Photoresponsive Elastic Properties of Azobenzene-Containing Poly(ethylene-glycol)-Based Hydrogels

Article abstract of DOI:10.1021/bm501710e

The elastic modulus of the extracellular matrix is a dynamic property that changes during various biological processes, such as disease progression or wound healing. Most cell culture platforms, however, have traditionally exhibited static properties, mak

Full text of DOI:10.1021/bm501710e

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Article
Photoresponsive Elastic Properties of Azobenzene-
Containing Poly(ethylene-glycol)-based Hydrogels
Adrianne M Rosales, Kelly M Mabry, Eric Michael Nehls, and Kristi S. Anseth
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501710e • Publication Date (Web): 28 Jan 2015
Downloaded from http://pubs.acs.org on February 7, 2015
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Photoresponsive elastic properties of azobenzene-containing poly(ethylene-
glycol)-based hydrogels
1,2
2
2
1,2,*
Adrianne M. Rosales, Kelly M. Mabry, Eric Michael Nehls, and Kristi S. Anseth
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Howard Hughes Medical Institute and the BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA
Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80309, USA
To whom correspondence should be addressed: kristi.anseth@colorado.edu
2
*
Abstract
The elastic modulus of the extracellular matrix is a dynamic property that changes
during various biological processes, such as disease progression or wound healing. Most
cell culture platforms, however, have traditionally exhibited static properties, making it
necessary to replate cells to study the effects of different elastic moduli on cell phenotype.
Recently, much progress has been made in the development of substrates with mechanisms
for either increasing or decreasing stiffness in situ, but there are fewer examples of
substrates that can both stiffen and soften, which may be important for simulating the
effects of repeated ECM injury and resolution. In the work presented here, poly(ethylene
glycol)-based hydrogels reversibly stiffen and soften with multiple light stimuli via
photoisomerization of an azobenzene-containing crosslinker. Upon irradiation with
2
cytocompatible doses of 365 nm light (10 mW/cm , 5 min), isomerization to the
azobenzene cis configuration leads to a softening of the hydrogel up to 100-200 Pa (shear
storage modulus, G’). This change in gel properties is maintained over a timescale of
several hours due to the long half-life of the cis isomer. The initial modulus of the gel can be
recovered upon irradiation with similar doses of visible light. With applications in
mechanobiology in mind, cytocompatibility with a mechanoresponsive primary cell type is
demonstrated. Porcine aortic valvular interstitial cells were encapsulated in the developed
hydrogels and shown to exhibit high levels of survival, as well as a spread morphology. The
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developed hydrogels enable a route to the noninvasive control of substrate modulus
independent of changes in the chemical composition or network connectivity, allowing for
investigations of the effect of dynamic matrix stiffness on adhered cell behavior.
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Keywords: biomaterials, azobenzene, matrix mechanics, dynamic hydrogel
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Introduction
In conjunction with biological ligands and proteins, the mechanical properties of the
native extracellular matrix (ECM) environment present powerful cues that direct cell
phenotype. To decouple the effects of matrix stiffness from other factors, synthetic
hydrogels have emerged as a facile route to presenting cells with a substrate of well-
defined elasticity.1, 2 However, the native ECM modulus is a dynamic property that
fluctuates as cells remodel their environment during disease progression, wound healing,
or development. While many hydrogel platforms can recapitulate the elasticity of a tissue
of interest, fewer provide handles to change this elasticity on demand and in the presence
of attached cells.
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Recently, several strategies have emerged to tune the elastic modulus of a substrate
in situ. For example, DNA-crosslinked hydrogels have been shown to modulate reversible
changes in stiffness over tens of kilopascals by hybridization with introduced DNA strands
that are complementary to the crosslinker.3, 4 Another strategy induces the formation of
reversible crosslinks through the addition of divalent cations to a collagen-alginate
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composite hydrogel. Both of these strategies show good reversibility; however, they also
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both rely on the addition of a component (DNA or cations such as Ca ) that can be uptaken
by cells and may alter cellular signaling or behavior. Changes in protein conformation can
also be used to confer dynamic properties to a hydrogel.6, 7 For example, Kong et al. took an
innovative approach to reversibly controlling modulus in protein hydrogels by using redox
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chemistry to fold and unfold the protein, thereby altering the crosslinker length. Effects of
the changes in oxidation state on cells have yet to be explored. As a complement to these
systems, photoreactions are an attractive route to controlling ECM elasticity in a non-
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invasive way. Kloxin et al. developed poly(ethylene glycol) (PEG) networks with a
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photodegradable moiety incorporated next to the crosslinking site. Using cytocompatible
doses of 365 nm light, the user can thus decrease the modulus of a hydrogel over several
kilopascals by decreasing the crosslinking density. Light-based chemistries can also be
used to stiffen a hydrogel, as demonstrated by Guvendiren et al. in a hyaluronic acid-based
system that uses sequential addition and radical polymerization crosslinking reactions.9
While both of these strategies offer spatial and temporal control, the changes they confer to
the network structure and matrix mechanics are irreversible.
