Rapid Diels-Alder Cross-linking of Cell Encapsulating Hydrogels

Article abstract of DOI:10.1021/acs.chemmater.9b02485

Recent efforts in the design of hydrogel biomaterials have focused on better mimicking the native cellular microenvironment to direct cell fate. To simultaneously control multiple material parameters, several orthogonal chemistries may be needed. However, present strategies to prepare cell-encapsulating hydrogels make use of relatively few chemical reactions. To expand this chemical toolkit, we report the preparation of hydrogels based on a Diels-Alder reaction between fulvenes and maleimides with markedly improved gelation kinetics and hydrolytic stability. Fulvene-maleimide gels cross-link up to 10-times faster than other commonly used DA reaction pairs and remain stable for months under physiological conditions. Furthermore, fulvene-maleimide gels presenting relevant biochemical cues, such as cell-adhesive ligands and proteolytic degradability, support the culture of human mesenchymal stromal cells. Finally, this rapid DA reaction was combined with an orthogonal click reaction to demonstrate how the use of selective chemistries can provide new avenues to incorporate multiple functionalities in hydrogel materials.

Full text of DOI:10.1021/acs.chemmater.9b02485

Article
Cite This: Chem. Mater. XXXX, XXX, XXX−XXX
pubs.acs.org/cm
Rapid Diels−Alder Cross-linking of Cell Encapsulating Hydrogels
,
†,‡
§
Christopher M. Madl*
and Sarah C. Heilshorn
†
Department of Bioengineering, Stanford University, Stanford, California 94305, United States
‡
Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University, 269 Campus Drive
CCSR 4215, Stanford, California 94305, United States
§
Department of Materials Science & Engineering, Stanford University, Stanford, California 94305, United States
S Supporting Information
*
ABSTRACT: Recent efforts in the design of hydrogel biomaterials have focused
on better mimicking the native cellular microenvironment to direct cell fate. To
simultaneously control multiple material parameters, several orthogonal chemistries
may be needed. However, present strategies to prepare cell-encapsulating hydrogels
make use of relatively few chemical reactions. To expand this chemical toolkit, we
report the preparation of hydrogels based on a Diels−Alder reaction between
fulvenes and maleimides with markedly improved gelation kinetics and hydrolytic
stability. Fulvene−maleimide gels cross-link up to 10-times faster than other commonly used DA reaction pairs and remain
stable for months under physiological conditions. Furthermore, fulvene−maleimide gels presenting relevant biochemical cues,
such as cell-adhesive ligands and proteolytic degradability, support the culture of human mesenchymal stromal cells. Finally, this
rapid DA reaction was combined with an orthogonal click reaction to demonstrate how the use of selective chemistries can
provide new avenues to incorporate multiple functionalities in hydrogel materials.
INTRODUCTION
reaction is carried out in a simple saline buffer as opposed to
■
16
cell culture medium. Furthermore, the reversibility of the DA
reaction pair can result in swelling and substantial degradation
Hydrogel biomaterials are commonly used as mimics of the
native extracellular matrix (ECM) due to their high water
content and biocompatibility. Numerous studies in recent
years have revealed the ability of several hydrogel properties to
regulate cellular behaviors, including matrix stiffness, viscoe-
lasticity, cell-adhesive ligand concentration, growth factor
19
1
,2
of furan−maleimide gels over time. Under physiological
conditions, the cross-links can undergo a retro-DA reaction to
yield the maleimide starting material, which is prone to
hydrolysis. Thus, the cross-links are gradually broken, resulting
in changing material properties such as hydrogel stiffness and
mesh size that can become confounding variables in cell
culture studies.
3
presentation, and matrix degradation. To facilitate cross-
linking and chemical functionalization of hydrogels for cell
encapsulation, reactions from the broad class of “click
chemistry” reactions have grown in popularity due to their
robust and selective reactivity in the presence of water and
Previous mechanistic studies have revealed correlations
between the electronic structure of substituted furan dienes
and the rates of both DA and retro-DA reactions with
4
−6
living cells.
These reactions are characterized by rapid
20−23
reaction kinetics and good hydrolytic stability, both of which
are essential qualities for cross-linking reactions used to
generate cell-encapsulating hydrogels.
The classic Diels−Alder (DA) reaction represents an
underutilized cross-linking chemistry in regards to cell-
encapsulating hydrogels. While DA hydrogels have shown
maleimides.
While previous studies focused on altering
the substituents of the furans, for instance, by adding an
electron-donating methyl group, we reasoned that replacing
the furan diene with an even more electron-rich cyclic diene,
such as a fulvene, would further enhance the reaction rate of
the DA reaction (Figure 1). Fulvenes are commonly used as
7
−14
promise as drug delivery platforms,
their use as three-
dimensional (3D) tissue engineering scaffolds has been limited
to a small subset of polymeric hydrogel materials and cell
15,16
culture applications.
This is likely due in part to the
relatively slow gelation kinetics of the commonly-used DA
reaction pair that employs furan as the diene and maleimide as
the dienophile. Despite the substantial acceleration of the DA
1
7
Figure 1. Dienes used to prepare DA cross-linked hydrogels.
reaction in water, furan−maleimide gels can take hours to
cross-link under physiological conditions that are suitable for
7
,11,16,18
cell encapsulation.
Such long gelation times often result
Received: June 25, 2019
in an inhomogeneous cell distribution within the hydrogels due
to sedimentation and decreased cell viability if the cross-linking
Revised: September 16, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.chemmater.9b02485
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Chemistry of Materials
Article
dienes in organic synthesis and have been previously used to
maleic anhydride (490 mg, 5.0 mmol) in 25 mL of anhydrous
dimethylformamide (DMF) in a 100 mL round-bottom flask was
stirred at room temperature for 6 h. The resulting solution was cooled
to 0 °C, and sym-collidine (1.4 mL, 10.5 mmol) was added dropwise.
