of 9
Food Polyelectrolytes Compress the Colonic Mucus Hydrogel by a
Donnan Mechanism
Asher Preska Steinberg,
Zhen-Gang Wang,
and Rustem F. Ismagilov
*
,
,
Division of Chemistry and Chemical Engineering and
Division of Biology and Biological Engineering, California Institute of
Technology, 1200 E. California Blvd., Pasadena, California 91125, United States
*
S
Supporting Information
ABSTRACT:
Systems consisting of a polyelectrolyte solution
in contact with a cross-linked polyelectrolyte network are
ubiquitous (e.g., bio
fi
lms, drug-delivering hydrogels, and
mammalian extracellular matrices), yet the underlying physics
governing these interactions is not well understood. Here, we
fi
nd that carboxymethyl cellulose, a polyelectrolyte commonly
found in processed foods and associated with in
fl
ammation
and obesity, compresses the colonic mucus hydrogel (a key
regulator of host
microbe interactions and a protective
barrier) in mice. The extent of this polyelectrolyte-induced
compression is enhanced by the degree of polymer negative
charge. Through animal experiments and numerical calcu-
lations, we
fi
nd that this phenomenon can be described by a Donnan mechanism. Further, the observed behavior can be
quantitatively described by a simple, one-parameter model. This work suggests that polymer charge should be considered when
developing food products because of its potential role in modulating the protective properties of colonic mucus.
INTRODUCTION
In this work, we sought to understand how polymer charge
in
fl
uences polymer-driven mucus compression. The colonic
mucus hydrogel is a critical barrier in the colon
it is the nexus
of host
microbe interactions and it protects against microbial
in
fi
ltration and physical insults.
1
This hydrogel, which lines the
walls of the colon, is composed primarily of high-molecular-
weight (MW) glycoproteins (
1.2 MDa) known as mucins
and is held together by physical entanglements, chemical cross-
links, and electrostatic interactions.
2
,
3
Although the micro-
biology and chemical biology communities have exhaustively
studied how microbes interact with this hydrogel and its
biochemical composition,
1
,
2
,
4
,
5
the underlying physics that
governs the structural features of the colonic mucus hydrogel
has only recently begun to be explored.
6
In particular, it is vital
to understand what in
fl
uences the de-swelling or compression
of this hydrogel because several studies have found correlations
between changes in the mesh size and thickness of colonic
mucus and changes in host health.
7
,
8
Our recent work has
found that neutral or uncharged polymers can compress the
colonic mucus hydrogel by a mechanism that can be described
using a simple,
fi
rst-principles thermodynamics model based
on Flory
Huggins solution theory.
6
It was shown that for
these uncharged polymers, the extent of polymer-induced
mucus compression is increased by either increasing the
polymer concentration or increasing the polymer MW at a
given polymer concentration. However, the human diet
contains many charged polymers (i.e., polyelectrolytes),
which are predominately negatively charged.
9
,
10
One polyelectrolyte that is commonly placed in food and is
generally regarded as safe
(GRAS) by the U.S. Food & Drug
Administration (FDA) is carboxymethyl cellulose (CMC).
11
This polyelectrolyte is a cellulose derivative that has a negative
charge in the gut due to carboxymethyl groups attached to
some of its monomer units.
12
Interestingly, although many
charged versions of CMC exist, the FDA allows only up to a
degree of substitution (DS) of 9 charged groups per 10
monomers (abbreviated as
DS 0.9
). There is no existing
literature explaining how changing the charge of these
polymers a
ff
ects the design of food products. CMC is added
to processed foods because of its ability to enhance the
viscosity of food and to stabilize emulsions by slowing droplet
coalescence,
9
,
13
which leads to it often being mistakenly called
an
emulsi
fi
er
even though it is not a surfactant but a high-
MW polyelectrolyte. Recent biological studies found that
feeding mice CMC resulted in low-grade in
fl
ammation and
obesity. CMC feeding was also correlated with a thin mucus
layer that allowed for microbial encroachment upon the
host.
14
,
15
In addition, it has been shown that acute exposure to
CMC (by direct injection into the small intestine) can alter the
structure of the small intestine mucus layer in rats.
16
However,
mechanistic understanding of these e
ff
ects is lacking; it is
unclear if, in vivo, colonic mucus is thinner because it is
disrupted or compressed. We hypothesize that the thin colonic
Received:
March 29, 2019
Revised:
May 23, 2019
Published:
June 19, 2019
Article
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2019, 20, 2675
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© 2019 American Chemical Society
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DOI:
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mucus layer in mice fed CMC was the result of mucus
compression.
Many studies have covered the physical chemistry of
polyelectrolyte solutions,
17
,
18
polyelectrolyte hydrogels,
19
,
20
complex coacervation between oppositely charged polyelec-
trolytes,
21
,
22
and complexation between polyelectrolytes with
oppositely charged objects.
23
,
24
In contrast, the interactions
between systems composed of polyelectrolyte solutions and
polyelectrolyte gels remain vastly understudied,
25
both
experimentally and theoretically. Here, we seek to untangle
the physical interactions between colonic mucus (a biological
polyelectrolyte gel) and CMC (a polyelectrolyte).
MATERIALS AND METHODS
Details of Animals Used.
All mice were 2
6 months old, male or
female speci
fi
c-pathogen-free (SPF) C57BL/6 mice (RRID: IM-
SR_JAX:000664). In our previous study, we did not observe any
di
ff
erences in mucus compression related to age (in the same age
range as this study) or gender.