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One of the most enticing platforms for a reversible, photo-based reaction to control
matrix mechanics is the use of a photoisomer, especially azobenzene. Azobenzenes have
been used to control properties of polymeric networks, such as the gel-sol transition10-12
and the swelling ratio.13 They have emerged as an effective photoswitch for use in
biomaterials because they absorb light in a region that is compatible with many biological
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systems (350-550 nm). In small molecule systems, azobenzene converts from the trans
isomer to the cis isomer upon irradiation with 360 nm light in a remarkably efficient
reaction (~80%-95%). Removal of the light or irradiation in the visible range regenerates
the trans isomer almost completely (>99%) because it is the more thermodynamically
stable conformation. These properties can be recapitulated in azobenzene-containing
hydrogel networks to control matrix properties.
One of the main advantages of controlling hydrogel mechanics with azobenzene is
the lack of initiator required to complete photoisomerization. Because this photoreaction
proceeds with light alone, no free radicals are created during irradiation. Furthermore,
light is a non-invasive stimulus that allows for changes without altering the chemical
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environment of the cells. Azobenzene undergoes a structural change, and it is therefore
anticipated that gel mechanics will be controlled without affecting the overall network
connectivity. However, because the reverse isomerization of azobenzene occurs thermally,
there is likely to be some reversal of the modulus over time. For this reason, it is critical to
choose an azobenzene with stable cis isomer and a long half life.
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In the work presented here, azobenzene is incorporated into peptide crosslinkers of
PEG hydrogels to modulate the matrix elasticity in a reversible manner with both 365 nm
(UV) and 400-500 nm (visible) light. The photoisomerization disrupts hydrogen bonds
from the surrounding network, leading to a decrease in matrix elasticity that is fully
recovered upon thermal relaxation or the reverse isomerization of the azobenzene. The
incorporated photoswitch was carefully chosen to maximize the lifetime of the cis state at
37°C, and cytocompatibility was demonstrated with encapsulated primary porcine aortic
valvular interstitial cells (VICs). This work establishes an accessible route to the reversible
control of matrix elasticity in the presence of primary cells cultured in three dimensions.
Future efforts will focus on maximizing the elastic response to direct cell phenotype.
Materials and Methods
Synthesis
of
3-[[4-([[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl)phen-
yl]diazenyl]benzoic Acid (3,4’-Fmoc-AMPB) 3,4’-Fmoc-AMPB was synthesized in two
steps according to previously published methods.15, 16 Briefly, 3-nitrosobenzoic acid was
first synthesized by dissolving 3-aminobenzoic acid (2 g, 14.6 mmol) in DCM (40 mL).
Oxone (18 g, 29.3 mmol, 2 equivalents) dissolved in 160 mL water was added to the
reaction flask to create a biphasic solution. The solution was stirred under argon at room
temperature for 3 hours. The precipitate was separated by first evaporating the DCM,
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followed by centrifuging. The precipitate was washed two times with water and two times
with methanol, then dried in a vacuum oven overnight at 40 °C. 3-nitrosobenzoic acid
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(
Figure S1): H NMR (DMSO-d
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, 500 MHz): δ= 7.88 (m, 1H), 8.19 (d, 1H), 8.39 (m, 2H),
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13.52 (s, br., 1H).
In the second step, the 3-nitrosobenzoic acid (2.0 g, 13.4 mmol) was sonicated in a
:1 mixture of acetic acid and DMSO (50 mL:50 mL). To this green solution, 4-(N-Fmoc-
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aminomethyl)aniline (2.3 g, 6.69 mmol, 0.5 equivalents) was added, and the reaction
solution was stirred under argon for three days at room temperature. The precipitated
orange product was centrifuged to separate it from the reaction solution and then
redissolved in ethyl acetate (~ 4L) and washed with water. The organic layers were dried
over MgSO
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and the solvent was evaporated. The product was then used without further
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purification. 3,4’-Fmoc-AMPB (Figure S2): H NMR (DMSO-d
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, 500 MHz): δ= 4.26 (t, 1H),
4.29 (d, 2H), 4.40 (d, 2H), 7.35 (t, 2H), 7.43 (m, 4H), 7.73 (m, 3H), 7.90 (d, 2H), 7.91 (d, 2H),
.99 (t, 1H), 8.14 (m, 2H), 8.39 (s, 1H).
