993) Treatment of diseases of the eye with
mucoadhesive delivery systems. Adv Drug Deliv Rev 11: 349–383.
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org6 June 2011 | Volume 6 | Issue 6 | e21547
7. Hummer G, Rasaiah JC, Noworyta JP (2001) Water conduction through the Download full-text
hydrophobic channel of a carbon nanotube. Nature 414: 188–190.
8. Lai SK, O’Hanlon DE, Harrold S, Man ST, Wang YY, et al. (2007) Rapid
transport of large polymeric nanoparticles in fresh undiluted human mucus. Proc
Natl Acad Sci USA 104: 1482–1487.
9. Li B, Logan BE (2004) Bacterial adhesion to glass and metal-oxide surfaces.
Colloids Surf B Biointerfaces 36: 81–90.
10. Yang M, Lai SK, Wang YY, Zhong W, Happe C, et al. (2011) Biodegradable
Nanoparticles Composed Entirely of Safe Materials that Rapidly Penetrate
Human Mucus. Angew Chem Int Ed Engl 50: 2597–2600.
11. King MV (1991) Dimensional changes in cells and tissues during specimen
preparation for the electron microscope. Cell Biophys 18: 31–55.
12. Mollenhauer HH (1993) Artifacts caused by dehydration and epoxy embedding
in transmission electron microscopy. Microsc Res Tech 26: 496–512.
13. Vanhecke D, Graber W, Studer D (2008) Close-to-native ultrastructural
preservation by high pressure freezing. Methods Cell Biol 88: 151–164.
14. Lai SK, Wang YY, Hida K, Cone R, Hanes J (2010) Nanoparticles reveal that
human cervicovaginal mucus is riddled with pores larger than viruses. Proc Natl
Acad Sci USA 107: 598–603.
15. Suk JS, Lai SK, Wang YY, Ensign LM, Zeitlin PL, et al. (2009) The penetration
of fresh undiluted sputum expectorated by cystic fibrosis patients by non-
adhesive polymer nanoparticles. Biomaterials 30: 2591–2597.
16. Boskey ER, Moench TR, Hees PS, Cone RA (2003) A self-sampling method to
obtain large volumes of undiluted cervicovaginal secretions. Sexually Transmit-
ted Diseases 30: 107–109.
17. Wang YY, Lai SK, Suk JS, Pace A, Cone R, et al. (2008) Addressing the PEG
mucoadhesivity paradox to engineer nanoparticles that ‘‘slip’’ through the
human mucus barrier. Angew Chem Int Ed Engl 47: 9726–9729.
18. Lai SK, Wang YY, Cone R, Wirtz D, Hanes J (2009) Altering mucus rheology to
‘‘solidify’’ human mucus at the nanoscale. PLoS ONE 4: e4294.
19. OSHA Occupational Safety and Health Guidelines.
20. EPA (1997) Exposure Factors Handbook (Final Report). Washington, D.C.: U.S.
Environmental Protection Agency.
21. ICRP (1994) 5. Deposition model. Annals of the ICRP 24: 36–54.
22. Mercer RR, Russell ML, Roggli VL, Crapo JD (1994) Cell number and
distribution in human and rat airways. Am J Respir Cell Mol Biol 10: 613–624.
23. Apgar J, Tseng Y, Fedorov E, Herwig MB, Almo SC, et al. (2000) Multiple-
particle tracking measurements of heterogeneities in solutions of actin filaments
and actin bundles. Biophys J 79: 1095–1106.
24. Valentine MT, Perlman ZE, Gardel ML, Shin JH, Matsudaira P, et al. (2004)
Colloid surface chemistry critically affects multiple particle tracking measure-
ments of biomaterials. Biophys J 86: 4004–4014.
25. Amsden B (1998) Solute diffusion within hydrogels. Mechanisms and models.
Macromolecules 31: 8382–8395.
26. Amsden B (1999) An obstruction-scaling model for diffusion in homogeneous
hydrogels. Macromolecules 32: 874–879.
27. Olmsted SS, Padgett JL, Yudin AI, Whaley KJ, Moench TR, et al. (2001)
Diffusion of macromolecules and virus-like particles in human cervical mucus.
Biophys J 81: 1930–1937.
28. Shen H, Hu Y, Saltzman WM (2006) DNA diffusion in mucus: effect of size,
topology of DNAs, and transfection reagents. Biophys J 91: 639–644.
29. Gipson IK, Moccia R, Spurr-Michaud S, Argueso P, Gargiulo AR, et al. (2001)
The Amount of MUC5B mucin in cervical mucus peaks at midcycle. J Clin
Endocrinol Metab 86: 594–600.
30. Ceric F, Silva D, Vigil P (2005) Ultrastructure of the human periovulatory
cervical mucus. J Electron Microsc (Tokyo) 54: 479–484.
31. Odeblad E (1968) The functional structure of human cervical mucus. Acta
Obstet Gynecol Scand 47: 57–79.
32. Singh EJ (1975) Oral contraceptives and human cervical mucus lipids.
Am J Obstet Gynecol 123: 128–132.
33. Lai SK, Wang YY, Wirtz D, Hanes J (2009) Micro- and macrorheology of
mucus. Adv Drug Deliv Rev 61: 86–100.
34. Jordan N, Newton J, Pearson J, Allen A (1998) A novel method for the
visualization of the in situ mucus layer in rat and man. Clin Sci (Lond) 95:
35. Matsuo K, Ota H, Akamatsu T, Sugiyama A, Katsuyama T (1997)
Histochemistry of the surface mucous gel layer of the human colon. Gut 40:
36. Verkman AS, Song Y, Thiagarajah JR (2003) Role of airway surface liquid and
submucosal glands in cystic fibrosis lung disease. Am J Physiol Cell Physiol 284:
37. King M (2006) Physiology of mucus clearance. Paediatr Respir Rev 7(Suppl 1):
38. Gardel ML, Shin JH, MacKintosh FC, Mahadevan L, Matsudaira P, et al.
(2004) Elastic behavior of cross-linked and bundled actin networks. Science 304:
39. MacKintosh FC, Kas J, Janmey PA (1995) Elasticity of semiflexible biopolymer
networks. Paediatr Respir Rev 75: 4425–4428.
40. Palmer A, Xu J, Kuo SC, Wirtz D (1999) Diffusing wave spectroscopy
microrheology of actin filament networks. Biophys J 76: 1063–1071.
42. Kim WD (1997) Lung mucus: a clinician’s view. Eur Respir J 10: 1914–1917.
43. Puchelle E, Bajolet O, Abely M (2002) Airway mucus in cystic fibrosis. Paediatr
Respir Rev 3: 115–119.
44. Voynow JA, Rubin BK (2009) Mucins, mucus, and sputum. Chest 135:
45. Wickstrom C, Davies JR, Eriksen GV, Veerman EC, Carlstedt I (1998) MUC5B
is a major gel-forming, oligomeric mucin from human salivary gland, respiratory
tract and endocervix: identification of glycoforms and C-terminal cleavage.
Biochem J 334(Pt 3): 685–693.
Synthetic Nanoparticles Disrupt Mucus Barrier
PLoS ONE | www.plosone.org7 June 2011 | Volume 6 | Issue 6 | e21547