6
We justi
fi
ed the use of both male and
female mice because an experimental study found that
3-month-old
C57BL/6 mice had similar mucus thickness and morphology
regardless of sex.
26
We justi
fi
ed the use of a range of ages of mice
because, although it has been reported that 19-month-old C57BL/6
mice had thinner colonic mucus compared to 2.5
3-month-old
C57BL/6 mice, 19 months old is well outside the age range of this
study.
26
,
27
Mice used in ex vivo experiments in
Figures 2
4
and
Figure S1
were maintained on a solid chow diet (PicoLab Rodent
Diet 20) and were given food and water ad libitum. Mice used in
experiments in
Figure 1
were maintained on a chow diet until the day
of the experiment. Starting 23 h before euthanization, these mice were
restricted (no chow or water) to a solution of 1% w/v carboxymethyl
cellulose (carboxymethylcellulose sodium, USP grade, medium
viscosity, PN: C9481-500G) with 5% w/v sucrose (USP grade, PN:
S3929) in water or a solution of 1% v/v Tween (polysorbate 80, food
grade, Sigma-Aldrich, PN: W291706) with 5% sucrose in water. For
these 23 h, mice were kept on mesh-bottom cages to prevent re-
ingestion of chow-derived polymeric contents from fecal matter. All
mice were obtained from Jackson Labs (The Jackson Laboratory, Bar
Harbor, ME, USA) and were then housed at Caltech
s animal facility.
All animal experiments were approved by the California Institute of
Technology (Caltech) Institutional Animal Care and Use Committee
(IACUC; protocol no. 1691) and the U.S. Army
s Animal Care and
Use Review O
ffi
ce (ACURO; protocol no. 70905-LS-MUR.03). Mice
were euthanized via CO
2
inhalation as approved by the Caltech
IACUC in accordance with the American Veterinary Medical
Association Guidelines on Euthanasia.
28
Details of Microscopy.
Images were acquired by taking z-stacks
on a Zeiss LSM 880 upright confocal microscope using confocal
fl
uorescence to image particles (488 nm excitation/505
736 nm
band pass
fi
lter), confocal re
fl
ectance to image the epithelium (561
nm excitation/505
736 nm band pass
fi
lter), bright
fi
eld for the
epithelium and particles, or two photon for the FC oil layer and
epithelium (700 or 750 nm excitation/650
758 nm band pass
fi
lter).
Imaging of Samples Using
FC Oil Approach
.
Sample
preparation and imaging were carried out as described previously in
ref
6
(in ref
6
, see Supporting Information Materials and Methods,
section
Imaging of Unwashed Tissue
).
Imaging of Samples Using
Microparticle Approach
.
Sample preparation and imaging were carried out as described
previously in ref
6
(in ref
6
, see Supporting Information Materials and
Methods, sections
Imaging of Washed Tissue
and
Thickness
Measurements of Washed Mucus Hydrogel
). The protocol was
modi
fi
ed such that the
fl
uorescent 1
μ
m diameter polystyrene beads
coated with poly(ethylene glycol) (PEG) with a molecular weight
(MW) of 5 kDa were used as the microparticles (created as described
in ref
29
). These were imaged using
fl
uorescence in addition to
confocal re
fl
ectance (488 nm excitation/505
736 nm band pass
fi
lter). For the thickness measurements obtained using the micro-
particle approach shown in
Figure 1
, determination of the mucus
thickness was done in the same way as the FC oil approach.
Compression Measurements.
Compression measurements were
carried out as described in ref
6
(in ref
6
, see Supporting Information
Materials and Methods, section
Quantifying Polymer-induced
Compression of Washed Mucus Hydrogel
). In this work, we de
fi
ne
% compression
as % compression = [
Δ
t
/
t
0
]
*
100%, where
t
0
is the
initial mucus thickness, and
Δ
t
=
t
0
t
f
, where
t
f
is the
fi
nal mucus
thickness. We modi
fi
ed the protocol such that each compression
measurement in this work represents the mean of compression
measurements taken on colonic explants from three separate mice.
The compression value from each individual explant is the average of
compression measurements in
fi
ve di
ff
erent positions on that explant.
The error bars are the standard error of the mean with
n
= 3. For
measurements done with 1
×
phosphate-bu
ff
ered saline (PBS), we
diluted 10
×
PBS (Corning 10
×
PBS, pH 7.4
±
0.1, without calcium
and magnesium, RNAse-/DNAse- and protease-free, product no. 46-
013-CM) 10-fold with Milli-Q water. In the compression experiments
in
Figure 4
with polymers in 10
×
PBS, the tissue was incubated with
microparticles in 1
×
PBS for
1 h before placing on the polymers in
10
×
PBS. The
fi
nal thickness was then measured after 10 min. This
was done to prevent prolonged exposure (1 h or longer) to 10
×
PBS
(which after long times could cause tissue deterioration due to the salt
imbalance) while the microparticles sedimented down on the top of
the mucus hydrogel.
Figure 1.
Carboxymethyl cellulose (CMC) compresses the colonic
mucus hydrogel in vivo. (A) Cartoon side view depicting the
fl
uorocarbon (FC) oil imaging setup, which retains polymeric
contents in contact with colonic mucus, prevents dehydration, and
maintains mucus at a similar thickness (
t
) to that of initial in vivo
thickness (
t
0
). (B) In vivo, the mucus hydrogel is in contact with
polymeric contents that can compress mucus. (C) Cartoon side view
depicting the microparticle imaging setup in which polymeric
contents are washed away with bu
ff
er and particles with a diameter
(
d
) greater than the mucus mesh size (
ξ
) are used to measure mucus
thickness. The mucus thickness increases (
Δ
t
) from in vivo when
compressive
polymers are absent. (D) Mucus thickness measure-
ments from mice fed either a solution of 1% CMC + 5% sucrose (1%
CMC) or a solution of 1% Tween + 5% sucrose (1% Tween) for 23 h.