In a second round-bottom flask (50 mL), N-hydroxysuccinimide (2.3
g, 20 mmol) was dissolved in anhydrous DMF (25 mL) and cooled to
24,25
prepare self-healing, polymeric materials.
In this work,
fulvenes are paired with maleimide dienophiles to rapidly
cross-link multi-arm poly(ethylene glycol) (PEG) hydrogels
and hybrid PEG-engineered protein hydrogels with markedly
improved stability for cell encapsulation (Scheme 1).
0
°C. To the N-hydroxysuccinimide solution stirred at 0 °C, 2.8 mL
20 mmol) of trifluoroacetic anhydride was added dropwise, and the
mixture was allowed to stir for 10 min. To this mixture, sym-collidine
2.7 mL, 20 mmol) was added dropwise, and the mixture was allowed
(
Scheme 1. Fulvene−Maleimide DA Reaction
(
to stir for 10 min. This mixture was then added dropwise to the first
solution over a course of 1−2 h by cannula, maintaining both flasks at
0
°C. The combined reaction mixture was allowed to return to room
temperature, and the reaction was allowed to proceed overnight. The
reaction mixture was diluted with 60 mL of DCM and washed with 1
M aqueous hydrochloric acid (3 × 25 mL). The organic layer was
dried over magnesium sulfate, filtered, and concentrated by rotary
evaporation. The residue was resuspended in minimal diethyl ether,
and the white precipitate was collected by vacuum filtration. The
precipitate was washed with diethyl ether (2 × 50 mL) and dried
under vacuum to yield succinimidyl-4-(N-maleimidomethyl)-
EXPERIMENTAL SECTION
■
General Considerations. All reagents were purchased from
Sigma-Aldrich or Fisher Scientific and used without further
purification, unless otherwise noted. Multi-arm PEG starting materials
were purchased from JenKem Technology. NMR spectroscopy was
performed on a Varian Inova 500 MHz spectrometer in the Stanford
University NMR Facility. Chemical shifts were referenced to the
residual solvent peak. For NMR of modified elastin-like proteins
cyclohexane-1-carboxylate (SMCC) as a white solid (1.0 g, 3.0
1
mmol, 60% yield). H NMR (500 MHz, CDCl ) δ ppm: 6.71 (s, 2H),
3
3
2
1
.39 (d, J = 7.1 Hz, 2H), 2.82 (m, 4H), 2.57 (tt, J = 12.2, 3.5 Hz, 1H),
.16 (m, 2H), 1.79 (m, 2H), 1.72 (ddd, J = 11.3, 7.6, 3.8 Hz, 1H),
1
3
.53 (qd, J = 13.0, 3.4 Hz, 2H), 1.06 (qd, J = 12.8, 3.4 Hz, 2H);
C
NMR (126 MHz, CDCl ) δ ppm: 170.94, 170.55, 169.13, 133.98,
3
4
3.34, 40.34, 36.02, 29.25, 27.95, 25.54.
Synthesis of Diene- and Dienophile-Functionalized Multi-
(
ELPs), a drop of D O was added to the solution of ELP in DMSO-d
2
6
to suppress the signals from the amide protons.
Arm PEGs. Multi-arm PEG macromers were functionalized with
furan, methylfuran, and fulvene dienes and maleimide dienophiles by
standard amide coupling chemistries using HATU- or NHS-activated
carboxylic acid precursors, as described in detail in the Supporting
Information. PEG−maleimide/tetrazine was prepared by reacting 8-
arm PEG-amine with SMCC and methyltetrazine−NHS ester (Click
Chemistry Tools), as described in the Supporting Information. The
polymers were purified by precipitation in diethyl ether followed by
either dialysis against Milli-Q-grade water or aqueous washes.
Detailed synthetic protocols for each multi-arm PEG variant are
included in the Supporting Information. Successful PEG functional-
Synthesis of 4-(Cyclopenta-2,4-dien-1-ylidene)pentanoic
Acid. This synthetic protocol was adapted from previously published
24,26
procedures.
Cyclopentadiene was freshly distilled from dicyclo-
pentadiene. Briefly, dicyclopentadiene was heated to 170 °C with
stirring in a round-bottom flask equipped with a Vigreaux distilling
column that was attached via a glass joint to a Liebig condenser. The
condenser was further equipped with a second, round-bottom flask
submerged in a dry ice/isopropanol bath to prevent re-dimerization of
the cracked cyclopentadiene. The collected cyclopentadiene was used
immediately. Levulinic acid (4.0 g, 35 mmol) and cyclopentadiene
(
7.3 mL, 5.9 g, 87.5 mmol) were dissolved in 35 mL of methanol.
1
ization was confirmed by H NMR (Supporting Information).
This mixture was degassed with nitrogen for 10 min. A catalytic
amount of pyrrolidine (20 mol %, 0.5 g, 7 mmol) was dissolved in
triethylamine (5.1 g, 50 mmol) and added dropwise to the stirring
reaction mixture over the course of 10 min. The resulting mixture was
degassed for an additional 5 min, and the reaction was allowed to
proceed at room temperature under a nitrogen atmosphere. The
reaction progress was monitored by thin-layer chromatography, which
indicated that the starting material was consumed within 1 h. At that
point, the reaction mixture was diluted with diethyl ether (100 mL)
and washed with 0.5 M aqueous hydrochloric acid (100 mL). The
organic layer was then extracted with 0.1 M aqueous sodium
hydroxide (2 × 100 mL). The combined aqueous layers were acidified
to pH ≈ 1 with hydrochloric acid and then extracted into
dichloromethane (3 × 200 mL). The combined organic layers were
dried over magnesium sulfate, filtered, and concentrated by rotary
evaporation to yield a yellow oil. The crude product was purified by
silica chromatography. Silica gel (high-purity grade, pore size 60 Å,
Preparation of Fulvene-Modified ELP. Engineered ELPs
containing the integrin-binding RGD motif were produced by
standard recombinant protein expression techniques and purified as
28,29
previously described.
