SCIENCE ADVANCES | RESEARCH ARTICLE
BIOMEDICAL ENGINEERING
Nanoparticles that do not adhere to mucus provide
uniform and long-lasting drug delivery to airways
following inhalation
Craig S. Schneider,1,2,3* Qingguo Xu,1,4* Nicholas J. Boylan,1,3* Jane Chisholm,1,3
Benjamin C. Tang,1,3† Benjamin S. Schuster,1,5‡ Andreas Henning,1,3 Laura M. Ensign,1,2,3,4
Ethan Lee,3 Pichet Adstamongkonkul,5 Brian W. Simons,6,7 Sho-Yu S. Wang,1,3 Xiaoqun Gong,1,8
Tao Yu,1,5 Michael P. Boyle,9 Jung Soo Suk,1,4,5§ Justin Hanes1,2,3,4,5§
2017 © The Authors,
some rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).
INTRODUCTION
Inhalation of drug-loaded nanoparticles (NP) is a promising approach
for the treatment of diseases that affect the lungs, including asthma,
chronic obstructive pulmonary disease, cystic fibrosis (CF), and lung
cancer (1–4). Biodegradable NP may be delivered into the lungs via
nebulization or as a dry powder to achieve high airway deposition,
where they may then provide (i) protection of drug payloads against
enzymatic and/or hydrolytic degradation or inactivation (5–7), (ii)
controlled release of drugs over prolonged periods of time locally
(8, 9), (iii) reduced local and systemic side effects by lowering total dose,
dose frequency, and systemic exposure (10, 11), and (iv) potential to
overcome a variety of extracellular and intracellular barriers due to
tailorable geometry and nanometric size (12–16).
In an attempt to enhance drug residence time in the lungs following
inhalation, drugs are typically packaged into biodegradable NP that
1
The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. 2Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA. 3Department of Chemical and Biomolecular
Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. 4Wilmer Eye
Institute, Johns Hopkins Medical Institute, Baltimore, MD 21287, USA. 5Department of Biomedical Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA. 6Department of Pathology, Johns Hopkins University
School of Medicine, Baltimore, MD 21287, USA. 7Department of Molecular and
Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore,
MD 21287, USA. 8School of Life Sciences, Tianjin University, Tianjin 300072, People‘s
Republic of China. 9Division of Pulmonary and Critical Care Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD 21205, USA.
*These authors contributed equally to this work.
†Present address: Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA.
‡Present address: Department of Bioengineering, University of Pennsylvania,
Philadelphia, PA 19104, USA.
§Corresponding author. Email: [email protected] (J.S.S.); [email protected] (J.H.)
Schneider et al., Sci. Adv. 2017; 3 : e1601556
5 April 2017
adhere to mucus, namely, mucoadhesive particles (MAP) (17–20). MAP
have been typically engineered to possess cationic (21, 22) or thiolated
(23, 24) surfaces that interact with negatively charged glycosylated or
cysteine-rich hydrophobic domains of airway mucin fibers, respectively.
However, those highly cationic or thiol-decorated NP have been formulated with hydrophobic core polymers (21, 24), and thus, the mucoadhesion may have been attributed to hydrophobic interactions as well (25,
26). The dogma is that MAP may be retained for longer duration in the
lungs by decreasing mucociliary clearance (MCC) rates (27), through
rheological changes of mucus via multivalent mucus-particle interactions (28–31). However, by definition, MAP adhere to the most superficial mucus gel layer and, thus, are not expected to reach the underlying
periciliary layer (PCL) that is cleared less rapidly (32). Moreover, we
hypothesized that MAP would not spread uniformly within the mucus
gel layer or PCL, potentially leading to uneven delivery of inhaled drugs
within the airways that may reduce drug efficacy.
We and others have demonstrated that airway mucus forms an
adhesive and steric barrier to conventional NP (that is, MAP) regardless
of particle diameter (25, 33–36). Using multiple-particle tracking
(MPT), we found that particles as large as 200 nm can rapidly penetrate
freshly collected human respiratory mucus, obtained from healthy
donors (26) and CF patients (25) ex vivo, but only when the NP surface was passivated with dense coatings of poly(ethylene glycol)
[PEG; that is, mucus-penetrating particles (MPP)]. However, a recent
publication argued that although MPP are diffusive over short distances
and time scales in airway mucus (as confirmed via MPT ex vivo), transport
across the mucus gel layer in the airways in vivo, with a thickness of up
to a few to tens of micrometers, is not feasible (37). Thus, we sought to
directly compare the in vivo behavior of MPP to MAP of various sizes
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Mucoadhesive particles (MAP) have been widely explored for pulmonary drug delivery because of their perceived
benefits in improving particle residence in the lungs. However, retention of particles adhesively trapped in airway
mucus may be limited by physiologic mucus clearance mechanisms. In contrast, particles that avoid mucoadhesion
and have diameters smaller than mucus mesh spacings rapidly penetrate mucus layers [mucus-penetrating particles
(MPP)], which we hypothesized would provide prolonged lung retention compared to MAP. We compared in vivo
behaviors of variously sized, polystyrene-based MAP and MPP in the lungs following inhalation. MAP, regardless of
particle size, were aggregated and poorly distributed throughout the airways, leading to rapid clearance from the
lungs. Conversely, MPP as large as 300 nm exhibited uniform distribution and markedly enhanced retention compared
to size-matched MAP. On the basis of these findings, we formulated biodegradable MPP (b-MPP) with an average
diameter of <300 nm and examined their behavior following inhalation relative to similarly sized biodegradable
MAP (b-MAP). Although b-MPP diffused rapidly through human airway mucus ex vivo, b-MAP did not. Rapid
b-MPP movements in mucus ex vivo correlated to a more uniform distribution within the airways and enhanced
lung retention time as compared to b-MAP. Furthermore, inhalation of b-MPP loaded with dexamethasone sodium phosphate (DP) significantly reduced inflammation in a mouse model of acute lung inflammation compared to
both carrier-free DP and DP-loaded MAP. These studies provide a careful head-to-head comparison of MAP versus MPP
following inhalation and challenge a long-standing dogma that favored the use of MAP for pulmonary drug delivery.