The mucus thickness is plotted on the vertical axis (in
μ
m) for
di
ff
erent groups of mice. Measurements using the microparticle
approach (
washed (gut contents removed)
) are in blue; thickness
measurements obtained using the FC oil approach (
unwashed
) are
in orange (see ref
6
for details and validation of approach). Thickness
measurements represent the average thickness measured on explants
from individual mice. Error bars are the standard error of the mean
(SEM) where
n
= the number of mice. All groups contained at least
three mice.
P
values were obtained using Welch
s
t
-test. The gray bar
across the
fi
gure indicates mucus thickness measured for chow-fed
mice using the FC oil approach from our previous study,
6
where we
measured
t
=67
±
7
μ
m (mean
±
SEM). The bottom of the bar is
t
=
60
μ
m, and the top of the bar is
t
=74
μ
m.
Biomacromolecules
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DOI:
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Polymers Used for Compression Measurements.
We used
carboxymethyl cellulose (CMC) with a degree of substitution (DS) of
7 charged monomers per 10 monomers (DS 0.7) (Sigma-Aldrich, PN:
419311), CMC DS 0.9 (Sigma-Aldrich, PN: 419303), and
hydroxyethyl cellulose (HEC) (Sigma, PN: 308633).
Gel Permeation Chromatography (GPC) of Polymers.
GPC
was used to measure the MW and hydrodynamic radii (
R
h
). This was
used to con
fi
rm that CMC used in the mouse-feeding experiments in
Figure 1
, and CMC and HEC used in all other
fi
gures were
approximately the same MW and
R
h
(measurements shown in
Figure
S3 and Table S1
). GPC measurements were conducted as described
in ref
29
. CMC derivatives were analyzed using a refractive index
increment (d
n
/d
c
)of
0.163
n
c
d
d
=
.
30
HEC was analyzed using
0.150
n
c
d
d
=
.
31
Curve Fitting in
Figure 3
.
For the curve
fi
tting presented in
Figure 3
, we used the
scipy.optimize.curve_
fi
t
function in Python
3.6.4, which is included as a supplemental
fi
le to the manuscript.
RESULTS AND DISCUSSION
Carboxymethyl Cellulose (CMC) Compresses Mucus
Reversibly in Vivo.
We
fi
rst sought to test two hypothesis:
(1) the colonic mucus hydrogel is thin when mice are fed
CMC because the mucus hydrogel is compressed; (2) the
mechanism by which CMC interacts with mucus is di
ff
erent
than that of an emulsi
fi
er
polysorbate 80 (Tween 80)
because of the di
ff
erences in their physicochemical properties
(CMC is a high-MW polyelectrolyte, whereas Tween is a low-
MW, nonionic surfactant).
To test these two hypotheses, we devised a simple
experiment in which we fed one group of SPF mice a solution
of 1% w/v CMC and another group 1% w/v Tween 80 for 23
h, and then measured the thickness of the mucus hydrogel. We
justi
fi
ed the removal of the standard chow diet because our
previous work with di
ff
erent dietary polymers suggested that
the components of chow do not contribute to mucus
compression in SPF mice.
6
We tested this in ref
6
by
measuring mucus compression on colonic explants using
polymers in bu
ff
er and comparing it to compression induced
by the same polymers prepared in extracted luminal
fl
uid from
chow-fed SPF mice. We fo
und similar amounts of
compression. Additional evidence supporting this in ref
6
was that adding the luminal
fl
uid from chow-fed SPF mice to
colonic explants did not induce mucus compression and that,
for chow-fed SPF mice, the mucus thickness on explants
remained the same when luminal contents were removed. In
this work, for our experiment to test the di
ff
erences between
feeding 1% w/v CMC and 1% w/v Tween 80, we
fi
rst
measured the thickness of the mucus hydrogel using our
fl
uorocarbon (FC) oil approach (
Figure 1
A,B; see ref
6
for
further details). Brie
fl
y, this method allows us to avoid washing
colonic explants with bu
ff
er (which could cause the loss of
polymeric contents that are in contact with the mucus
hydrogel), and it eliminates the use of a
fi
xative (which
could alter mucus structure). Instead, we remove luminal
contents with FC-40 oil, which is immiscible with and denser
than water, and coat the explant with FC-40 oil, which sits on
the top of mucus. The FC oil approach has the further
advantage of preventing dehydration of the mucus layer,
allowing us to measure the extent of compression as it would
be in vivo. The thickness is then obtained by measuring the
di
ff
erence in the position of the epithelial cells under mucus
(identi
fi
ed using bright-
fi
eld and confocal re
fl
ectance) and the
position of the FC oil
hydrogel interface (identi
fi
ed using
confocal re
fl
ectance). We found that both the CMC and
Tween 80 groups had a thin mucus layer (
Figure 1
D, gold
bars) compared with previous thickness (
t
) measurements we
had done with groups of mice fed a standard chow diet, where
we measured
t
=67
±
7
μ
m (ref
6
;
Figure 1
D, gray bar).