The primary amines in the ELPs were
sequentially modified with cysteic acid (to increase solubility) and 4-
(cyclopenta-2,4-dien-1-ylidene)pentanoic acid (fulvene) using stand-
1
ard peptide synthesis approaches. Experimental details and H NMR
characterization of the functionalized proteins are provided in the
Supporting Information.
Rheological Characterization. Functionalized multi-arm PEGs
were dissolved separately into phosphate-buffered saline (PBS) at a
concentration of 50 mg/mL. Hydrogels were formed by mixing
maleimide−PEGs with furan−, methylfuran−, or fulvene−PEGs at a
1:1 volume ratio. Hydrogels were cast directly on the rheometer stage.
Rheological characterization was performed using an ARG2
rheometer fitted with a 20 mm, 1° cone geometry. To prevent
dehydration, a solvent trap was used for all experiments. For runs
longer than a few hours, the samples were additionally surrounded by
mineral oil to further prevent drying. Gelation time sweeps were
performed at 1 Hz oscillatory frequency, 5% strain, and 37 °C. The
gelation point was identified by finding the time at which tan(δ)
reached a local maximum (Figure S1). Following complete gelation,
frequency sweeps from 0.1 to 10 Hz were performed at 10% strain
and 37 °C. For ELP−fulvene hydrogels, ELP−fulvene was dissolved
to 50 mg/mL in PBS on ice and mixed with 4-arm maleimide−PEG
(50 mg/mL) at a 1:1 volume ratio. Hydrogels were cast directly on
the rheometer stage pre-cooled to 4 °C. During the gelation time
sweep, the stage was held at 4 °C for 10 min to prevent thermal
aggregation of the ELP, and then the temperature was increased to 37
2
30−400 mesh) was wet-packed in a flash chromatography column in
ethyl acetate. The crude product was loaded directly onto the column,
and the desired product was eluted with ethyl acetate. Fractions
containing the product were combined and concentrated in vacuo to
afford 4-(cyclopenta-2,4-dien-1-ylidene)pentanoic acid as a yellow
1
solid (1.8 g, 11 mmol, 31.4% yield). H NMR (500 MHz, CDCl ) δ
3
ppm: 10.78 (br s, 1H), 6.51 (m, 4H), 2.88 (t, J = 7.6 Hz, 2H), 2.61 (t,
1
3
J = 7.6 Hz, 2H), 2.22 (s, 3H); C NMR (126 MHz, CDCl ) δ ppm:
3
1
2
78.64, 149.69, 143.40, 131.57, 131.41, 120.75, 120.16, 33.38, 31.48,
0.56.
Synthesis of SMCC. This synthetic protocol was based on a
2
7
previously published procedure.
A suspension of trans-4-
(
aminomethyl)cyclohexane carboxylic acid (786 mg, 5.0 mmol) and
B
DOI: 10.1021/acs.chemmater.9b02485
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Chemistry of Materials
Article
°
C at a rate of 3 °C/min and held at 37 °C for the remainder of the
pixel intensity as a function of the z-axis position using ImageJ. The
image was divided into 4 equal segments, and the cell density per
segment was given by summing the pixel intensity values within that
segment.
run. As above, gelation time sweeps were performed at 1 Hz
oscillatory frequency and 5% strain.
Hydrogel Degradation Study. PEG components were dissolved
into PBS and mixed to form hydrogels as described above. Hydrogels
Cell Encapsulation in DA ELP Hydrogels. After trypsinization,
(
50 μL) were cast into pre-weighed microcentrifuge tubes and cross-
hMSCs were resuspended in a 5% (w/v) solution of ELP−fulvene in
6
linked in a 37 °C incubator. Fulvene gels were given 4 h to ensure
complete cross-linking, while furan and methylfuran gels were cross-
linked for ∼24 h. PBS (1 mL) was added to the microcentrifuge
tubes, and the samples were maintained at 37 °C. To assess hydrogel
degradation, PBS was carefully aspirated and excess liquid wicked
away with a Kimwipe prior to measuring the wet mass of hydrogels.
After measuring the wet mass, the samples were frozen at −80 °C and
lyophilized. The dry mass of the hydrogels was then measured. Mass
loss over time was determined from the hydrogel dry mass
measurements. Hydrogel mass swelling ratios were calculated by
dividing the wet mass by the corresponding dry mass for each gel. For
PBS to a concentration of 4 × 10 cells/mL and maintained on ice.
The cell suspension was mixed in a 1:1 volumetric ratio with a 5% (w/
v) solution of 4-arm PEG−maleimide in PBS to afford a final cell
6
concentration of 2 × 10 cells/mL. To enable facile handling of
hydrogels in culture and during fluorescence microscopy analysis, gels
were cast into custom cylindrical silicone molds (8 mm diameter ×
0.5 mm height) prepared from 0.5 mm thick silicone insulator
(Electron Microscopy Sciences) that was oxygen plasma bonded to
31
no. 1 glass coverslips, as previously described. The pregel mixture
was dispensed into the molds in 24-well tissue culture plates and
maintained on ice for 10 min to prevent thermal aggregation of the
ELP. The plates were then moved to room temperature for 5 min
before being transferred to a 37 °C tissue culture incubator for 20 min
to complete gelation. The wells were then filled with cell culture
4
2
-arm PEG−fulvene gels, time points were collected at 0, 1, 3, 7, 14,
1, and 28 days and at 2, 3, and 4 months. For 4-arm PEG−furan gels,
time points were collected at 0, 1, 2, and 3 days, and for 4-arm PEG−
methylfuran gels, time points were collected at 0, 1, 2, 3, and 4 days.