SCIENCE ADVANCES | RESEARCH ARTICLE
following inhalation. Additionally, we compared the efficacy of a corticosteroid, dexamethasone sodium phosphate (DP), delivered either via
a biodegradable MPP formulation (b-MPP), a biodegradable MAP formulation (b-MAP), or the carrier-free soluble drug in a mouse model of
lipopolysaccharide (LPS)–induced acute lung inflammation.
RESULTS
Table 1. Physicochemical characterization of PS NP. Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS).
z-potential was measured at pH 7.4 in 10 mM NaCl. Mucus penetration indicates the ability of the NP to penetrate CF sputum, as determined by MPT in our
previous work (23, 42, 43) and fig. S3. X, no penetration; O, penetration.
Particle type
Diameter (nm)
PDI
z-Potential (mV)
Mucus penetration
60
60-nm PS
58 ± 1
0.12
−34 ± 2
X
60
60-nm PS-PEG
66 ± 3
0.18
−12 ± 1
O
100
100-nm PS
90 ± 5
0.06
−44 ± 3
X
100
100-nm PS-PEG
107 ± 3
0.03
−5 ± 1
O
300
300-nm PS
282 ± 2
0.04
−50 ± 2
X
300
300-nm PS-PEG
307 ± 9
0.03
−3 ± 1
O
1000
1000-nm PS
1025 ± 13
0.13
−83 ± 4
X
1000
1000-nm PS-PEG
1045 ± 20
0.11
−5 ± 1
X
Nominal size (nm)
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Characterization of NP
To test whether the more rapid diffusion of MPP compared to MAP in
mucus ex vivo would correlate to a more uniform airway distribution
and/or a prolonged airway retention, we first produced model polystyrene (PS) NP with dense surface coatings of PEG (PS-PEG MPP)
by conjugating 5-kDa PEG chains to carboxyl groups on the particle
surfaces, as previously described (34, 38, 39). Because the average mucus
mesh spacing for mouse respiratory mucus is unknown, we investigated
a broad range of NP sizes (60 to 1000 nm in diameter). In agreement
with our previous findings (34), dense PEG coverage of NP resulted
in marginal increases in particle diameters and significantly increased
z-potential (to near neutral) regardless of particle size (Table 1). In
comparison, PS NP without PEG coatings (PS MAP) exhibited negative
z-potentials, ranging from −34 to −83 mV, reflecting the presence of
unconjugated carboxyl groups on the NP surface.
We also synthesized b-MAP and b-MPP in a size range of 100 to
200 nm, because well-PEGylated NP with particle diameters <200 nm
are known to penetrate human respiratory mucus and CF sputum
(25, 26). First, we fabricated b-MPP using a diblock copolymer composed of poly(lactic-co-glycolic acid) (PLGA) and PEG (PLGA-PEG),
namely, PLGA-PEG MPP, for mechanistic in vivo studies of lung
distribution and retention. Uncoated PLGA NP were used as a MAP
control (PLGA MAP). Both formulations exhibited hydrodynamic diameters of 130 to 140 nm, but whereas the average z-potential of PLGA
MAP was strongly negative (−73 mV), that of PLGA-PEG MPP was
near neutral (−6 mV) due to the dense PEG coating (Table 2). Second,
we synthesized b-MPP from all generally-regarded-as-safe (GRAS)
components, using PLGA as a core and Pluronic F127 (PLGA/F127
MPP) to provide a noncovalent muco-inert surface coating. Pluronics
are a class of triblock copolymers composed of poly(ethylene oxide)–
poly(propylene oxide)–poly(ethylene oxide) (PEO-PPO-PEO),
where PPO adsorbs onto hydrophobic surfaces, whereas PEO (also
known as PEG) imparts nonadhesive surface coatings (40). We have
previously shown that dense F127 coatings lead to rapid penetration
of PLGA/F127 MPP through human cervicovaginal mucus (41). Pluronic F68 coating was used as a MAP control (PLGA/F68 MAP), because
it does not provide sufficient shielding of the surface to prevent mucusNP adhesive interactions (41). The hydrodynamic diameters of PLGA/
F127 MPP and PLGA/F68 MAP were comparable (180 to 200 nm)
(Table 2). As a control, we formulated another uncoated, PLGA NP
free of Pluronics (PLGA/PF MAP) having a similar diameter (190 nm).
Similar to PLGA-PEG MPP, the z-potential of PLGA/F127 MPP was
near neutral (−8 mV). In contrast, z-potentials of PLGA/F68 MAP
and PLGA/PF MAP were −23 and −64 mV, respectively, underscoring
insufficient and absent surface shielding.
It is imperative that NP retain their physicochemical properties
under relevant physiological conditions. We thus investigated whether
dense PEG coatings on NP promote their colloidal stability in mouse
bronchoalveolar lavage fluid (BALF) (Fig. 1, A and B). Unlike uncoated
PLGA/PF MAP, particle diameters and polydispersity indices were well
preserved for both PLGA/F127 MPP and PLGA/F68 MAP following
24 hours of incubation in BALF. We next assessed the in vivo safety of
these GRAS NP following inhalation on the basis of histological analysis
(fig. S1) and immune cell profiling in BALF (fig. S2). Whereas lungs of
mice dosed with PLGA/F127 MPP were virtually indistinguishable
compared to saline-treated controls, mice treated with either PLGA/
PF MAP or PLFA/F68 MAP displayed signs of acute inflammation.
We also formulated PLGA/F127 MPP and PLGA/F68 MAP loaded
with a corticosteroid. To increase the drug loading level, we adapted
a previously reported method of coordinate complexation of the watersoluble corticosteroid DP with zinc, followed by encapsulation into NP
(42, 43). The physicochemical properties of DP loaded into PLGA/F127
MPP (DP/PLGA/F127 MPP) and PLGA/F68 MAP (DP/PLGA/F68
MAP) are shown in Table 2. DP/PLGA/F127 MPP exhibited nearneutral z-potential (−4 mV), whereas DP/PLGA/F68 MAP possessed a
highly negative surface charge (−23 mV). Morphological examination via
transmission electron microscopy revealed spherical NP geometry with
no evidence of drug precipitation (Fig. 1C). Both DP/PLGA/F127 MPP
and DP/PLGA/F68 MAP exhibited DP loading levels of ~8.5% w/w and
sustained in vitro DP release over 10 days (Fig. 1D).