The FC oil approach allows us to measure the mucus
thickness in an environment that approximates the
native
state
of the adherent, colonic mucus hydrogel when it is in
contact with in vivo gut contents (see ref
6
for further details
and validation of this approach). However, we wanted to test
whether the mucus was thin because it was disrupted or
whether it was compressed. We therefore used a di
ff
erent
tissue preparation approach that allowed us to measure the
mucus thickness after washing out the in vivo gut contents
(including polymers and other molecules that could disrupt or
compress mucus). We took two more groups of mice and fed
them with the same solutions, but this time, before imaging, we
washed the tissue with phosphate-bu
ff
ered saline (PBS) to
remove any colonic polymeric contents that could compress
mucus. We then quanti
fi
ed the mucus thickness using the
microparticle approach
(
Figure 1
B,C;
Materials and
Methods
). This and similar approaches have been used
previously to quantify the thickness of the adherent, inner
colonic mucus layer ex vivo.
6
,
7
,
32
Brie
fl
y, in the microparticle
approach, after removing all gut contents, a solution of
microparticles (in PBS) with a diameter (
d
) larger than the
mucus mesh size (
ξ
) is allowed to sediment down on the top
of the mucus hydrogel. These microparticles were coated with
polyethylene glycol (PEG
), as PEG coating has been
previously shown to reduce the mucoadhesivity of particles.
33
Because
d
>
ξ
, the microparticles are excluded from the
hydrogel (which we con
fi
rmed in our previous work
6
), and we
can determine the thickness by measuring the di
ff
erence in the
position of the epithelium (using confocal re
fl
ectance and
bright-
fi
eld) and the position of the microparticles (using
fl
uorescence). Using the microparticle approach, we observed
that the mucus layer was substantially thicker in the CMC
group than in the Tween group (
Figure 1
D, blue bars).
Furthermore, we observed that the mucus in the
washed
CMC-fed group was substantially thicker than the mucus in
the
unwashed
CMC-fed group. The reversibility of the e
ff
ect
suggests that the mucus hydrogel in CMC-fed groups is
compressed in vivo, springing back when
compressive
polymeric contents are washed out with bu
ff
er. The
reversibility also suggests that the alternative hypothesis, that
the polymer itself degrades mucus, is incorrect; degradation
would likely disrupt the integrity of the mucus hydrogel and
not allow it to recover its thickness. Another potential factor is
that the gut microbiota has been shown to degrade colonic
mucus in di
ff
erent contexts.
4
However, because the mucus
thickness in the
washed
CMC-fed group agreed with our
previous measurements of the inner mucus layer in chow-fed
mice (i.e., the
normal
mucus thickness in healthy, SPF mice),
it suggests that the colonic mucus hydrogel is not degraded by
the gut microbiota over the course of our experiments. For the
Tween-fed groups, our data showed that both the washed and
unwashed Tween-fed groups had thinner mucus compared
with the washed CMC-fed group (
Figure 1
D) and our
previous measurements of mucus in chow-fed mice. Because
the
normal
thickness of the inner mucus layer could not be
recovered, it suggests that in the Tween-fed groups, mucus was
irreversibly
thinned
. In total, these experiments suggest that
not only does CMC compress the colonic mucus hydrogel
Biomacromolecules
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DOI:
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2019, 20, 2675
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reversibly in vivo, but the physical mechanism by which it
interacts with mucus is di
ff
erent than that of Tween. This
observation was unexpected because polyelectrolytes and
emulsi
fi
ers have been considered to be similar in previous
gut studies.
14
,
15
,
34
We therefore sought to understand the
mechanism by which CMC compresses the colonic mucus
hydrogel.
Carboxymethyl Cellulose (CMC) Degree of Charge
Increases Extent of Mucus Compression.
We next sought
to understand the mechanism by which CMC compresses the
colonic mucus hydrogel. Here, we aimed to test whether
modulating the amount of charge on CMC could in
fl
uence the
extent of mucus compression. We
fi
rst tested if CMC
compressed mucus ex vivo. We used our microparticle
approach to measure the initial thickness of mucus (
t
0
)ona
colonic explant, then placed the explant in a solution of 1% w/
v CMC DS 0.7 (
Figure 2
A), waited 10 min, and measured the
thickness (
t
f
)(
Figure 2
B). We found that CMC compressed
the mucus hydrogel (
Figure 2
A
B) and that the extent of
compression remained constant over the course of 30 min
(
Figure S2
), suggesting that the system had reached a steady
state. Similarly, in our previous study, we found compression
by an uncharged polymer (polyethylene glycol) was constant
over the course of 60 min.
6
Additionally, for CMC, the
compression was reversible
by washing out the CMC
solution with bu
ff
er, the hydrogel returned to its initial
thickness (
Figure S2
). As with the in vivo experiments, the ex
vivo reversibility suggests that the observed phenomenon is a
form of compression as opposed to degradation of the mucus
hydrogel because in the latter case, the hydrogel would likely
be unable to recover its initial thickness.
To understand how negative polymer charge a
ff
ects the
extent of compression, we next compared how mucus
compression di
ff
ered as a function of polymer concentration
for CMC DS 0.7, CMC DS 0.9 (a derivative of CMC that is
more charged than CMC DS 0.7), and hydroxyethyl cellulose
(HEC, a cellulose derivative with the same chemical backbone
but no charge). Each polymer was added in a range of
concentrations that are approved by the FDA for addition to
food
11
and commonly used in processed foods.
9
Generally, the extent of mucus compression increased with
increased polymer concentration for all three polymers (
Figure
2
C). We found that at most polymer concentrations, the more
highly negatively charged polymer (CMC DS 0.9) induced the
most compression (
Figure 2
C). In contrast, the neutral
polymer (HEC) generally induced the least compression at
any given polymer concentration (
Figure 2
C). These data
suggest that, generally, the negative charge of the polymer
increases the extent of mucus compression.