For 8-arm PEG−fulvene gels, time points were collected at 1, 7, 14,
and 28 days and at 2, 3, and 4 months. For 8-arm PEG−furan gels,
time points were collected at 1, 7, 14, 28, and 49 days.
medium and maintained in a 37 °C, 5% CO incubator. Culture
2
medium was changed every two days.
Cell Viability Assay. The viability of hMSCs encapsulated in DA
ELP hydrogels was assessed both 1 h and 7 days after encapsulation.
At each time point, 4 gels were stained with Live/Dead reagents
(Thermo Fisher Scientific) diluted into phenol red-free culture
medium (1:2000 dilution for calcein-AM, 1:500 for ethidium
homodimer). After incubating in the dye solution for 40 min at 37
°C, the gels were washed twice with phenol red-free culture medium
and imaged on a Leica SPE confocal microscope. The images were
processed using ImageJ software (NIH).
Cell Morphology Assay. The morphology of hMSCs encapsu-
lated in DA ELP hydrogels was assessed 7 days after encapsulation.
Samples were fixed with 4% paraformaldehyde in PBS, washed twice
with PBS, and then permeabilized with 0.25% Triton X-100 in PBS
(PBST). Samples were stained with FITC-phalloidin to label F-actin
and Hoechst to label DNA and then washed three times with PBST.
Samples were mounted onto coverslips by placing a drop (∼20 μL) of
antifade mounting medium (Thermo Fisher Scientific) on the
coverslip and inverting the silicone molds containing gels onto the
drop of mounting medium. The mounted samples were imaged on a
Leica SPE confocal microscope.
Orthogonal Chemical Functionalization Assay. To demon-
strate that fulvene-based DA cross-linking is amenable to the use of
other “click” reactions to subsequently functionalize hydrogels, gels
were prepared by mixing a 5% (w/v) solution of ELP−fulvene in PBS
in a 1:1 volumetric ratio with a 5% (w/v) solution of 8-arm PEG−
maleimide (negative control) or 8-arm PEG−maleimide/tetrazine in
PBS. Gels (15 μL) were cast in microcentrifuge tubes and maintained
on ice for 10 min to prevent thermal aggregation of the ELP. The
samples were then moved to room temperature for 5 min before
being placed at 37 °C for 1 h to complete gelation. The gels were
allowed to equilibrate in PBS (0.5 mL) for 30 min at 37 °C. The gels
were then incubated for 1 h in PBS with or without a bicyclononyne
1
The mechanism of hydrogel degradation was assessed by H NMR
analysis. Deuterated phosphate buffer (pD ≈ 7.4; 100 mM; 10×) was
prepared by dissolving sodium phosphate dibasic (Na HPO ; 51.1
mg) and sodium phosphate monobasic (NaH PO ; 16.8 mg) in
deuterium oxide (5 mL). The phosphate buffer was diluted to 10 mM
in deuterium oxide and used to dissolve the diene- and dienophile-
modified 4-arm PEGs to 50 mg/mL. The furan, methylfuran, or
fulvene PEG solution was mixed in a 1:1 volumetric ratio with the
2
4
2
4
1
maleimide PEG solution and added to an NMR tube. The H NMR
spectra were immediately collected to obtain pre-reaction data. The
tubes were subsequently maintained at 37 °C, and NMR spectra were
collected at 1, 7, and 14 days to assess gelation and subsequent
degradation.
Cell Culture. Human bone marrow-derived mesenchymal stromal
cells (hMSCs) were purchased from Lonza. The cells were expanded
in high glucose Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum, 1% GlutaMAX, and 1% penicillin/
streptomycin (Gibco) on tissue culture-treated plastic in a 37 °C, 5%
CO incubator. Culture medium was changed every two days and the
cells were passaged approximately weekly using trypsin/EDTA
(
2
Gibco). The cells were used at passage 8.
Cell Settling Assay. The protocol to assess cell settling in DA
cross-linked hydrogels was based on a previously published
30
procedure. hMSCs were lifted by trypsinization, washed with PBS,
and lightly fixed with 1% paraformaldehyde in PBS. The cells were
stained with Hoechst DNA dye to visualize nuclei by fluorescence
microscopy. The cells were pelleted by centrifugation, washed to
remove unbound dye, and pelleted again. The cell pellet was
resuspended in a 5% (w/v) solution of 4-arm PEG−maleimide in PBS
to a concentration of 4 × 10 cells/mL. The cell suspension was then
mixed in a 1:1 volumetric ratio with a 5% (w/v) solution of either 4-
arm PEG−furan, 4-arm PEG−methylfuran, or 4-arm PEG−fulvene in
PBS to afford a final cell concentration of 2 × 10 cells/mL. 70 μL of
the pregel solution was then added to a microcuvette (n = 3 per
condition) and allowed to gel upright at 37 °C overnight. The
following day, the cuvette was laid on its side and imaged on a Leica
SPE confocal microscope. The degree of cell settling was quantified
by dividing the images into 4 segments along the vertical axis,
counting the number of cells per segment, and then calculating the
6
(BCN)-functionalized far-red dye (CF640R-BCN; Biotium; 10 μM)
at 37 °C. The supernatant was discarded, and the gels were washed
with 0.5 mL PBS (3 × 15 min). The gels were then degraded by
incubation with 0.5% trypsin/EDTA (Gibco) at 37 °C and
mechanical disruption by sonication. The fluorescence intensity of
dye bound in the hydrogel network was measured with a Molecular
Devices SpectraMax M2 plate reader.