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Synthesis of azobenzene-containing peptide
An azobenzene-containing peptide
crosslinker (KCGGPQG-3,4’-AMPB-IWGQGCK) was synthesized on a solid phase peptide
synthesizer (Tribute Protein Synthesizer, Protein Technologies, Inc.) using Rink Amide
MBHA resin (Novabiochem, 0.59 mmol/g). Briefly, Fmoc deprotection proceeded via 20%
piperidine/2% 1,8-diazabicyclo[5.4.0]undec-7-ene in NMP for 20 minutes. Fmoc-protected
amino acids were coupled in 4-fold molar excess in the presence of 0.4M N-
methylmorpholine in DMF for 30 minutes, except for the 3,4’-Fmoc-AMPB, which was used
in a 2-fold molar excess. The N-terminus of the peptide was acetylated using 5% acetic
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anhydride and 6% lutidine in NMP, and the peptide was cleaved from the resin using
trifluoroacetic acid (TFA), phenol, triisopropylsilane, and water (95/2.5/1.25/1.25 v/v) for
at least 2 hours. Crude peptides were precipitated into chilled diethyl ether, then purified
by semi-preparative reverse phase HPLC (AutoPurification HPLC, Waters) using a 10
minute gradient of acetonitrile in water (5-50%, 30 mL/min). The peptide eluted at 36.5%
acetonitrile, and the expected mass was confirmed by matrix assisted laser desorption
ionization time-of-flight mass spectrometry (Figure S3: MALDI-TOF, Applied Biosystems
DE Voyager, exp/obs: 1698.9/1697.8). A matrix metalloproteinase (MMP)-degradable
sequence (KCG-3,4’-AMPB-GPQGIWGQGCK) was made in the same fashion.
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Hydrogel formation
Photoresponsive hydrogels were formed using a Michael-type
addition of dithiol peptides (described above) onto multi-arm vinyl sulfone-functionalized
poly(ethylene glycol)s (PEG-VS, JenKem Technology USA). PEG-VS was dissolved in either
0.1 M HEPES buffer (pH 7.8) or 0.4 M triethanolamine in PBS (pH 8) at the desired w/v
concentration. The peptide crosslinker was dissolved in 3:1 DMSO:H O at a concentration
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of 80 mM. Prior to making the peptide stock solutions, the free thiol content of the
photoresponsive peptide was measured using an Ellman’s assay. Only peptides with a free
thiol content of 70% or greater were used, and adjustment of the functional group
concentration with this assay enabled consistent gel formation between peptide batches
(Table 1). However, for best results, gels prepared with peptide from the same batch were
compared, as in the data presented in Figure 5. The PEG-VS and peptide components were
mixed at the desired stoichiometric ratio, quickly vortexed, and reacted in a 0.5 mm thick
rubber mold between two SigmaCoted glass slides. The gels were allowed to react at 37°C
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for 2 hours at pH 8 or 30 minutes at room temperature in the HEPES buffer, as noted. After
gelation, the gels were gently removed from the molds and placed into PBS to swell
overnight at room temperature.
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UV/Vis spectroscopy
Absorbance spectra were collected with a Synergy H1
microplate reader (BioTek) using a wavelength range of 280 nm to 700 nm. For swollen
hydrogel measurements, 20 μl hydrogels were directly formed in a clear 96-well plate, and
they were allowed to swell overnight in 200 μl of PBS before taking absorbance readings.
Two high pressure mercury light sources were used for irradiation experiments: Exfo
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Omnicure S1000, 365 nm, 10 mW/cm and Exfo Acticure 4000, 400-500 nm, 10 mW/cm .
Rheology
For rheological measurements of swollen hydrogels, the gel was loaded onto
a rheometer (TA Instruments, Discovery HR-3) with an 8 mm parallel plate geometry and a
UV light guide accessory. A 5 mm liquid light guide was manually switched between two
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high pressure mercury light sources (Exfo Omnicure S1000, 365 nm, 10 mW/cm and Exfo
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Acticure 4000, 400-500 nm, 10 mW/cm ) at the desired time points while measuring the
modulus of the gel at 25°C using an oscillatory strain of 1% and a frequency of 1 rad/s
(linear viscoelastic regime). The gel was surrounded with water to maintain equilibrium
swelling and avoid drying at the gel-air interface. The starting axial force for all
measurements was between 0.2 and 0.4 N.
For in situ gel evolution measurements, 20 μl of the gel precursor solution was
quickly vortexed then added to the plate. Modulus measurements were taken immediately
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after the precursor solution was added to the rheometer, and the formulation was
surrounded with dimethylpolysiloxane (Sigma Aldrich) to prevent evaporation of water.