Mucus Compression due to Charged Polymers Is
Consistent with a Donnan Mechanism.
We knew from
previous studies with the colonic mucus hydrogel (speci
fi
cally,
the strati
fi
ed, cross-linked mucus hydrogel, which is
fi
rmly
adhered to the epithelium) and the periciliary brush that the
polymer-induced compression of biological polymer networks
can be driven by the di
ff
erences in osmotic pressure between
the external polymer solution and the solution phase internal
to the cross-linked polymer network.
6
,
35
In such scenarios, the
osmotic pressure di
ff
erence (
ΔΠ
) drives the
fl
ux of water out
of the polymer network, causing the network to shrink or
compress; the equilibrium gel volume is determined by the
balance between
ΔΠ
on the one hand and the mixing pressure
(due to the change in free energy from mixing the gel with
solvent and free polymer) and the pressure associated with the
elastic deformation of the network chains (i.e., elastic
contributions) on the other hand.
36
An alternative, equivalent
conceptualization is that at equilibrium, the osmotic pressure
of the external solution is equal to the following contributions
from the hydrogel phase: the osmotic pressure of the solution
internal to the hydrogel, the mixing pressure, and elastic
contributions. It has been well established that polyelectrolyte
solutions and gels can also preferentially partition ions between
phases,
19
,
37
,
38
causing an increase in the osmotic pressure of
the polyelectrolyte phase compared with that of the external
solution phase with which it is in contact. This is what is
known as Donnan partitioning or a Donnan mechanism. Given
that both CMC and the adherent, cross-linked colonic mucus
hydrogel itself are both negatively charged, we therefore
hypothesized that the theory of Donnan partitioning could be
used to explain the enhancement of mucus compression we
observed with increased polymer charge. In previous studies
with synthetic hydrogels, polymer-induced compression has
been experimentally quanti
fi
ed by visualizing changes in
hydrogel volume, and an explanation of these results has
been o
ff
ered using well-established theoretical frameworks
such as the Flory
Huggins theory and the Flory
Rehner
theory.
39
45
We therefore sought to use a similar methodology
to understand if polyelectrolytes compress the colonic mucus
hydrogel by a Donnan mechanism.
Before testing our hypothesis with numerical calculations,
we
fi
rst wanted to understand if mucus exhibits Donnan
partitioning in a simple scenario when the colonic mucus
hydrogel is placed in a bu
ff
ered solution without CMC. First,
we write down the condition of electroneutrality for both the
external bu
ff
er solution (ext) and inside mucus (int)
46
,
47
ccc
ext
ext
0
==
+−
(1)
Figure 2.
Negatively charged CMC compresses mucus ex vivo more
than uncharged polymers. (A, B) Cartoons (left) and images (right)
in the side view show the 1
μ
m diameter particles (purple) sitting on
the top of the mucus (A) before and (B) after the addition of 1% w/v
CMC solution. The epithelium is shown in green. (C) Plot of mucus
compression (where % compression = [
Δ
t
/
t
0
]
*
100%, further details
in
Materials and Methods
) as a function of polymer concentration (%
w/v). Each data point represents the average of compression
measured on three independent replicates (three explants from
di
ff
erent mice), where the compression from an individual replicate is
the average of
fi
ve compression measurements at lateral positions on
the explant. Error bars are SEM with
n
= 3. HEC = hydroxyethyl
cellulose, CMC DS 0.7 = CMC with a degree of substitution of 7
negatively charged groups per 10 monomers, and CMC DS 0.9 =
CMC with a degree of substitution of 9 negatively charged groups per
10 monomers. Images shown in side views were processed as
described in ref
6
and
Figure S1
.
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cc
m
int
int
=+
+−
(2)
where
c
+
denotes the molar concentration of mobile cations,
c
is the molar concentration of mobile anions,
c
0
is the
concentration of monovalent salt, and
m
is the molar
concentration of charges on mucus (this analysis assumes
that the polyelectrolyte counterions are the same as the salt
cations). In this case, the cation for CMC is Na
+
, and the
cation in the bu
ff
er is predominantly Na
+
, as explained below.
Invoking the equality of electrochemical potential for the
mobile ions and combining
eqs 1
and
2
give us
c
c
m
c
m
c
22
1
int
00 0
2
=+
+
+
i
k
j
j
j
j
j
y
{
z
z
z
z
z
(3)
Equation 3
gives the fractional increase of positively charged
ions inside the mucus hydrogel due to Donnan partitioning. In
our experiments, we use PBS as the bu
ff
er, which by the molar
concentration is
90% NaCl. Therefore, we approximated the
ionic strength to be equal to the molar concentration of NaCl:
c
0
= 137 mM. We can estimate the molar concentration of
negative charges on mucus by estimating the volume fraction
of mucus (
φ
m
)tobe
φ
m
1% (this is consistent with results
from the literature: refs
6
,
48
50
), which, combined with the
amount of charged groups per mucin,
47
yields
m
5 mM. This
yields
1.02
c
c
int
0
+
. We can therefore assume that any di
ff
erential
salt partitioning by the colonic mucus hydrogel itself at
physiological ionic strengths is negligible.