Statistical Analysis. For comparisons between two experimental
groups, two-tailed Student’s t-tests were used to determine statistical
significance. For comparisons among more than two experimental
groups, one-way analysis of variance (ANOVA) with Bonferroni post-
hoc testing to account for multiple comparisons was used to
determine statistical significance. For the gelation point experiment
using 8-arm PEGs, a single sample, two-tailed Student’s t-test was
used to test the null hypothesis that the furan gel formed at the same
rate as the fulvene gel (i.e., that the gelation point for the furan gels
6
30
sedimentation coefficient (δ in eq 1) as previously described
ci2
∑
∑ c
δ = n(
)
(1)
2
i
where n is the number of image segments and c is the cell density per
segment. Cell density was approximated by measuring the grayscale
i
C
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Chemistry of Materials
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Figure 2. Rapid cross-linking kinetics of fulvene−maleimide DA hydrogels. (A) 4-arm PEG hydrogels are prepared by mixing diene (furan,
methylfuran, or fulvene) PEG with maleimide PEG. (B) Gelation time sweeps of 4-arm PEG-based DA gels. Arrows indicate the critical gelation
point. (C) Time to critical gelation point and (D) time to half-maximal storage modulus for 4-arm PEG-based DA gels. (E) 8-arm PEG hydrogels
are prepared by mixing diene (furan or fulvene) PEG with maleimide PEG. (F) Gelation time sweeps of 8-arm PEG-based DA gels. The arrow
indicates the critical gelation point for the furan gel. (G) Time to critical gelation point and (H) time to half-maximal storage modulus for 8-arm
PEG-based DA gels. In C and D, **P < 0.01, ***P < 0.001, one-way ANOVA with Bonferroni post-hoc test. In G, ****P < 0.0001, one-sample
Student’s t-test with the null hypothesis that the gelation time for the furan gel is equivalent to the approximate gelation time for the fulvene gel
(∼30 s). In H, ****P < 0.0001, two-tailed Student’s t-test. Data are mean ± s.d., n = 3−4.
occurred within the ∼30 s it took to start the rheometer after mixing
the precursor solutions). A P-value of less than 0.05 was considered to
be statistically significant.
gels in ∼40 min, compared to greater than 10 h for the furan
and methylfuran gels (Figure 2D). Consistent with previous
reports, the methylfuran-based gels cross-link more rapidly
22
than unsubstituted furan-based gels (Figure 2B,C).
RESULTS AND DISCUSSION
■
Gelation kinetics are a function of multiple parameters in
addition to the reaction rate of the cross-linking groups, such
as the concentration of the polymeric components and the
number of functional groups per component. Percolation
theory predicts that for a given concentration of polymeric
components, an increase in the number of reactive groups per
polymer will achieve a percolating network more rapidly
during cross-linking, thereby decreasing the time required to
cross-link the network. Accordingly, we also measured the
gelation kinetics of DA hydrogels based on 8-arm PEGs. To
assess the two extremes of reaction rates, we compared 8-arm
PEGs functionalized with furan and fulvene dienes (Figure
Improving the Gelation Kinetics of DA Cross-linking
for Cell Encapsulation. The first limitation with current DA
reaction pairs used to cross-link hydrogels is their relatively
slow gelation kinetics. To assess whether substituting
commonly used furan moieties with fulvenes would enhance
the gelation rate of hydrogels, separate 4-arm PEG
components were modified with maleimide dienophiles and
either furan, methylfuran, or fulvene dienes by standard amide
coupling chemistry (Figure 2A). The diene and dienophile
PEGs were mixed together in a 1:1 molar ratio with a final
polymer content of 5% (w/v) and subjected to oscillatory
shear rheology to determine gelation kinetics (Figure 2B).
Gelation kinetics were assessed by two metrics: time until the
critical gelation point (i.e., the point at which a percolating
network is first formed; Figure S1) and time until the
hydrogels reached half their maximal storage modulus.
Whereas the furan and methylfuran-based hydrogels took
2
E). Consistent with our results with 4-arm PEGs, fulvene-
based gels cross-linked much more rapidly than furan-based
gels. The furan gels reached the critical gelation point in ∼2 h,
whereas the fulvene gels crossed the gelation point before the
rheometer could start collecting data (less than ∼30 s) (Figure
2F). Similarly, furan gels reached their half-maximal storage
modulus in ∼5.5 h, while fulvene gels reached a half-maximal
storage modulus in less than 10 min (Figure 2G). As expected,
for both furan and fulvene gels, 8-arm PEGs reached their
∼
10 and ∼7 h, respectively, to reach the critical gelation point,
fulvene-based hydrogels reached the critical gelation point in
less than 20 min, a greater than 10-fold improvement (Figure
2
C). Similarly, half-maximal modulus was achieved in fulvene
D
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percolation threshold much faster than the corresponding 4-
arm PEGs due to the increased number of reactive groups per
PEG macromer. Indeed, previous studies have shown that
heavily functionalized polysaccharides can enable sufficiently
rapid gelation even with furan and methylfuran dienes to be
15,16,22
useful for cell encapsulation.
The addition of fulvene-
based DA cross-linking now expands the utility of this
chemistry to other hydrogel network architectures, such as 4-
arm PEGs, that are commonly used as 3D tissue engineering
scaffolds and drug delivery platforms.