SCIENCE ADVANCES | RESEARCH ARTICLE
Ex vivo diffusion of biodegradable MPP and MAP in human
CF sputum
To ensure that the b-MPP formulations used in this study, specifically
PLGA-PEG MPP and PLGA/F127 MPP, efficiently penetrated human
mucus ex vivo, their transport rates were measured in CF sputum freshly expectorated by CF patients. The greater diffusion of PLGA-PEG
MPP and PLGA/F127 MPP compared to PLGA MAP and PLGA/F68
MAP in airway mucus was first qualitatively confirmed (movies S1 to S4)
Table 2. Physicochemical characterization of biodegradable NP.
Hydrodynamic diameter and PDI were measured by DLS. z-potential
was measured at pH 7.4 in 10 mM NaCl. Mucus penetration indicates
the ability of the NP to penetrate CF sputum, as determined by MPT
in Fig. 2.
Particle type
Diameter (nm) PDI z-Potential
(mV)
Mucus
penetration
141 ± 15
0.14
−73 ± 4
X
PLGA-PEG
128 ± 6
0.09
−6 ± 1
O
PLGA/PF
190 ± 7
0.09
−64 ± 2
X
PLGA/F68
180 ± 17
0.09
−23 ± 4
X
PLGA/F127
200 ± 8
0.14
−8 ± 2
O
DP/PLGA/F68
150 ± 10
0.08
−23 ± 1
X
DP/PLGA/
F127
260 ± 21
0.10
−4 ± 2
O
A
B
– BALF
600
0.6
**
+ BALF 2 h
+ BALF 24 h
400
**
PDI
Diameter (nm)
Lung distribution and retention of variously sized
polystyrene MPP and MAP
To examine whether rapid mucus penetration over short time scales
ex vivo correlates with particle transport across thick mucus layers in
vivo, we investigated the distribution of variously sized PS MAP and
PS-PEG MPP in the mouse conducting airways following intranasal
administration. We found that PS-PEG MPP with diameters of 60,
100, and 300 nm uniformly distributed throughout the mucus layer
in the airways, showing little to no aggregation and penetration into
folds found in the airway epithelium (Fig. 3, A to C). In contrast, similarly sized PS MAP were usually found in large, clumped aggregates
positioned away from the epithelium (Fig. 3, A to C). The findings
correlated well with the diffusion behavior of NP on the lumen of freshly
*
+ BALF 24 h
0.2
200
0
0.4
– BALF
+ BALF 2 h
PLGA/
F127
PLGA/
F68
PLGA/
PF
0
PLGA/
F127
PLGA/
F68
D 120
Drug release (%)
C
DP/PLGA/F127
PLGA/
PF
DP/PLGA/F68
DP/PLGA/F127
100
80
60
40
20
0
0
2
4
6
8
10
Time (days)
Fig. 1. Characterization of GRAS-based biodegradable NP. Ex vivo stability of NP in BALF at room temperature for 2 or 24 hours, as measured by (A) hydrodynamic
diameter and (B) PDI. (C) Transmission electron micrographs of DP/PLGA/F127 and PLGA/F68 NP. Scale bars, 100 nm. (D) Release kinetics of DP from DP/PLGA/F127 and
DP/PLGA/F68 NP. Data represent means ± SD. *P < 0.05, **P < 0.01.
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PLGA
and then quantified using MPT. PLGA MAP and PLGA/F68 MAP
both exhibited confined particle trajectories, whereas PLGA-PEG
MPP and PLGA/F127 MPP exhibited Brownian-like particle trajectories (Fig. 2A). The median mean-squared displacement (MSD) of
particles at a time scale of 1 s was nearly 800-fold greater for PLGAPEG MPP compared to PLGA MAP and 25-fold greater for PLGA/
F127 MPP compared to PLGA/F68 MAP (Fig. 2B).
We have previously shown via MPT that PS-PEG MPP with diameters as large as 200 nm are capable of diffusing rapidly in human
airway mucus, whereas 500-nm PS NP are unable to do so regardless
of dense surface PEG coatings, suggesting that they are too large to fit
through the mucus pores (4, 7, 25, 26). However, we had not investigated the diffusion behaviors of PS NP having an intermediate diameter
of 300 nm. Thus, we have confirmed here that 300-nm PS-PEG MPP
exhibit significantly greater median MSD compared to non-PEGylated
counterparts (that is, 300 PS MAP) in CF sputum (fig. S3).
SCIENCE ADVANCES | RESEARCH ARTICLE
excised mouse tracheas (movies S5 and S6), where PS-PEG MPP 60 to
300 nm in diameter diffused rapidly in mouse tracheal mucus, whereas
the movement of PS MAP of all sizes was strongly hindered or immobilized. In contrast to smaller NP (≤300 nm), there was no
difference in the airway distributions of 1000-nm PS MAP and PSPEG MPP (Fig. 3D); 1000-nm MAP and MPP were both found
poorly distributed and away from the epithelial surface, similar to
the observations with smaller PS MAP, suggesting that 1000-nm
particles are too large to fit through the mucus mesh in the airways
regardless of surface coating.
Next, we measured the time course retention of MPP versus MAP in
the lumen of mouse lungs by quantifying the amount of NP in the
BALF. The retention of 60-nm PS-PEG MPP was enhanced compared
to that of 60-nm PS MAP at each time point studied (Fig. 3E). After
2 hours, more than 85% of the initially deposited 60-nm PS-PEG MPP
were retained in the lung lumen, whereas only ~45% of PS MAP remained (P < 0.01). Similarly, 100- and 300-nm PS-PEG MPP showed
enhanced retention compared to similarly sized PS MAP at each
time point studied. At least 70% of 100- and 300-nm PS-PEG
MPP were retained at 2 hours after administration, but only ~30%
of 100- and 300-nm PS MAP remained in the lung at the same time
point (P < 0.01; Fig. 3, F and G). In contrast, the retention of 1000-nm
PS MAP and PS-PEG MPP was not statistically different at any time
point studied (Fig. 3H), with 2-hour retention values of ~45 and ~50%,
respectively.