Our previous numerical results for polymer-induced mucus
compression
6
suggested that an uncharged polymer of a similar
MW, and the radius of gyration (
R
g
) to the polymers used in
this study (PEG 200 kDa with
R
g
22 nm) is mostly excluded
from mucus
the ratio of polymers inside mucus to the
polymers in the external solution was at most
0.3 and
approached 0 as the polymer concentration increased. The
HEC and CMC used in this study are slightly larger than PEG
200 kDa; the measured hydrodynamic radius (
R
h
) of HEC and
CMC from our gel permeation chromatography (GPC)
measurements (see
Table S1 and Figure S3
)is
R
h
20
nm
,
which we can use in conjunction with the Kirkwood
Riseman
relation
51
to estimate
R
g
30 nm. In addition, the charged
polymers should experience electrostatic repulsions with the
mucin strands (which also have some negative charge). We
would therefore expect that HEC and CMC should be even
more excluded from mucus than PEG 200 kDa. If we then take
as a second simplifying assumption that the polymer is
completely excluded from mucus, we can write down
ΔΠ
(in
units of Pa) as
17
ion
pol
Δ
Π=ΔΠ +Π
(4)
where
ΔΠ
ion
is due to Donnan partitioning of the small ions
between the external polyelectrolyte solution and the mucus
network and can be written as (see
Supporting Information
for
derivation)
RT
cp ccp
22()
ion
000
ΔΠ
=+−
+
(5)
where
R
is the gas constant,
T
is the temperature (in kelvin),
and
p
is the molar concentration of charges from the charged
polymer (which we know because the number of charges per
monomer is given by the manufacturer and we determined the
polymer MW by GPC;
Table S1 and Figure S3
). The polymer
osmotic pressure (
Π
pol
) for an uncharged polymer can be
written as
52
RT
cc
c
MW
1
pol
p
p
p
1.3
Π
=+
*
i
k
j
j
j
j
j
j
j
j
j
i
k
j
j
j
j
j
j
y
{
z
z
z
z
z
z
y
{
z
z
z
z
z
z
z
z
z
(6)
where
c
p
is the polymer concentration (in kg/m
3
), MW is the
polymer molecular weight (in Da), and
c
p
*
is the polymer
overlap concentration (in kg/m
3
), which can be estimated as
52
c
NR
MW
p
4
3
avo g
3
*
=
π
(7)
where
N
avo
is the Avogadro number and
R
g
is the polymer
radius of gyration (in m). The polymer MW, based on our
GPC measurements, is
150 kDa (
Table S1 and Figure S3
).
We can use this along with the polymer
R
g
and
eq 7
to
calculate
c
p
*
1.9 kg/m
3
. This justi
fi
es the use of
eq 6
for the
polymer osmotic pressure instead of the osmotic pressure for a
dilute polymer solution (which would simply be the
fi
rst term
of
eq 6
) because the polymer concentrations we test in this
study all exceed the polymer overlap concentration, meriting
the inclusion of the second term in
eq 6
, which accounts for
the behavior above overlap concentration. Using
eq 4
7
,we
estimated
ΔΠ
for both the neutral and charged polymers used
in
Figure 2
C. For the charged polymers, the ionic contribution
to the osmotic pressure (
eq 5
) is substantially greater than the
polymer contribution (
eq 6
) at all polymer concentrations,
suggesting the Donnan mechanism contributes more to
ΔΠ
(see
Figure S4
). We plotted the extent of mucus compression
against
ΔΠ
in
Figure 3
B. We found that the extent of
compression generally increases with
ΔΠ
. Furthermore, the
relationship between mucus compression and
ΔΠ
has a similar
functional form to the classical stress
elongation relation for
uniaxial deformations from the a
ffi
ne network model, which
Figure 3.
Extent of mucus compression plotted against the di
ff
erence
in osmotic pressure (
ΔΠ
) due to the added polymer. (A) Cartoon
depicting the theoretical picture of Donnan partitioning by charged
polymers (labeled
CMC
). Mobile ions are preferentially partitioned
outside of mucus by the charged polymers. (B) Extent of mucus
compression plotted against the theoretical calculation of
ΔΠ
.
Compression values are the same experimental data as
Figure 2
C
.
The dashed line is a
fi
t to the classical stress
elongation relation,
where
()
G
1
2
λ
ΔΠ =
λ
and
1
% mucus compression
100
λ
=−
.
G
(the
compression modulus) was used as a free parameter, and in the
fi
t,
G
= 749 Pa. HEC = hydroxyethyl cellulose, CMC DS 0.7 =
carboxymethyl cellulose with a degree of substitution of 7 charged
monomers per 10 monomers, and CMC DS 0.9 = carboxymethyl
cellulose with a degree of substitution of 9 charged monomers per 10
monomers.
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has been used previously to describe the compression of
hydrogels composed of biopolymers
53
,
54
and synthetic
polymers
55
,
56
and can be written as
52
G
1
eng
2
σλ
λ
=−
i
k
j
j
j
y
{
z
z
z
(8)
where
σ
eng
is the engineering stress or the applied stress on the
network (which, in this case, is
ΔΠ
),
G
is the compression
modulus of the network (in Pa), and
λ
is the deformation
factor, which is related to % mucus compression through:
1
% mucus compression
100
λ
=−
. The negative sign is included
because the stress is compressive. A
fi
tto
eq 8
is plotted as
the dashed line in
Figure 3
B. We take
G
as the one free
parameter in this
fi
t, which yields
750 Pa. We are not aware
of directly measured values for
G
for colonic mucus. However,
this
fi
tted value is of the same order of magnitude as that
estimated using available literature data (see
Supporting
Information
for details). Ultimately, it is both the collapse of
the mucus compression data largely onto a single curve in
Figure 3
B and the functional form of this curve that suggest
that the mucus hydrogel is undergoing a form of uniaxial
deformation induced by
ΔΠ
.