To demonstrate that fulvene-based DA hydrogels exhibit
sufficiently rapid gelation kinetics to enable homogeneous cell
encapsulation, a cell sedimentation assay was performed using
human mesenchymal stromal cells (hMSCs, also commonly
referred to as mesenchymal stem cells) and 4-arm PEG
macromers. The cells were lightly fixed, stained with Hoechst
to label the nuclei, and suspended in a 1:1 volumetric mixture
of maleimide PEG and diene PEG at 5% (w/v) total polymer
content. The mixture was transferred to a microcuvette and
allowed to cross-link for 24 h at 37 °C. The gels were imaged
by confocal microscopy, and cell sedimentation was quantified
30
as a “sedimentation coefficient” as defined previously. This
sedimentation coefficient is the second moment average
divided by the first moment average, which is analogous to
the dispersity index for polymeric materials. Thus, a coefficient
of 1 indicates a perfectly homogeneous distribution of cells
throughout the volume of the gel, whereas coefficients greater
than 1 indicate that sedimentation has occurred. As expected
from their slow gelation kinetics, furan and methylfuran gels
resulted in significant cell sedimentation, with coefficients of
Figure 3. Rapid cross-linking of fulvene-based DA gels prevents cell
sedimentation. (A) Confocal fluorescence micrographs along the z-
axis of 4-arm PEG-based DA hydrogels with encapsulated hMSCs.
Prior to encapsulation, the hMSCs were lightly fixed with
paraformaldehyde and stained with Hoechst to label the nuclei
(white). (B) Cell sedimentation quantified as the sedimentation
coefficient for hMSCs encapsulated in 4-arm PEG-based DA gels
cross-linked with furan, methylfuran, or fulvene dienes. Dashed line
represents a perfectly homogeneous cell distribution. ***P < 0.001,
one-way ANOVA with Bonferroni post-hoc test. Data are mean ± s.d.,
n = 3.
∼3 (Figure 3). In contrast, sedimentation coefficients for
fulvene gels were ∼1, indicating a homogeneous cell
distribution throughout the hydrogels (Figure 3). Thus,
fulvene gels cross-link rapidly enough to maintain a
homogeneous cell distribution, even in hydrogels prepared
from polymers with low degrees of diene and dienophile
functionalization.
multi-arm PEGs functionalized with maleimide dienophiles
and furan, methylfuran, or fulvene dienes as described above.
The gels were maintained in PBS at 37 °C, and samples were
collected at defined time points. Consistent with previous
Improving the Stability of DA Cross-linked Hydrogels
for Cell Culture. A second significant limitation in using DA-
based hydrogels as 3D cell culture platforms is the relatively
rapid degradation rate of maleimide−furan gels under
19
results, gels formed from 4-arm PEG modified with furan and
methylfuran dienes exhibited substantial changes in swelling
behavior and were completely degraded by 3 and 4 days after
cross-linking, respectively (Figure 4B,C). Strikingly, fulvene-
based DA hydrogels exhibited markedly improved stability,
with only modestly increased swelling and negligible mass loss
after 4 months under physiological conditions (Figure 4B,C).
Accordingly, rheological characterization of the three hydrogels
revealed significant time-dependence in the mechanical
properties of freshly prepared furan and methylfuran hydrogels,
but not in fulvene hydrogels, suggesting that cross-links formed
by furan-based moieties are more dynamic and thus expected
to be less stable over time (Figure S2).
21,32−34
physiological conditions. DA reactions are reversible,
with retro-DA reactions liberating the original maleimide
groups at cross-link points. While these maleimides can react
again with the corresponding furans to re-form the cross-link,
the maleimide groups are also susceptible to hydrolysis and can
degrade to a maleamic acid that does not appreciably
participate in network cross-links due to its significantly slower
19
DA reaction kinetics. Thus, over time, the number of cross-
links is decreased, resulting in increased hydrogel swelling that
eventually results in dissolution of the network. Previous
approaches to decrease hydrolysis involved adding hydro-
phobic spacers to the maleimide to decrease water accessibility
and increasing the number of reactive groups per macromer to
The mechanism of hydrogel degradation via a retro-DA
reaction and subsequent hydrolysis of the maleimide
dienophile was confirmed by NMR (Figures S3 and S4).
Consistent with the gelation kinetics data obtained from
rheological analysis (Figure 2), NMR reveals that the DA
cross-linking reaction approaches completion within 24 h for
all hydrogel formulations, as indicated by the disappearance of
the maleimide resonance peak. After further maintaining the
gels in phosphate buffer at 37 °C, the peaks corresponding to
the DA adduct decreased from 1 to 7 to 14 days in the furan-
and methylfuran-based gels (Figure S3), while the adduct
signal was stable in the fulvene gels (Figure S4), indicating
11
maintain a percolating network for a longer duration.
Nevertheless, such gels still exhibited significant increases in
swelling, indicating a decrease in cross-link density over time
that is directly linked to changes in other important hydrogel
properties including stiffness and nutrient transport that can
alter cell phenotype.
To assess the stability of fulvene-based DA hydrogels, the
mass swelling ratio and dry mass of hydrogels were monitored
over time (Figure 4). Hydrogels were prepared by mixing
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Figure 4. Fulvene gels remain stable long-term under physiologically relevant conditions. (A) Schematic of 4-arm PEG network. (B) Mass swelling
ratio and (C) dry mass of 4-arm PEG-based DA hydrogel networks over time for samples maintained in PBS (pH 7.4) at 37 °C. (D) Schematic of
8
-arm PEG network. (E) Mass swelling ratio and (F) dry mass of 8-arm PEG-based DA hydrogel networks over time for samples maintained in
PBS (pH 7.4) at 37 °C. Data are mean ± s.d., n = 3 gels per time point.