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VIC isolation and culture Using a collagenase digestion, primary VICs were isolated from
porcine aortic leaflets, which were obtained from fresh hearts acquired from Hormel
within 24 hours of slaughter.17, 18 Aliquots were frozen in a liquid nitrogen cooled tank until
needed, then thawed and expanded on tissue culture polystyrene (TCPS) plates. VICs were
thawed in Media M199 supplemented with 15% fetal bovine serum (FBS), 100 U/mL
penicillin, 100 ug/mL streptomycin, and 1 ug/mL fungizone at 37°C and 5% CO . VICs were
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expanded for two days, then passaged once and expanded for an additional day before
encapsulation into the hydrogels.
VIC encapsulation VICs were trypsinized from TCPS plates, pelleted from 15% FBS
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media, and resuspended in PBS at a concentration of 25 x 10 cells/mL. The appropriate
amount of the cell suspension was added to the gel precursor solution to achieve an
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encapsulation density of 10 x 10 cells/mL. The gel precursor solution contained PEG-VS at
a concentration of 10 wt. % in sterile-filtered 0.1 M HEPES buffer at pH 7.8, the peptide
crosslinker (80 mM) in 3:1 DMSO:H O, and the appropriate amount of sterile PBS to
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achieve the desired final concentration of PEG-VS and peptide. The cell-containing
precursor solution was quickly vortexed, and 40 µL were pipetted into a rubber mold (0.5
mm thick, 8 mm diameter) between two SigmaCoted slides. The gels were allowed to form
at room temperature for 30 minutes in a sterile tissue culture hood; then they were gently
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removed from the molds and placed into an ultra-low adhesion well plate with 2 mL of 1%
FBS M199 media. Media was changed the next day.
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Cell viability analysis VIC viability was quantified using a Live/Dead membrane integrity
assay (Live/Dead, Life Technologies). Samples were incubated in PBS containing 1 μM
calcein AM and 4 μM ethidium homodimer for 30 minutes. Cells were imaged on a 710 LSM
NLO confocal microscope (Zeiss), and images were stacked using maximum intensity
projections. Cells in which the membrane-permeable calcein AM was cleaved by esterases
within the cell were counted as live, while cells permeable to ethidium homodimer were
counted as dead. Over 100 cells were counted per sample. For each condition, 3 or more
biological replicates were performed. Error bars represent standard error of the mean.
Immunostaining for α-smooth muscle actin (αSMA) and f-actin
Samples were fixed
in 10% formalin and then washed with PBS. Permeabilization with 0.1% TritonX in PBS
was followed by blocking of non-specific staining with 1% BSA in PBS. Mouse anti-αSMA
diluted 1:200 in PBS was applied to the samples. Gels were then rinsed with PBS prior to
application of AlexaFluor 488 goat-anti-mouse (1:300) and TRITC-phalloidin (1:300) to
stain for f-actin. Next, nuclei were stained with 4',6-diamidino-2-phenylindole (0.01
mg/mL). Cells were imaged on a 710 LSM NLO confocal microscope (Zeiss).
Results and Discussion
Here, a three-dimensional hydrogel platform with photoresponsive mechanical
properties was developed. Because the elasticity of biological tissues is a dynamic property,
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a cell culture substrate with reversible properties could lend insight on the effects of
changing stiffness on cell phenotype without confounding factors such as replating cells.
Furthermore, a three-dimensional geometry better recapitulates the natural extracellular
matrix environment compared to a two-dimensional substrate.
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Gel Formation
To obtain a hydrogel with photoresponsive mechanical properties, a peptide
crosslinker containing a photoisomer (3,4’-AMPB) was first synthesized. Although the
photoisomer could also be incorporated at the chain ends of a PEG crosslinker (Figure S4),
the use of a peptide crosslinker enabled facile incorporation with solid-phase synthesis
methods, as well as site-specific incorporation into the crosslinker. Here, the peptide
sequence was based on that of native collagen, and the location of the photoisomer either
conferred degradability or non-degradability to the peptide (KCG-3,4’-AMPB-
GPQGIWGQGCK
or KCGGPQG-3,’4-AMPB-IWGQGCK, respectively). The degradable
photoresponsive peptide was only used in the cell encapsulation studies detailed below.
To form the hydrogel, a Michael addition reaction was used under slightly basic
conditions with a PEG-VS macromer and the photoresponsive peptide (Figure 1).