Overall, this analysis, which relies on well-established
theoretical frameworks,
17
,
19
,
46
,
52
suggeststhatitisthe
di
ff
erence in the osmotic pressure between the external
polyelectrolyte solution phase and the solution phase within
the mucus hydrogel, which drives the compression of mucus by
CMC. It further suggests that the di
ff
erence in the osmotic
pressure and the concomitant compression is increased for
polyelectrolytes via a Donnan mechanism.
Increasing Ionic Strength Decreases Mucus Com-
pression by Polyelectrolytes.
Because our data in
Figure 3
B
suggested that the increase in the amount of compression we
see for polyelectrolytes is due to a Donnan mechanism, we
devised a simple set of experiments to test this hypothesis
further. It is known that the amount of Donnan partitioning
decreases with increasing salt concentration (this can be seen
by inspection of
eqs 3
and
5
). We therefore formulated two
hypotheses: (i) Polyelectrolyte-induced compression will be
reduced by high ionic strength because
ΔΠ
ion
is reduced (see
Figure 4
A,B). (ii) For uncharged polymers, the amount of
compression will remain the same when the ionic strength is
increased because there is no contribution from
ΔΠ
ion
at any
ionic strength.
We expect the most signi
fi
cant increase in compression due
to Donnan partitioning to occur in the 1% w/v CMC DS 0.9
solution, which has the highest molar concentration of charges.
By solving
eq 5
,we
fi
nd that for 1% CMC DS 0.9 in a 1
×
PBS
solution (
c
0
0.137 M),
Π
ion
6000 Pa. If we increase the
ionic strength 10-fold to
c
0
1.37 M by using a 10
×
PBS
solution, this decreases to
Π
ion
700 Pa. We would therefore
anticipate that such an increase in the ionic strength would
reduce the compression caused by 1% CMC DS 0.9.
We then tested our hypothesis experimentally by comparing
the measured compression for 1% CMC DS 0.9 in 1
×
PBS to
that of 1% CMC DS 0.9 in 10
×
PBS and found, consistent
with our
fi
rst hypothesis, that there was more compression in
the 1
×
PBS solution (
Figure 4
C). We then tested if the high
ionic strength treatment (10
×
PBS) a
ff
ected the amount of
compression for 1% HEC (an uncharged polymer) and found,
consistent with our second hypothesis, that compression was
the same for 1
×
and 10
×
PBS treatments (
Figure 4
C). As a
control, to ensure that the high ionic strength was not
disrupting the integrity of the mucus hydrogel and eliminating
its compressibility, we tested for compression on colonic
explants with 10
×
PBS at a high concentration of HEC (5% w/
v). We found the mucus compressed to equal amounts in both
1
×
and 10
×
PBS treatments (
Figure 4
C), suggesting that high
ionic strength does not disrupt the integrity of the colonic
mucus hydrogel.
Overall, these data suggest that the increase in mucus
compression observed in response to polyelectrolytes,
compared with uncharged polymers, is due to the preferential
partitioning of mobile ions into the external solution (i.e., a
Donnan mechanism). The concomitant increase in the
osmotic pressure di
ff
erence between the solution and mucus
hydrogel results in this increase in compression.
CONCLUSIONS
There is considerable interest in understanding how diet
impacts the composition and spatial structure of the gut
microbiota and any concomitant e
ff
ects that may impact the
physical structure of the gut (e.g., mucus) and its
physiology.
4
,
14
,
15
However, few studies have focused on
understanding the underlying physics behind how polymeric
additives in food directly interact with gut structure and
Figure 4.
Increasing the ionic strength decreases the extent of
polyelectrolyte-induced mucus compression, consistent with a
Donnan mechanism. (A, B) Schematic depicting the decrease in
polyelectrolyte-induced mucus compression in bu
ff
er solutions with
high ionic strength: (A) When ionic strength is low, there is a greater
di
ff
erence in the concentrations of mobile ions in the external phase
(the polymer solution) and internal phase (the mucus gel).
Subsequently, there is a greater di
ff
erence in the external osmotic
pressure (
Π
ext
) compared to the internal osmotic pressure (
Π
int
). (B)
When ionic strength is high, polyelectrolytes still partition mobile
ions, but there is a smaller di
ff
erence in the concentrations of mobile
ions between the polymer solution and the mucus hydrogel.
Therefore, there is a smaller di
ff
erence in
Π
ext
compared to
Π
int
.
(C) Extent of mucus compression as determined via the microparticle
imaging approach. Each bar represents the mean of compression
measurements from three biological replicates (each replicate is a
colonic explant from a mouse). The compression value from each
individual replicate is the average of compression measurements
acquired at
fi
ve di
ff
erent lateral positions on that explant. Error bars
are SEM with
n
=3.
P
values were computed using Welch
s
t
-test; 1%
CMC = 1% w/v carboxymethyl cellulose with DS 0.9, 1% HEC = 1%
w/v hydroxyethyl cellulose, 5% HEC = 5% w/v hydroxyethyl
cellulose, 10
×
PBS = phosphate-bu
ff
ered saline at 10-fold its normal
concentration, and 1
×
PBS = phosphate-bu
ff
ered saline at its normal
concentration.
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physiology.
6
Food science has traditionally focused more
narrowly on aspects of food design such as the packaging,
preservation, processing, and safety of food.
9
,
57
However,
research is showing that, at least in animal models, even at
approved concentrations, some GRAS food additives are
correlated with markers of disease (such as in
fl
ammation and
obesity
14
,
15
). Thus, it is important to improve our quantitative
understanding of how these food additives interact with the
host and modify gut physiology. In particular, there is a need to
understand how food additives interact with mucus and the
critical barrier in the colon that mediates host
microbe
interactions and protects the host against physical damage.