Lung distribution and retention of biodegradable MPP
and MAP
On the basis of the findings with model PS NP, we investigated the
distribution of b-MPP and b-MAP in the mouse conducting airways
Fig. 3. The distribution and retention of model PS-based NP in the mouse lungs. Airway distribution of (A) 60-, (B) 100-, (C) 300-, and (D) 1000-nm PS and PS-PEG
NP at 30 min after administration. PS (green) and PS-PEG (red) NP were coadministered except for 100-nm NP, which were independently dosed to different mice. Cell
nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). The white and yellow arrows indicate PS NP aggregated in the mucus blanket and PS-PEG NP near
the surface of epithelium, respectively. Unlike smaller PS-PEG, 1000-nm PS-PEG sparsely distributed throughout the airway similar to PS (red arrows). The retention of PS
(white bars) and PS-PEG (gray bars) NP over time is reported as a percentage of the initial deposited dose for (E) 60-, (F) 100-, (G) 300-, and (H) 1000-nm NP (n > 5). Data
represent means ± SEM. **P < 0.01.
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Fig. 2. The diffusion of biodegradable NP in human CF sputum. MPT was performed on NP administered ex vivo in freshly expectorated CF sputum. (A) Representative trajectories of particles. Scale bar, 1 mm. (B) Median MSD at a time scale of 1 s (n = 4). Data represent means ± SEM. *P < 0.05.
SCIENCE ADVANCES | RESEARCH ARTICLE
following inhaled administration. Similar to the model PS-PEG MPP as
large as 300 nm (Fig. 3, A to C), PLGA-PEG MPP and PLGA/F127
MPP each displayed uniform distribution throughout the bronchial
surfaces in close proximity to the airway epithelium (Fig. 4, A and B).
In contrast, b-MAP formulations (that is, PLGA MAP and PLGA/F68
In vivo efficacy comparison of DP-loaded biodegradable
MPP and MAP
To determine whether the improved particle uniformity and
enhanced retention of b-MPP yield improved efficacy of DP in a
model of acute lung inflammation, we intranasally dosed LPSchallenged mice with DP/PLGA/F127 MPP, DP/PLGA/F68 MAP,
or carrier-free soluble DP (Fig. 5). At a dose of 1 mg/kg, soluble DP
had little anti-inflammatory effect (Fig. 5, A and B). In contrast,
treatment with the identical DP dose of DP/PLGA/F127 MPP significantly reduced the recruitment of inflammatory cells to the lung, as
evidenced by the decrease in total BALF cell count compared to treatment with soluble DP or saline (P < 0.01) (Fig. 5A). Treatment with
DP/PLGA/F68 MAP also moderately reduced the inflammatory cell
infiltration compared to saline-treated controls (P < 0.05); however,
the effect was not statistically significant compared to soluble DP. In
addition, only the DP/PLGA/F127 MPP provided significant reduction
of a proinflammatory cytokine, tumor necrosis factor–a (TNF-a) (P <
0.01) (Fig. 5B).
Fig. 4. The distribution and retention of PLGA-based biodegradable NP in
the mouse lung. Bronchial distribution of (A) PLGA (green) and PLGA-PEG
(red) NP and (B) PLGA/F68 (left panel) and PLGA/F127 (right panel) NP at 30 min
after administration. Cell nuclei were stained blue with DAPI. The white and yellow arrows indicate PLGA NP aggregated in the mucus blanket and PLGA-PEG NP
near the epithelial surface, respectively. (C and D) Tracheal distribution of PLGA
(green) and PLGA-PEG (red) NP at 30 min after administration. The yellow dashed
box in (C) highlights a submucosal gland covered with PLGA-PEG NP. The white
arrows in (C) indicate PLGA NP stuck in the mucus blanket. The same gland can
be observed at higher magnification in (D) where the yellow arrow points out
deep penetration of PLGA-PEG NP into the gland. (E) The retention of PLGA
(white bars) and PLGA-PEG (gray bars) NP over time in BALF. (F) The retention
of PLGA and PLGA-PEG NP in the entire lung over time (n = 5). Representative
images for PLGA and PLGA-PEG NP exposed lungs over time are shown. The percentage of retention is reported as the percentage of the initial fluorescence (FL)
for PLGA (open squares, solid line) and PLGA-PEG (open circles, dotted line) NP.
Data represent means ± SEM. *P < 0.05, **P < 0.01.
Schneider et al., Sci. Adv. 2017; 3 : e1601556
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DISCUSSION
MAP have been widely used to improve drug delivery to the lung
airways (17–21), including demonstration of improved sustained
drug levels in the lungs compared to carrier-free drug formulations
(17, 18, 20). However, we found that inhaled NP capable of efficiently
penetrating human airway mucus ex vivo (that is, MPP ≤300 nm in
diameter) exhibited greatly improved particle distribution and retention
in the lungs in vivo compared to MAP of similar size, and, further, that a
drug loaded into MPP was more effective than the same drug loaded
into MAP. The improved retention of these MPP is most likely
attributed to their ability to overcome MCC by rapidly diffusing deep
into the mucus layer, as evidenced by the presence of MPP in epithelial
folds as well as a submucosal gland. MPP exhibited enhanced retention in
the lung airways compared to MAP, suggesting that a direct correlation
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MAP) were again found in large clumps away from the airway epithelium (Fig. 4, A and B). We observed that PLGA-PEG MPP penetrated
deep (approximately 200 mm from the epithelial surface) into a submucosal gland found in the mouse trachea (Fig. 4, C and D, yellow
arrow). PLGA MAP were exclusively located in large aggregates in
the luminal mucus layer (LML) of the trachea (Fig. 4C, white arrow),
similar to that found with MAP in the bronchi.
We next studied the retention of PLGA-PEG MPP and PLGA MAP
via the same lavage method used for the model NP study. PLGA-PEG
MPP were retained longer in the lung lumen compared to PLGA
MAP at each time point studied (Fig. 4E). PLGA-PEG MPP showed
a similar behavior to PS-PEG MPP of similar size, with more than
80% retained in the lung at 2 hours after administration. PLGA MAP
were rapidly cleared from the lung, similar to PS MAP having similar
particle diameter, with only ~25% retained in the lung after 2 hours
(P < 0.01). To measure the retention of PLGA-PEG MPP and PLGA
MAP in the entire lung (including NP that cannot be collected via
lavage), we measured the fluorescence of NP retained in excised
whole-lung tissues at various time points after inhalation using an
in vivo imaging system (Fig. 4F). The retention of PLGA-PEG MPP
was significantly greater at each time point up to 24 hours compared
to that of PLGA MAP. At 6 hours, approximately 75% of PLGA-PEG
MPP were retained, whereas only ~50% of PLGA MAP were retained
at the same time point (P < 0.01).