Previously, Michael addition-type reactions have been shown to be an effective route to
encapsulating cells within a hydrogel.19, 20 In addition, the use of Michael addition
chemistry avoids the use of a photoinitiator. At 365 nm, the molar absorptivity of the
-1
-1
photoresponsive peptide is approximately 2300 M cm , which is at least an order of
-1
-1 21
-1
-
magnitude higher than that of other initiators such LAP (218 M cm ) or I2959 (4 M cm
1)21
. The photoresponsive peptide also demonstrates significant absorption in the visible
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Figure 1. Photoresponsive hydrogels are formed using Michael addition reactions
between multi-arm PEG vinylsulfones (PEG-VS) and thiol-containing peptides in 0.1 M
HEPES buffer at pH 7.8. The schematic above represents an off-stoichiometry
formulation. The crossover of the storage (G’) and the loss (G”) modulus occurs within
minutes, and the gel evolution plateaus within 15 minutes.
-1
-1
light range (e.g., molar absorptivity at 420 nm ~ 270 M cm ); thus, attempts to
photoinitiate gel formation resulted in poor or no gelation with a variety of PEG macromers
and photoinitiators. In contrast, the Michael addition reactions allowed for gelation within
minutes. Figure 1 illustrates one example of the rapid gelation that occurs for the PEG-
VS/peptide gels in 0.1M HEPES buffer at pH 7.8. Crossover of the storage modulus, G’, with
the loss modulus, G”, occurs within 2 minutes, and 95% of the final modulus is achieved
within 15 minutes. Furthermore, by varying the initial macromer concentration or
crosslinking density, a range of initial storage moduli, G’, were achieved that spanned 500
Pa – 8000 Pa.
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Because the Michael addition reaction occurs at basic pH, the reaction competes
with disulfide formation between the peptide crosslinkers. For this reason, the measured
moduli of the resulting gels were lower than the theoretical moduli. Although the addition
of a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) would not interfere with
the Michael addition and thus facilitate
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complete gelation, its presence could
potentially be detrimental to cellular proteins
during encapsulation. For this reason, the gel
properties were examined without addition of
a reducing agent.
Gel Mechanical Properties
To
understand
the
viscoelastic
properties of the hydrogel, the swollen shear
modulus was first measured over frequencies
ranging from 0.005 rad/s to 100 rad/s at a
constant strain of 1% (Figure 2). Over the
entire frequency range, G’ (Figure 2a) was
approximately two orders of magnitude larger
than the loss modulus, G” (Figure 2b),
indicating that the elastic properties of the gel
dominate over the viscous losses. Furthermore,
Figure 2. Typical frequency sweeps of
photoresponsive hydrogels with and
without UV irradiation (365 nm, 10
2
mW/cm ) showing both the storage (a)
and the loss (b) modulus. The gel shown
here is formulated with 8-arm, 20 kDa
PEG macromer at 5 wt% and 1.0 mM
crosslinking density.
the
hydrogels
showed
a
frequency-
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independent modulus characteristic of an elastic Hookean solid between frequencies of
approximately 0.1 – 10 rad/s. Therefore, a frequency of 1 rad/s was chosen for all
subsequent studies. Next, to understand the response of the hydrogel’s mechanical
properties to light, the modulus was examined with the UV light either on (open symbols)
or off (closed symbols). When the UV light was on, G’ was smaller in the linear viscoelastic
range (by approximately 50 Pa for the example shown in Figure 2a), indicating a change in
gel structure. Interestingly, at very low frequencies outside of the linear viscoelastic range,
there was significant overlap between the irradiated and non-irradiated gels. No significant
change was observed in the loss modulus.
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The change in G’ reflects changes in the gel structure conferred by the isomerization
of azobenzene between the planar, trans configuration (UV off) and the bent, cis
conformation (UV on). Confirmation of the isomeric state of the azobenzene-containing
crosslinker was obtained by measuring the absorbance spectrum of the swollen hydrogel
(Figure 3a). Azobenzene exhibits two characteristic bands that shift depending on the
substituents of the phenyl rings: one in the UV region (~320-360 nm) that corresponds to
*
the π-π transition and one in the visible region (~420-450 nm) that corresponds to the n-
*
*
π transition. Due to symmetry constraints, the n-π transition is not allowed in the planar
trans configuration, therefore making the visible range absorbance very weak. However,
*
upon isomerization, the n-π transition is allowed, and the absorbance in the visible range
*
increases because the molar extinction coefficient for the cis n-π transition is larger than
*
22
*
that of the trans n-π transition. Simultaneously, the cis π-π transition in the UV range is
weaker than that of the trans configuration. These characteristics are observed in the
swollen hydrogels formed here. Before exposure to UV light, the gel displays an absorbance
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Figure 3. a) Representative UV/Vis of an equilibrium swollen 5 wt% 8-arm PEG-VS gel
with 5.0 mM azobenzene-containing peptide crosslinker. UV/Vis shows isomerization of
the azobenzene-containing crosslinker to the cis form after irradiation with 365 nm light
2
(
(
10 mW/cm , 2 min) and back to the trans form after irradiation with 400-500 nm light
2
10 mW/cm , 2 min). Rheology shows a cyclic modulus change with alternating
2
2
irradiation at 365 nm (10 mW/cm , 5 min) and 400-500 nm (10 mW/cm , 2 min) for an
equilibrium-swollen 5 wt% 8-arm PEG-VS hydrogel crosslinked at (b) 0.5 mM, (c) 1.0
mM, and (d) 5.0 mM. The magnitude of the change in modulus increases with increasing
azobenzene content. All rheological data here is presented on the same scale for ease of
comparison.