Figure 5. Engineered protein−PEG hybrid DA hydrogels support the culture of hMSCs. (A) Schematic of fulvene-modified ELPs containing a cell-
adhesive RGD domain (green) and a structural elastin-like domain (blue). Upon mixing with a 4-arm PEG−maleimide crosslinker, the fulvene
ELPs form hydrogel networks. (B) Gelation time sweep for ELP−PEG fulvene DA gels. Gelation point data are mean ± s.d., n = 3. Viability of
hMSCs encapsulated in ELP−PEG fulvene DA gels after (C) 1 h and (D) 7 days, measured by a live/dead cytotoxicity assay. Viability data are
mean ± s.d., n = 4. (E) Confocal fluorescence micrograph showing spreading of hMSCs cultured in ELP−PEG fulvene DA gels for 7 days. The
actin cytoskeleton was stained with phalloidin (green) and the nuclei were stained with Hoechst (blue).
reversibility of the furan and methylfuran DA cross-links.
Further, NMR peaks corresponding to the hydrolyzed
maleimide appeared and increased in intensity throughout
this time interval for the furan and methylfuran gels (Figure
S3), indicating that the maleimides existing in equilibrium with
the DA adduct were hydrolyzing and thereby decreasing the
number of available cross-link sites over time.
Consistent with this degradation mechanism, increasing the
degree of macromer functionalization by using 8-arm PEG had
the expected effect of increasing stability by increasing network
connectivity (Figure 4D−F). 8-arm PEG hydrogels formed
from furan macromers exhibited more gradual swelling
compared to 4-arm gels and were fully degraded by 7 weeks
(Figure 4E,F). 8-arm fulvene gels continued to demonstrate
excellent stability, with negligible changes in both swelling and
dry mass after 4 months (Figure 4E,F). Thus, fulvene-based
DA gels are attractive as 3D cell culture platforms to culture
cells for long durations with consistent microenvironmental
parameters.
These results demonstrate that the choice of DA reaction
pair and the degree of functionality of the polymeric
components dictate several crucial hydrogel properties that
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Figure 6. Orthogonal functionalization of DA cross-linked hydrogels using tetrazine ligation. (A) Schematic depicting covalent modification of DA
hydrogels prepared from ELP−fulvene and 8-arm PEG functionalized with maleimides (black) and tetrazines (purple) with a tetrazine-reactive dye
(
CF640R-BCN, red). (B) Quantification of fluorescence intensity in gels with either excess maleimide or tetrazine moieties and either treated or
untreated with tetrazine-reactive dye. ***P < 0.001, one-way ANOVA with Bonferroni post-hoc test. Data are mean ± s.d., n = 4.
determine the suitability of the gel system for a given
application. For instance, rapidly cross-linking fulvene gels
best maintain a homogenous cell distribution, making them
useful as 3D cell culture materials. The 8-arm fulvene gels
cross-link on a very rapid time scale (seconds to minutes),
suggesting that highly functionalized fulvene-polymers may be
useful in a dual-component bio-ink for 3D printing of cell-
laden constructs that require fast gelation kinetics to retain
their printed shape. The chemical stability of the cross-links in
the fulvene gels makes these materials suitable for cell culture
applications in which the stiffness must be maintained within a
specific range to expand or differentiate mechanosensitive stem
To assess the suitability of fulvene DA cross-linked gels as
3D cell culture platforms, hMSCs were encapsulated within
hydrogels prepared from fulvene-functionalized ELPs and 4-
arm PEG−maleimide. Cell viability was assessed both acutely
at 1 h after encapsulation and longer-term at 7 days after
encapsulation by a live/dead cell membrane integrity assay
(
Figure 5C,D). The cells remained highly viable (>96%)
following encapsulation, indicating that the DA cross-linking
mechanism is well tolerated by cells. After 7 days in culture, the
cells remained ∼90% viable, consistent with previous results
culturing hMSCs in hydrogels with similar concentrations of
28,38
RGD peptide.
Furthermore, after 7 days, the hMSC
3
5,36
cultures showed evidence of cell-mediated remodeling. Cell
morphology was assessed by fixing the cell-laden hydrogels and
staining the actin cytoskeleton with fluorescent phalloidin.
Protrusions were observed extending from the cell bodies into
the surrounding hydrogel material, indicative of cell adhesion
and local degradation of the gel network (Figure 5E). This
behavior is consistent with hMSCs cultured in other hydrogel
cells.
In contrast, other cell types that require gradual
degradation of the hydrogel material in order to grow, such as
37
intestinal stem cells, may benefit from the addition of furan-
based cross-links that are more susceptible to degradation.
Furan-based networks may additionally be useful to control the
release of cells or drugs for therapeutic applications by
programming the rate of degradation into the network through
selecting the identity and number of DA reaction pairs per
polymeric component.
43−45
systems with cell-remodelable adhesive ligands.
Orthogonal Functionalization of DA Cross-linked
Hydrogels. Finally, to demonstrate that the DA cross-linking
reaction can be used in combination with an orthogonal “click”
reaction to selectively functionalize hydrogels, a tetrazine
ligation (an inverse electron demand DA reaction) was used to
covalently attach a fluorescent dye to the polymer network.
Hydrogels were prepared from fulvene ELP and an 8-arm PEG
that was modified with both maleimides and tetrazines (Figure
Hybrid Protein−PEG DA Hydrogels for Cell Culture.