1
Changes to the thickness and mesh size of the colonic mucus
barrier have been associated with dramatic changes in host
health.
7
,
8
,
14
In this work, we found that a polyelectrolyte, CMC,
compresses mucus reversibly in vivo, in contrast to an
emulsi
fi
er (Tween), which appeared to irreversibly disrupt
mucus. We found that the amount of mucus compression
induced by CMC increased as a function of the degree of
polymer charge, which is a characteristic that has not been
considered in the design of food products. Furthermore, we
found that the increase in the amount of compression due to
polymer charge is consistent with a Donnan mechanism. A
simple, one-parameter mo
del that combines the well-
established theoretical frameworks of Flory
Huggins solution
theory and Donnan partitioning was found to be su
ffi
cient to
quantitatively capture the observed behavior. We have o
ff
ered
a potential explanation for the phenomenon observed in this
work using the theoretical framework of Donnan partitioning;
however, more comprehensive theoretical models need to be
developed and tested to completely understand this mecha-
nism and explicitly account for the possible penetration of
polyelectrolytes into the mucus hydrogel.
Our work so far has not considered how
fl
uid
fl
ow, possible
rheological e
ff
ects such as viscous relaxation of mucus on
longer timescales,
49
,
58
and possible anisotropy in the structure
of the colonic mucus hydrogel
2
a
ff
ect polymer-induced
compression. Additionally, another factor in vivo is the
regulation of isotonicity between the gut lumen and epithelium
by the active transport of water and salts.
59
It is unclear how
much this last factor would impact the observed phenomenon
for two reasons: (i) small changes in the
fl
ux of water and salts
will a
ff
ect the base osmotic pressure (i.e., the osmotic pressure
both inside the hydrogel and in the lumen) but not the
di
ff
erence in osmotic pressure inside and outside the hydrogel
and (ii) our experiments and calculations suggest that the
described Donnan e
ff
ects disappear at tenfold physiological
ionic strength, which is unlikely to occur in vivo. Finally, in our
previous work, we found that the extent of mucus compression
generally increased with the molecular weight of the polymer,
6
but we have yet to consider if this relationship is further
complicated by the polymer charge. All of these e
ff
ects will be
important areas to investigate in future work to understand
how di
ff
erent polymers compress mucus in vivo
.
The system we have considered in this work is an example of
a class of systems consisting of a polyelectrolyte solution
directly interacting with a biological, polyelectrolyte network.
Such systems can be found throughout nature; other examples
include bio
fi
lms in contact with extracellular DNA,
60
medical
hydrogels in contact with gut polymers,
61
ECM in contact with
interstitial
fl
uid,
62
and hyaluronic acid
lubricin networks
(which lubricate our joints) in contact with synovial
fl
uid.
63
Our work begins to unravel this physics in the context of
polyelectrolyte-induced mucus compression, which could lead
to new, safer design of food products that do not alter the
structure of colonic mucus.
ASSOCIATED CONTENT
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website
at DOI:
10.1021/acs.bio-
mac.9b00442
.
Derivation of the ionic contribution to osmotic pressure
due to Donnan partitioning, estimation of the
compression modulus for the colonic mucus hydrogel,
fi
gure depicting image processing, ex vivo compression
reversibility data, gel permeation chromatography
measurements of charged and uncharged polymers,
and comparison of polymer and ionic contributions to
the osmotic pressure (
PDF
)
Code to perform curve
fi
tting in Figure 3B (
ZIP
)
AUTHOR INFORMATION
Corresponding Author
*
E-mail:
rustem.admin@caltech.edu
. Tel: +1 626 395 2333.
Fax: +1 626 568 8743.
ORCID
Asher Preska Steinberg:
0000-0002-8694-7224
Zhen-Gang Wang:
0000-0002-3361-6114
Rustem F. Ismagilov:
0000-0002-3680-4399
Author Contributions
A.P.S. and R.F.I. designed the research; A.P.S. performed the
research; A.P.S. analyzed the data. Z.-G.W. guided the
theoretical analysis. All authors wrote the paper.
Funding
Army Research O
ffi
ce (ARO) no. W911NF-17-1-0402; Jacobs
Institute for Molecular Engineering for Medicine; Center for
Environmental Microbial Interactions (CEMI); NSF Graduate
Research Fellowship DGE-144469; Caldwell CEMI Graduate
Fellowship.
Notes
Theauthorsdeclarethefollowingcompeting
fi
nancial
interest(s): The technology described in this publication is
the subject of a patent application
fi
led by Caltech.
ACKNOWLEDGMENTS
This work was supported in part by Army Research O
ffi
ce
(ARO) Multidisciplinary University Research Initiative
(MURI) contract no. W911NF-17-1-0402, the Jacobs Institute
for Molecular Engineering for Medicine, the Center for
Environmental Microbial Interactions (CEMI), an NSF
Graduate Research Fellowship DGE-144469 (to A.P.S.), and
a Caldwell CEMI Graduate Fellowship (to A.P.S.). We
acknowledge Michael Porter for useful discussion and
providing feedback on the manuscript; Andres Collazo and
Caltech
s Beckman Institute Biological Imaging Facility, the
Caltech O
ffi
ce of Laboratory Animal Resources, and the
Caltech veterinary technicians for technical support; Justin
Rolando for providing microparticles; and Natasha Shelby for
contributions to writing and editing this manuscript.
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