*
maximum at 330 nm, which is indicative of the π-π transition and the azobenzene unit in
2
the trans state. Immediately after irradiation at 365 nm (10 mW/cm , 2 min), the peak at
30 nm largely decreases and a new peak emerges at 425 nm, corresponding to the cis n-π*
3
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transition and the emergence of a photostationary state that is predominantly composed of
the cis isomer. The trans state can be recovered with minimal hysteresis by irradiation with
2
a visible light source (400-500 nm, 10 mW/cm , 2 min). These light doses are comparable
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to those used previously in cytocompatible systems,8, 23, 24 thus allowing for non-invasive
manipulation of molecular properties in the presence of cells. At this molecular level, the
isomerization is very fast; to probe changes on a bulk level, we turned to rheology.
The dynamic response of the swollen gel storage modulus G’ to both UV and visible
light was measured in situ as shown in Figure 3, for three different gels (Figure 3a
corresponds to 0.5 mM crosslinking density, Figure 3b corresponds to 1.0 mM crosslinking
density, and Figure 3c corresponds to 5.0 mM crosslinking density). Upon irradiation with
2
UV light (light gray shading, 365 nm, 10 mW/cm , 5 min), the gel modulus decreased in an
exponential fashion. After removal of the light, G’ stayed constant for 4 minutes, after which
2
a visible light cue was introduced (dark gray shading, 400-500 nm, 10 mW/cm , 2 min) and
G’ quickly increased back to the initial modulus. Again, after removal of the light, G’ stayed
constant until the next light cue was introduced. Depending on the hydrogel, sometimes a
steady decrease in the modulus was observed (as in Figure 3c). This artifact is due to a
decreasing axial force during the measurement, which may arise from small changes in the
swelling of the gel during isomerization. Subsequent measurements on the same gel
showed similar starting moduli, confirming that no degradation had occurred. Although
only two cycles are shown in Figure 3, the gel retained its dynamic switching
characteristics over numerous cycles. Furthermore, because of its small absolute value, no
significant changes were observed in the loss modulus, G”.
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One interesting characteristic of these gels is the decrease in modulus upon
isomerization of the crosslinker to the cis state. Previously, Petr et al. observed a decrease
in modulus as the cis state disrupted liquid crystalline order in a polymeric network.25, 26 In
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addition, several studies have observed gel-sol transitions upon the disruption of hydrogen
bonds between neighboring azobenzene units in supramolecular systems.27, 28 Although the
hydrogen bond structure in this system is still under investigation, the planar trans
conformation may better be able to stabilize hydrogen bonds between the peptide
crosslinkers, thereby rigidifying the substrate. Such a structure would then correspond to
the “stiff” trans state and “soft” cis state observed here. Heating the hydrogels confirms the
presence of hydrogen bonding, as the modulus sharply decreases at higher temperatures
(Figure S5). This result is in direct contrast to that predicted by rubber elasticity theory,
which states that the modulus of a swollen network should scale with temperature:
G = ρxlRTQ1/3
(1)
where ρxl is the crosslinking density and Q is the swelling ratio. The magnitude of the
switch in modulus was dictated by the crosslinking density, or the content of azobenzenes
within the gel (gels without azobenzene showed no switch in modulus, Figure S6). As
shown in Figure 3 b-d, the magnitude of the change in modulus increased from 12 Pa for a
crosslinking density of 0.5 mM to 121 Pa for a crosslinking density of 5 mM. Table 1 lists
the change in modulus for other gel configurations and concentrations. Both the 8-arm, 20
kDa PEG gels and the 4-arm, 10 kDa PEG gels have the same crosslinker length, but the 8-
arm, 20 kDa gels have a higher theoretical crosslinking density for the same gel
concentration, which corresponds to a greater number of azobenzenes per gel. In addition,
the 8-arm configuration increases the local concentration of the azobenzene units, which
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may allow for more hydrophobic interactions. As a result, the equilibrium swelling for the
8-arm gels is less than that predicted. This difference in swelling may also be related to the
observed magnitude change per mmol of azobenzene, which is greater in the 8-arm gels
than the 4-arm gels. Interestingly, attempts to further increase the azobenzene content by
increasing the concentration of the gel (higher weight %) or decreasing the size of the
crosslinker (lower molecular weight) resulted in no switching (Figure S7). Past a critical
concentration of azobenzene, the gel structure is likely altered or further stabilized by
interactions between neighboring azobenzene units.