Hydrogels used in 3D cell culture should present bioactive
signals, such as cell-adhesive ligands and peptides that enable
proteolytic degradation, to maintain high cell viability and to
38,39
allow cell spreading and migration.
To incorporate this
additional functionality in DA cross-linked hydrogels, en-
gineered ELPs were functionalized with fulvenes to enable
cross-linking with 4-arm PEG−maleimide (Figure 5A). The
ELPs used contain repeating cell-adhesive and structural
6
A). The tetrazines should not react to an appreciable degree
during the DA cross-linking reaction, as both fulvenes and
tetrazines are dienes and, due to the inverse electron demand
nature of the tetrazine ligation, the reaction between tetrazines
40
elastin-like domains. The cell-adhesive domains contain an
extended RGD peptide sequence derived from human
fibronectin to enable integrin-mediated cell adhesion, and the
structural domain consists of the elastin-like VPGXG peptide
sequence, wherein 1 out of 5 'X' residues are lysines to provide
primary amines for functionalization with the fulvenes for
cross-linking. Prior to the addition of the fulvene moieties, the
lysine residues were sulfated to increase the solubility of the
ELPs. The ELPs are additionally susceptible to proteolytic
degradation, enabling cell-mediated remodeling of the hydro-
gel matrix. While the elastin-like sequence is largely insensitive
46
and maleimides is very slow. Following cross-linking, the
remaining tetrazines are available to react with a fluorescent
dye bearing a BCN group. As a control, gels were also prepared
from 8-arm PEG modified only with maleimides. The excess
maleimides remaining after cross-linking should not appreci-
ably react with the BCN-bearing dye. Significant dye
incorporation was observed only in hydrogels containing
tetrazine moieties, as indicated by a ∼25-fold increase in
fluorescence intensity over background levels (Figure 6B).
Importantly, minimal residual dye remained in the hydrogels
with excess maleimides, demonstrating the selectivity of the
two-step, orthogonal click reaction approach.
4
1
to proteases other than dedicated elastases, we have
previously shown that the extended cell-adhesive sequence in
42
these materials can be cleaved by zinc metalloproteases. The
resulting gels cross-link rapidly, reaching their critical gelation
point in less than 25 min (Figure 5B). Thus, the rapid cross-
linking of the fulvene−maleimide DA reaction also applies to
hybrid protein-synthetic polymer hydrogels.
CONCLUSIONS
■
These results indicate that fulvene−maleimide DA reaction
pairs are a suitable chemistry for cross-linking cell-encapsulat-
ing hydrogels. Fulvene-based cross-linking overcomes the two
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Chemistry of Materials
Article
major limitations of previous DA gels by markedly increasing
the gelation rate of the materials to prevent cell sedimentation
and by significantly increasing the hydrolytic stability of the
gels to maintain consistent material properties over time.
Adding rapid DA cross-linking to the biomaterials science
toolkit will expand the range of functionalities that can be
incorporated into hydrogel mimics of the ECM. The
orthogonality of the DA reaction to other commonly used
click chemistries enables the development of well-controlled,
multicomponent hydrogel networks for use as tissue engineer-
ing scaffolds, cell transplantation vehicles, and drug delivery
systems.
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ASSOCIATED CONTENT
Supporting Information
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ACS Publications website at DOI: 10.1021/acs.chemma-
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■
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AUTHOR INFORMATION
Corresponding Author
E-mail: cmadl@stanford.edu.
Biodegradable Polysaccharide Derivative Hydrogels through Aqueous
■
Diels-Alder Chemistry. Macromol. Rapid Commun. 2011, 32, 905−
9
(
11.
*
14) Montiel-Herrera, M.; Gandini, A.; Goycoolea, F. M.; Jacobsen,
ORCID
N. E.; Lizardi-Mendoza, J.; Recillas-Mota, M.; Arguelles-Monal, W.
̈
M. N-(furfural) chitosan hydrogels based on Diels-Alder cyclo-
additions and application as microspheres for controlled drug release.
Carbohydr. Polym. 2015, 128, 220−227.
Christopher M. Madl: 0000-0003-3221-5041
Sarah C. Heilshorn: 0000-0002-9801-6304
Notes
(15) Nimmo, C. M.; Owen, S. C.; Shoichet, M. S. Diels−Alder Click
Cross-Linked Hyaluronic Acid Hydrogels for Tissue Engineering.
Biomacromolecules 2011, 12, 824−830.
The authors declare no competing financial interest.
(16) Yu, F.; Cao, X.; Li, Y.; Zeng, L.; Zhu, J.; Wang, G.; Chen, X.
ACKNOWLEDGMENTS
■
Diels-Alder crosslinked HA/PEG hydrogels with high elasticity and
fatigue resistance for cell encapsulation and articular cartilage tissue
repair. Polym. Chem. 2014, 5, 5116−5123.
C.M.M. was supported by a Ruth L. Kirschstein NRSA F31
fellowship (F31 EB020502) from the National Institutes of
Health (NIH), by the Siebel Scholars Program, and by the
Stanford ChEM-H Postdoctoral Training Program in Quanti-
tative Mechanobiology. This work was supported by funding
from the NIH (R21 HL138042, U19 AI116484, R01
HL142718), NSF (DMR 1808415), and the Stanford Wu
Tsai Neurosciences Institute. Part of this work was performed
at the Stanford Nano Shared Facilities (SNSF), supported by
the National Science Foundation under award ECCS-1542152.
(17) Rideout, D. C.; Breslow, R. Hydrophobic Acceleration of Diels-
Alder Reactions. J. Am. Chem. Soc. 1980, 102, 7816−7817.
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Cross-Linked Hydrogels Formed through Diels-Alder Coupling of
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