SCIENCE ADVANCES | RESEARCH ARTICLE
500
250
0
#
Saline
Saline
Free DP
0
DP/PLGA/F68
5
750
Free DP
10
∗
DP/PLGA/F68
∗∗
∗∗
1000
DP/PLGA/F127
15
B
TNF-α (pg/ml)
∗∗
DP/PLGA/F127
6
Total cell count (×10 )
A
exists between mucus penetration and improved retention. The rapid
clearance of MAP from the lung in the retention experiments reported
here agrees well with the time scale reported for MCC (44, 45) and was
faster than would be expected for clearance mediated via macrophages
[24 to 48 hours for airway macrophages and weeks to months for
alveolar macrophages (46)]. Notably, we have demonstrated similar
beneficial effects of MPP in the vagina (47, 48) and gastrointestinal
tract (49) in vivo. These findings altogether suggest that MPP are a
promising alternative to mucoadhesive formulations for therapeutic
delivery to the lung airways.
A recent study suggested that MPP, despite the rapid diffusion over
short time and length scales, are not able to penetrate physiologically
thick respiratory mucus layers (~10 to 100 mm) (37). They found that
PEG-coated magnetic dextran/iron oxide NP were unable to penetrate
horse respiratory mucus under the constant force of a magnet. These
results are in stark contrast to the wide distribution of MPP observed
in the present study, where MPP were well distributed throughout
airway lumens, including penetration into a submucosal gland that
was more than 100 mm deep from the epithelial surface. There are
several potential factors that may explain the discrepancy observed
in these two independent studies. First, the PEG-coated magnetic
dextran/iron oxide NP used in the previous study (37) were not confirmed to be muco-inert. Thus, the NP may have been hindered and/or
immobilized in horse tracheal mucus via adhesive interactions,
thereby preventing them from moving through the mucus even with
the aid of a magnetic force. As we have previously shown, a critical
PEG density on the surface of NP is crucial to achieve muco-inert
surface coatings, and insufficient PEG densities result in NP adhesion
to mucus (38, 50–52). Second, if the NP used were muco-inert, the magnetic force may have caused the NP to run into mucus bundles, causing
them to potentially deform around and trap the particles or to enter
mucus pores smaller than the particle diameter and rendered them
sterically trapped. We have previously shown that various mucus
samples exhibit highly heterogeneous pore size distributions, with
significant percentages of pores smaller than the size of conventional
NP (25, 26, 53–56). In addition, NP were under a constant uniSchneider et al., Sci. Adv. 2017; 3 : e1601556
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Fig. 5. In vivo anti-inflammatory effects of GRAS-based biodegradable NP
carrying DP in the lungs of mice challenged with Pseudomonas aeruginosa
LPS. Mice were challenged twice with LPS at 0 and 6 hours. At t = 24 hours, LPStreated mice received DP/PLGA/F127 or DP/PLGA/F68 NP at a dose of 1 mg/kg.
Control LPS-treated mice received either carrier-free DP or saline. Mice were sacrificed
at 48 hours for BALF analysis. (A) Total inflammatory cell counts. (B) Concentration of
TNF-a. Data represent means ± SD. *P < 0.05, **P < 0.01.
directional magnetic force, such that NP that entered into small mucus
pockets would be unlikely to escape. Under physiological conditions,
muco-inert NP would be able to diffuse back and forth in the pores,
escape from them, and continue their journey in the mucus.
The clinical implications of this work are significant, because improved lung distribution and retention achieved by MPP may enhance
the efficacy of other drugs that could treat a large number of lung
diseases. Mucus penetration of NP will also enhance the probability
that therapeutic NP may reach the underlying epithelial cells protected
by the mucus layer. Inhaled corticosteroids suffer from poor lung
pharmacokinetics and short duration of action because of their rapid
clearance by MCC as well as absorption into the blood (57). Repeated
dosing is often required to maintain the local drug concentration in
the therapeutic window, which may transiently expose patients to
elevated local and/or systemic drug concentrations, leading to toxicity/side effects. Delivery of corticosteroids in MPP formulations may
resolve these shortcomings by providing long-term therapeutic drug
concentrations in the lung without the need for frequent dosing. We
found that b-MPP loaded with DP improved the efficacy in reducing
inflammation in a mouse model of LPS-induced acute lung inflammation. Likewise, Nafee and co-workers recently demonstrated that inhaled ultrasmall lipid NP capable of penetrating mucus greatly
enhanced the antivirulence efficacy of an anti-infective, quorumsensing inhibitor (36). In the case of lung cancer, tumor cells are susceptible to chemotherapy only at distinct phases of the cell cycle (58).
The sustained therapeutic drug concentrations enabled by MPP may
augment the chance of targeting tumor cells at the appropriate phases
of the cell cycle in the lung airways. Prolonged retention of MPP may
also be helpful in CF gene therapy, which has been unsuccessful, to
date, in clinical trials (59, 60). We have recently reported that newly
engineered mucus-penetrating DNA NP significantly enhanced pulmonary gene transfer compared to conventional gene vectors (61),
including one that has shown efficacy comparable to a leading viral
gene vector widely tested in clinical trials (that is, adeno-associated
virus serotype 2) (59, 62).
MPP that rapidly penetrate the outer LML into the deeper PCL
may be retained significantly longer, because this layer is believed to
be nearly stationary (63, 64). This mechanism has been hypothesized
to explain the enhanced retention of human serum albumin compared to sulfur colloids in the canine lungs (32), as well as the ability
of certain viral particles to infect mucosal surfaces (65). However,
this mechanism has recently been challenged by the seminal findings
of Button and co-workers (66). In vitro experiments that used an
air-liquid interface culture of primary human bronchial epithelium
revealed that dextran probes larger than 40 nm were excluded from
the PCL, whereas smaller dextran molecules partitioned into the
layer closer to the epithelium (66). On the basis of this observation,
they postulated that the PCL may behave as a fine mesh rather than
a watery layer in which inhaled objects can freely diffuse. However,
because the observations were made in vitro only, it is unclear
whether this exact behavior would be replicated in vivo and, if so,
whether it is observed throughout the conducting airways regardless
of branching generation. Furthermore, this model does not take into
account the presence of nonciliated cells, including secretory cells,
which are often found in the airway epithelium (67). The presence
of these cells may create permeable gaps in PCL where secreted mucins
diffuse out to reach the LML. There are fewer ciliated cells in the lower
conducting airways, which may result in larger areas with a more
permeable PCL.