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Although the change in the hydrogel modulus upon exposure to UV light shows good
reversibility, the magnitude is quite modest (in the 10-100 Pa range of shear modulus).
Many 2D studies of the effects of matrix elasticity on cellular phenotype use a modulus
range that spans 10-100 kPa; however, numerous cell types have demonstrated
mechanosensitivity over a much smaller modulus range, especially in 3D geometries.29-34
Table 1. Properties of swollen photoresponsive hydrogels.
b
ΔG’ (Pa)c
PEG –VS
[PEG] (mM)
ρ
xl (mM)a
Q
exp
G’ (Pa)
Macromer
4
8
-arm, 10 kDa
5.2
0.2
2.0
6.0
0.5
1.0
2.5
5.0
19.8
190
7
11.1
15.5 ± 1.5
15.5 ± 1.5
11.8 ± 2.3
11.8 ± 2.3
11.8 ± 2.3
11.8 ± 2.3
1987 ± 530
6170 ± 410
500
1170 ± 155
2829 ± 422
5700 ± 200
25 ± 5
51 ± 1
12
48 ± 15
44 ± 6
121 ± 20
11.1
-arm, 20 kDa
2.6
2.6
2
2
.6
.6
a
Calculated crosslinking density, which corresponds to the concentration of azobenzenes in
the gel
b
Swelling ratio, calculated by Qexp = 1 + ρpolymer/ρH2O*(Mswollen/Mdry-1), where M is the mass of
the gel
c
2
Change in G’ upon irradiation with 365 nm light, 10 mW/cm , 5 min
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For instance, dermal fibroblasts exhibited
upregulated mediators of inflammation when
encapsulated in stiff hydrogels (1200 Pa)
compared to soft hydrogels (50 Pa).29 In
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another
example,
MMP
activity
of
encapsulated hMSCs increased with stiffness
when comparing soft (200 Pa), medium (500
Pa), and stiff (1200 Pa) hydrogels.34 This
range of moduli for 3D cell behaviors is
similar to that shown by the photoresponsive
gels developed here, and current efforts are
focused on tuning the parameters of this
system to study activation of fibroblasts in
3D.
Gel Relaxation Behavior
While Figure 3 demonstrates the
photoresponsive behavior of the hydrogels
on short time scales (minutes), the time scale
of interest for many important cell functions
is hours or days. After irradiation with UV
light, the azobenzene cis isomer should
slowly relax back to the more energetically
Figure 4. a) Thermal relaxation at 25 °C for
equilibrium swollen 5 wt% 8-arm PEG-VS
hydrogel containing the photoresponsive
peptide after irradiation with 365 nm light
2
(
10 mW/cm , 5 min); b) thermal
relaxation at 37 °C; c) kinetic plots show
that the relaxation behavior follows first-
order kinetics and yields a half life of
162.3 hours at 25°C and 9.2 hours at 37°C.
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stable trans state. Thus, the thermal relaxation behavior of the photoresponsive gel was
studied by monitoring the change in the absorbance spectra over time. After irradiation
2
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measured over several hours, and the gel was kept in the dark at the desired temperature
in between measurements. As shown in Figure 4a for a hydrogel at 25°C, the peak at 330
nm slowly increased over approximately a week, indicating an increasing population of
trans isomers. At 37°C (Figure 4b), this relaxation transition occurred much more quickly,
which is in agreement with Arrehenius-type behavior.
The thermal relaxation behavior follows first order kinetics (Figure 4c), which can
be described by Equation 235:
(2)
In this equation, A
irradiation, A is the absorbance at 330 nm for the photostationary state of the gel (t=0
after irradiation), and A is the absorbance at 330 nm at time t. Using this equation, the
reaction rate constant for the thermal relaxation to the trans isomer at 37°C is an order of
∞
is taken to be the absorbance of the trans isomer at 330 nm before
0
t
-1
-1
magnitude larger than that at 25°C: 0.0756 h compared to 0.0043 h , respectively. The
half time of the reaction can therefore be calculated and corresponds to 162.3 hours at
25°C and 9.2 hours at 37°C.