SCIENCE ADVANCES | RESEARCH ARTICLE
We found in this study that MAP, regardless of particle diameter,
were rapidly removed from the lumen of the lung in vivo. This suggests
that previously reported favorable outcomes achieved with a drug in
MAP compared to carrier-free soluble drug formulations may be partly
attributed to the benefits intrinsic to NP-based drug delivery systems
rather than NP mucoadhesion per se. In contrast, MPP were uniformly
distributed throughout the airway mucus layer and exhibited improved
retention, resulting in improved therapeutic efficacy compared to
carrier-free drug and drug delivered by a MAP formulation. These
findings suggest that MPP, at least those up to 300 nm in diameter,
provide an attractive alternative to the use of MAP to enhance pulmonary delivery of therapeutics.
MATERIALS AND METHODS
Characterization of NP
Hydrodynamic diameters and z-potentials of all NP were measured by
DLS and laser Doppler anemometry, respectively, using a Zetasizer
Nano ZS (Malvern Instruments) in 10 mM NaCl (pH 7.4) at 25°C, as
per the manufacturer’s instructions.
Fluorescent labeling of biodegradable polymers
PLGA2A (15 kDa; Lakeshore Biomaterials) and methoxy-PEG5kPLGA45k (Jinan Daigang Biomaterial Co.) were labeled with a carboxylreactive Alexa Fluor dye (AF488, AF555, or AF647), as previously
described (68). Reaction details are provided in the Supplementary
Materials.
Preparation of biodegradable NP
PLGA and PLGA-PEG NP were formulated by an emulsification solvent evaporation method, as previously described (68). Briefly, 40 mg of
polymer, including 20 mg each of fluorescently labeled and unlabeled
polymer, was dissolved in dichloromethane (DCM) (0.2 and 0.8 ml for
MAP and MPP, respectively). The organic DCM phase was then added
to an aqueous phase of 5 ml of 0.5% cholic acid sodium salt (CHA),
followed by probe sonication for 2 min (30% amplitude). The emulsion
was then added to a stirring solution of 35 ml of 0.5% CHA and allowed
to harden for 3 hours under magnetic stirring at 700 rpm. To ensure
complete removal of DCM, the beaker was transferred to a vacuum
chamber for 30 min. Biodegradable NP, either b-MPP or b-MAP,
were collected by centrifugation (10,000g, 30 min) and washed twice
in 35 ml of ultrapure water. NP were characterized as described above.
GRAS-based NP, including PLGA/PF, PLGA/F68, and PLGA/
F127 NP, were prepared by a solvent diffusion method, as previously
described (41). Ten milligrams of polymer was dissolved in 1 ml of
tetrahydrofuran (THF) and added dropwise to 40 ml of ultrapure
water and 5% F68 (BASF) and 5% F127 (BASF) aqueous solution
for PLGA/PF, PLGA/F68, and PLGA/F127, respectively, under
magnetic stirring at 700 rpm. After rotary evaporation for 30 min
to remove the THF, the NP were collected by centrifuging at 10,000g
for 25 min, washed twice with respective aqueous solutions, and resuspended in 0.4 ml of ultrapure water. NP were characterized as described above.
Schneider et al., Sci. Adv. 2017; 3 : e1601556
5 April 2017
Animals
All animal protocols were approved by the Institutional Animal Care
and Use Committee at the Johns Hopkins Medical Institutions. Female
CF-1 or Balb/c mice (6 to 8 weeks; Harlan) were allowed to acclimate for
1 to 2 weeks before handling, with access to food and water ad libitum
throughout the experiments.
Ex vivo stability of biodegradable NP in BALF
Mice were challenged twice with 100 mg of P. aeruginosa LPS (serotype
10; Sigma-Aldrich) at 0 and 6 hours by intranasal instillation. After
48 hours, animals were sacrificed, and the lungs were harvested. BALF
was collected by sequentially lavaging the lungs three times with 1 ml of
PBS, and the recovered fluids were pooled for each animal. Subsequently,
BALF samples collected from four animals were pooled, cells were
separated by centrifugation, and supernatant was collected and syringefiltered (0.2 mm). The protein content of the BALF supernatant was
measured using the BCA (bicinchoninic acid) Protein Assay Kit (Pierce).
Twenty microliters of stock NP solution (20 mg/ml) was added to 0.4 ml
of BALF (a protein concentration of 0.6 mg/ml) and incubated at room
temperature. At 2 and 24 hours after incubation, hydrodynamic diameters of NP were measured by DLS, as described above.
DP loading and release kinetics
To measure the DP content of Pluronic-coated biodegradable NP, the
NP were freeze-dried, weighed, and dissolved in 0.5 ml of acetonitrile.
Subsequently, 1 ml of 50 mM EDTA was added to chelate zinc and solubilize encapsulated DP, and the DP concentration was measured by
reverse-phase high-performance liquid chromatography (HPLC). Briefly,
isocratic separation was performed on a Shimadzu Prominence LC system equipped with a Pursuit 5 C18 column (Varian Inc.). The mobile
phase was composed of acetonitrile/water (35/65 v/v) containing 0.1%
trifluoroacetic acid (flow rate, 1 ml/min). Column effluent was monitored
by ultraviolet detection at 241 nm. The drug loading and encapsulation
efficiency were calculated according to the following equations
Drug loading ð%Þ ¼ ðamount of DP in NP=weight of NPÞ
100
ð1Þ
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Formulation of model MPP
Fluorescently labeled carboxyl-modified PS NP (Invitrogen) were
PEGylated via carbodiimide cross-linking chemistry, as previously
described, to generate MPP (39). Details are provided in the Supplementary Materials.
Preparation of DP-loaded biodegradable NP
DP was encapsulated into Pluronic-coated PLGA NP following a
modified solvent diffusion method, as previously described (42, 43).
Briefly, a DP-zinc complex was formed by adding 1 ml of 0.5 M zinc
acetate aqueous solution to 0.5 ml of an aqueous solution containing
10 mg of DP (Sigma-Aldrich). After centrifuging at 20,000g for 5 min,
the precipitated DP-zinc complex and 50 mg of PLGA1A (3.2 kDa;
Lakeshore Biomaterials) were dissolved in 2.5 ml of THF, followed by
the addition of 20 ml of triethanolamine. The mixture was added dropwise
into 100 ml of an aqueous solution containing either 5% F68 or 5% F127
while stirring to form DP-loaded b-MAP or b-MPP, respectively. After
rotor evaporation for 30 min to remove the THF, 1 ml of 0.5 M EDTA
aqueous solution (pH 7.5) was added to the NP suspension to chelate
excess zinc and solubilize any unencapsulated DP-zinc complexes. The
NP were then separated from unencapsulated DP, zinc, and EDTA by
centrifuging at 10,000g for 25 min, washed twice with 5% F68 or 5%
F127, and resuspended in 0.4 ml of ultrapure water. The hydrodynamic
diameters and z-potentials of NP were characterized as described above.
NP morphology was visualized using a Hitachi H-7600 transmission
electron microscope (Hitachi Co. Ltd.).
SCIENCE ADVANCES | RESEARCH ARTICLE
Encapsulation efficiency ð%Þ
¼ ðmeasured=theoretical loadingsÞ 100
ð2Þ
To measure the in vitro release of DP, 200 ml of the NP suspension (approximately 25 mg/ml) was sealed in a Spectra/Por 6 dialysis membrane
(molecular weight cutoff, 10 kDa; Spectrum Laboratories Inc.). The
sealed dialysis membrane was placed into a 50-ml conical tube containing
12 ml of PBS and incubated at 37°C on a platform shaker (140 rpm).
The entire release medium was collected at predetermined intervals and
replaced with 12 ml of fresh PBS. The concentration of DP in the release
medium was measured by HPLC, as described above.
MPT of biodegradable NP in CF mucus
Diffusion of fluorescently labeled (AF555) biodegradable NP in human
CF sputum was studied by MPT, as we have previously described (26).
Details are provided in the Supplementary Materials.
Ex situ tracking of model NP in freshly excised
mouse trachea
Fluorescently labeled PS-based MAP (excitation/emission maxima of 505/
515 nm; Invitrogen) and MPP (excitation/emission maxima of 660/
680 nm; Invitrogen) were mixed together at a concentration of 2 mg/
ml in sterile, hypotonic PBS (that is, diluted PBS). Female CF-1 mice
were anesthetized under continuous flow of 2% isoflurane (Baxter
Healthcare Corp), and 50 ml of the NP suspension was administered
via intranasal instillation. Mice were sacrificed 30 min after administration, and the tracheas were carefully resected and cut longitudinally
to generate “half-pipes” that were laid face-up on microscope slides.
The trachea was instantaneously flattened and sealed with a coverslip,
and movies of NP were captured at a temporal resolution of 66.7 ms
for 20 s with a Zeiss Axio Observer D1 inverted epifluorescence microscope (Zeiss) equipped with a Photometrics Evolve 512 camera
(Photometrics) and MetaMorph software (Molecular Devices) after
incubation for 1 hour. Areas where all NP were immobilized were
avoided to ensure that NP were located in regions with intact tracheal
mucus [some studies suggest that the mucus gel layer is discontinuous
(69–72)], and care was taken to avoid areas with convection. Fluorescently labeled MAP and MPP were imaged in the exact same field of
view to allow direct comparison of tracking results.
Distribution of NP in lung airways
Fluorescently labeled PS-based or PLGA-based MAP and MPP were
mixed together at a concentration of 2 or 15 mg/ml, respectively, in
sterile, hypotonic PBS. Female CF-1 mice were anesthetized under
a continuous flow of 2% isoflurane, and 50 ml of the NP suspension
was administered via intranasal instillation. Mice were sacrificed after
Schneider et al., Sci. Adv. 2017; 3 : e1601556
5 April 2017
Retention of NP in the lung
Retention of fluorescently labeled NP in the lungs was measured by two
complementary methods: the lavage method, which only measures NP
in the lung lumen, and the whole-lung method, which measures NP in
entire lung tissue. The whole-lung method accounts for particles
deposited in the conducting airways as well as the alveolar regions that
are not accessible via the lavage method.
For the lavage method, female CF-1 mice (6 to 8 weeks) were
anesthetized under a continuous flow of 2% isoflurane, and 50 ml of
the NP suspension was administered via intranasal instillation. Mice
were sacrificed at various time points via cervical dislocation, and the
lungs were resected, rinsed briefly in PBS, and lavaged three times
with 1 ml of PBS to collect BALF. Fluorescently labeled NP in the BALF
were measured on a 96-well fluorescent plate reader (BioTek Synergy
2, BioTek).
For the whole-lung method, similarly treated female CF-1 mice
were sacrificed at various time points via cervical dislocation, and the
lungs were resected, rinsed briefly in PBS, laid on a labeled petri dish,
and stored at −20°C until imaging. Imaging was performed with Xenogen IVIS Spectrum live animal imaging system (Caliper Life Sciences).
Fluorescence was monitored at excitation/emission wavelengths of 640/
680 nm with an exposure time of 1 s. Quantification of fluorescence was
performed with Living Image 2.5 software (PerkinElmer). Details are
provided in the Supplementary Materials.
In vivo anti-inflammatory efficacy of DP-loaded NP
Mice were challenged twice with 100 mg of LPS at 0 and 6 hours by
intranasal instillation. After 24 hours, carrier-free DP or DP-loaded NP
were administered in saline by oropharyngeal aspiration at a DP dose
of 1 mg/kg (n = 8). Mice in a control group received saline (n = 8).
After 48 hours, animals were sacrificed, and lungs were harvested.