The hydrogels presented here remain in a stiffened state over several hours after
only 5 minutes of UV irradiation. The stability of the cis isomer thus enables minimal
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exposure to UV light and avoids the creation of potentially harmful free radicals. However,
it is worthy to note that the stiffening or softening of biological tissues does not happen on
the time scale of minutes. One could better approximate a natural timescale by allowing the
cis isomer to thermally relax, which proceeds to completion in 18 hours at 37°C. As for
stiffening to intermediate moduli, this can be accomplished by irradiation with
wavelengths of light that are further away from the absorption maximum of the trans state.
Because the efficiency of the photoisomerization depends on the wavelength of light, the cis
content of the photostationary state, and therefore the change in modulus, can theoretically
be decreased with irradiation by longer wavelengths of UV light.
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VIC Viability
To demonstrate cytocompatibility with a mechanoresponsive cell type, valvular
interstitial cells (VICs) were encapsulated in the photoresponsive gels at gel compositions
of both 4 and 5 wt% using an 8-arm, 20 kDa PEG-VS macromer (Figure 5a-c) with 2 mM
RGD as an adhesive ligand (final modulus was between 400-800 Pa). Viability was highest
when the VICs could locally degrade their environment and spread, as demonstrated in the
gels that contained a fraction of MMP-degradable crosslinker. The degradability was
accomplished by simply moving the 3,4’-AMPB monomer out of the peptide cleavage site
(KCG-3,4’-AMPB-GPQGIWGQGCK), and the fraction of non-degradable crosslinks was
chosen using Flory-Stockmayer theory to calculate the theoretical critical amount of
crosslinks needed to form a gel (38%nd = 38% non-degradable crosslinker). Both the
degradable and non-degradable versions of the photoresponsive peptides maintained
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Figure 5. Encapsulated valvular interstitial cells (VICs) stained with calcein-AM (green,
live) and ethidium homodimer (red, dead) after 3 days in (a) 5wt% 8-arm PEG-VS, 100%
non-degradable linker (100%nd), (b) 5wt% 8-arm PEG-VS, 38%nd, and (c) 4wt% 8-arm
PEG-VS, 38%nd. (d) Quantitative live/dead results at 3 days of culture for 4 wt% and 5
wt% gels with and without MMP-degradable crosslinker (% denotes percentage of non-
degradable linker). (e) Immunostaining for αSMA (green), f-actin (red), and nuclei (blue)
in 4wt%PEG, 38% non-degradable linker after 5 days of culture. All scale bars = 100 μm.
similar reaction efficiencies and therefore affected the mechanical properties of the
hydrogels in the same way (Figure S8). As shown in Figure 5d, the VICs maintained high
levels of viability (~80%) in 4 wt% hydrogels and were able to achieve a spread fibroblast
morphology after three days in the gel. At this time point, cell-laden gels were treated with
2
2
both 365 nm irradiation (10 mW/cm , 5 min) and 400-500 nm irradiation (10 mW/cm , 2
min) and demonstrated high viability 24 hours post-irradiation (Figure S9). Matrix-
interactions and spreading are required for VIC survival and are also necessary for VICs to
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exhibit the activated phenotype of the myofibroblast. With longer culture times, VICs
continued to elongate, and after 5 days, many cells exhibited organized f-actin fibers
(Figure 5e). Some cells were also positive for αSMA stress fibers, a hallmark of the
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myofibroblast phenotype. Therefore, in these gels, there is a baseline level of fibroblast
activation that may be affected upon changes in gel modulus.
Conclusions
Using a crosslinker that responds to both UV and visible light, a PEG-based hydrogel
was designed for mechanically dynamic three-dimensional cell culture. The shear modulus
exhibited reversible changes without any alterations in network connectivity or monomer
composition. Furthermore, due to the use of a photoisomer, the changes in modulus could
be effected without the use of chemical initiators or other additives, making this strategy an
attractive route toward controlling the matrix environment in the presence of cells. Current
efforts are aimed toward tuning the photoresponsive material properties to maximize
changes in modulus for biological studies, such as mechanotransduction of cells in 3D.
Supporting Information
Supporting information includes heating curves for the azobenzene-containing
hydrogels, rheology of hydrogels without azobenzene, and additional rheology of various
hydrogel formulations. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Acknowledgments
This work was supported by an award from the American Heart Association in the
form of a postdoctoral fellowship (A.M. Rosales) and a summer undergraduate fellowship
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(E.M. Nehls). A.M. Rosales also gratefully acknowledges a Postdoctoral Enrichment
Program Award from the Burroughs Wellcome Fund. Finally, this work was also made
possible by funding from the Howard Hughes Medical Institute and was supported in part
by a grant from the National Science Foundation (CBET 1236662).
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