BALF was collected and analyzed for total and differential cell counts
and concentration of TNF-a as follows. The lungs were sequentially
perfused three times with 1 ml of PBS, and the recovered BALF was
pooled for each animal. Cells were collected by centrifugation, and the
supernatant was stored at −80°C for enzyme-linked immunosorbent
assay (ELISA). Red blood cells were lysed with ammonium-chloridepotassium (ACK) lysing buffer (Quality Biological Inc.). Total cell
counts were determined using a Vi-CELL XR automated cell viability
analyzer (Beckman Coulter Inc.), and a cytokine, TNF-a, was assayed
using a Quantikine ELISA kit (R&D Systems Inc.). Cytokine concentrations in BALF were adjusted for dilution by the urea method (73) and
expressed as a concentration of epithelial lining fluid (pg/ml).
In vivo safety profile of biodegradable NP
GRAS-based biodegradable NP were administered in saline by oropharyngeal aspiration at a dose of 1 mg per mouse (50 mg/kg) (n = 9).
Control mice were treated with saline (n = 9). After 24 hours, animals
were sacrificed, and lungs were harvested for either BALF (n = 7) or
tissue histology (n = 2). BALF was collected and analyzed for total
cell counts and TNF-a content, as described above. Differential cell
counts were performed via cytospin with a Shandon Cytospin 3 centrifuge (Shandon Scientific) after staining with hematoxylin and eosin
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Collection of CF sputum
Sputum samples spontaneously expectorated by male and female CF
patients aged 23 to 40 were collected at the Johns Hopkins Adult Cystic
Fibrosis Program. The procedures conformed to ethical standards,
and the sputum collection was performed under informed consent
on a protocol approved by the Johns Hopkins Medicine Institutional
Review Board. Two to three samples were acquired from the weekly
CF outpatient clinic, placed on ice upon collection and during transport,
pooled to minimize patient-to-patient variation, and studied the
same day. The total number of individual samples used for the present studies was 9.
30 min, and the lungs were carefully resected, added to plastic cryomolds
filled with optimum cutting temperature (OCT) compound (Sakura),
and frozen in liquid nitrogen. Lungs were sectioned, mounted, stained
with DAPI, and imaged on an epifluorescence microscope (Zeiss).
Details are provided in the Supplementary Materials.
SCIENCE ADVANCES | RESEARCH ARTICLE
(H&E). To prepare lungs for tissue sectioning, lungs were excised
completely from the chest, inflated with 1 ml of 10% formalin, and
then immersed in 10% formalin for 24 hours before embedding in
paraffin. Five-micrometer-thick sections were cut from each lung
and stained with H&E.
Statistical analysis
Statistical analysis of data was performed by one-way analysis of variance (ANOVA), followed by Tukey post hoc test or Games-Howell test
using SPSS 18.0 software (SPSS Inc.). Differences were considered to be
statistically significant at a level of P < 0.05.
SUPPLEMENTARY MATERIALS
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content/full/3/4/e1601556/DC1
Supplementary Materials and Methods
fig. S1. Diffusion of 300-nm PS and PS-PEG in human CF sputum.
fig. S2. Lung histology at 24 hours after administration.
fig. S3. Safety profile of GRAS-based biodegradable NP.
movie S1. Diffusion of PLGA NP in freshly collected CF sputum.
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comments. We thank M. Ramsay and S. Watts at the Johns Hopkins Adult Cystic Fibrosis
Center for CF sputum collection and the Integrated Imaging Center and Wilmer Imaging and
Microscopy Core Facility at Johns Hopkins University for assistance with microscopy. The
content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institute of Biomedical Imaging and Bioengineering, the National
Heart, Lung, and Blood Institute, or the NIH. Funding: Funding was provided by the NIH
(R01HL125169, R01HL136617, P01HL51811, R01HL127413, U54CA151838, and P30EY001765)
and the Cystic Fibrosis Foundation (CFF HANES08G0, HANES15G0, and HANES16XX0).
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Q.X., N.J.B., J.C., B.C.T., B.S.S., A.H., E.L., P.A., B.W.S., S.-Y.S.W., X.G., T.Y., and J.S.S. performed and/or
analyzed experiments. M.P.B. collected patient samples and managed clinical data. C.S.S., Q.X.,
N.J.B., J.C., L.M.E., J.S.S., and J.H. wrote the manuscript. Competing interests: The muco-inert
particle technology described in this publication is being developed by Kala Pharmaceuticals.
J.H. declares a financial, a management/advisor, and a paid consulting relationship with Kala
Pharmaceuticals. J.H. is a cofounder of Kala Pharmaceuticals and owns company stock, which is
subject to certain restrictions under Johns Hopkins University policy. The terms of this arrangement
are being managed by Johns Hopkins University in accordance with its conflict-of-interest policies.
The following patents involve some of the authors (Q.X., N.J.B., J.S.S., and J.H.) who contributed to
this publication and are related to the current manuscript: US 20100215580 A1 (Compositions and
methods for enhancing transport through mucus), CA 2816977 A1 (Compositions and methods
relating to reduced mucoadhesion), WO 2005072710 A3 (Drugs and gene carrier particles that
rapidly move through mucous barriers), WO 2012109363 A2 (Mucus penetrating gene carriers), WO
2013090804 A3 (Nanoparticles with enhanced mucosal penetration or decreased inflammation),
and US 9415020 B2 (Nanoparticle formulations with enhanced mucosal penetration: Drugs and
gene carrier particles that rapidly move through mucous barriers). Data and materials availability:
All data needed to evaluate the conclusions in the paper are present in the paper and/or the
Supplementary Materials. Additional data related to this paper may be requested from the authors.
Submitted 7 July 2016
Accepted 10 February 2017
Published 5 April 2017
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Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to
airways following inhalation
Craig S. Schneider, Qingguo Xu, Nicholas J. Boylan, Jane Chisholm, Benjamin C. Tang, Benjamin S. Schuster, Andreas
Henning, Laura M. Ensign, Ethan Lee, Pichet Adstamongkonkul, Brian W. Simons, Sho-Yu S. Wang, Xiaoqun Gong, Tao Yu,
Michael P. Boyle, Jung Soo Suk and Justin Hanes
Sci Adv 3 (4), e1601556.
DOI: 10.1126/sciadv.1601556
http://advances.sciencemag.org/content/3/4/e1601556
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