Biomacromolecules
Communication
phase HPLC. Chemical structures of the hydrogel precursors and
schematics of the cross-linking reaction and cellular encapsulation are
presented in Figure 1. Complete synthetic details are presented in the
Supporting Information.
reactive cyclooctyne moieties such as DIFO remain prohibitive
for many applications.
As an alternative synthetic approach, we hypothesized that
the inverse electron demand Diels−Alder click reaction
between tetrazine and an appropriate dienophile (e.g.,
norbornene, trans-cyclooctene) would have many of the same
benefits as SPAAC, but with simpler synthetic routes, if the
kinetics of the cross-linking chemistry were fast and compatible
with cellular encapsulation. Notably, numerous studies have
demonstrated tetrazine click reactions as powerful bio-
orthogonal chemistries for in vitro and in vivo cell labeling
and imaging.14−16 Tetrazine chemistry has also been leveraged
for the creation of block copolymers without requiring any
additional additives, initiators, or catalysts,17 suggesting that it
could also be useful for forming covalently cross-linked polymer
networks. Another attractive property of tetrazines is their
synthetic tractability. Karver et al. described the synthesis of 12
different tetrazine molecules, all of which were obtained in
fewer than three steps and in yields greater than 15%.18 In
contrast, the second-generation DIFO molecule that we
previously used (i.e., DIFO3) was synthesized in 12−13 steps
and obtained in approximately 8% yield.19,20
Here, we report the tetrazine−norbornene click reaction as a
new cross-linking chemistry suitable for the formation of cell-
laden hydrogels for 3D cell culture. We specifically used a PEG
functionalized with a benzylamino tetrazine moiety that was
previously shown to have high reactivity toward norbornene.14
Hydrogels were cross-linked with an ECM mimetic cell
degradable dinorbornene synthetic peptide. The kinetics of
hydrogel formation were evaluated by rheological character-
ization during in situ polymerization. The equilibrium modulus
and swelling ratio of hydrogels were also characterized. To
evaluate the suitability of this chemistry for 3D cell culture
applications, the postencapsulation viability of human mesen-
chymal stem cells (hMSCs) was evaluated. Finally, the potential
for photochemical modification of hydrogel networks was
explored.
Characterization of Hydrogel Properties. To evaluate the
kinetics of gel formation, hydrogels were prepared using 7.5 wt %
PEG-Tz (10.5 mM Tz) and 5.25 mM norb-KGPQGIWGQKK-norb in
phosphate buffered saline (PBS) and polymerized in situ on a TA
Instruments DHR-3 rheometer equipped with a 20 mm, 2° cone. A
peltier plate was used to maintain the temperature at 22 °C, and a
solvent trap was put in place to prevent the hydrogel from drying out
during testing. During polymerization, the hydrogels were subjected to
oscillatory shear at 10 rad/s and 10% strain and the evolution of the
storage and loss moduli (i.e., G′ and G″) was monitored for 30 min.
In addition, hydrogels were prepared with the following stoichio-
metrically balanced formulations (i.e., 1:1 total Tz:norb) and stored in
PBS at 37 °C for 2 days: (1) 10 wt % PEG-Tz (14 mM Tz), 6.5 mM
norb-KGPQGIWGQKK-norb, and 1 mM norb-AhxRGDS; (2) 7.5 wt
% PEG-Tz (10.5 mM Tz), 4.75 mM norb-KGPQGIWGQKK-norb,
and 1 mM norb-AhxRGDS; (3) 5 wt % PEG-Tz (7 mM Tz), 3 mM
norb-KGPQGIWGQKK-norb, and 1 mM norb-AhxRGDS. Equili-
brium modulus measurements were made by subjecting these
hydrogels to oscillatory shear, as described above, using an 8 mm
parallel plate geometry. The gels were tested while immersed in a 37
°C water bath. The 10 wt % and 7.5 wt % gels were tested at 10 rad/s
and 10% strain. The 5 wt % gels were tested at 1 rad/s and 1% strain in
order to be within the linear viscoelastic regime for this formulation.
Finally, wet and dry weights of the gels were recorded to calculate the
mass swelling ratio for each formulation. Calculated values for the
critical cross-link density required to achieve gelation, ρc, for each
formulation are provided in the Supporting Information.
Cell Encapsulation. To evaluate the cytocompatibility of the
tetrazine−norbornene cross-linking reaction, hMSCs isolated from
human bone marrow were encapsulated in PEG-peptide hydrogels,
and their viability was assessed via a membrane integrity assay at 24
and 72 h postencapsulation. Briefly, hMSCs were resuspended at a
final cell density of 5 × 106 cells/ml in a solution of 7.5 wt % PEG-Tz,
4.75 mM norb-KGPQGIWGQKK-norb, and 1 mM norb-RGDS. The
cell suspension was pipet mixed and then quickly transferred to sterile
syringe tip molds (i.e., 1 mL syringes that had been cut to remove the
tips and then inverted) in 30 μL aliquots. After allowing 15 min for
gelation, the cell-laden hydrogels were transferred to a 24-well plate
and cultured under standard conditions. hMSC viability was assessed
using the commercially available Live/Dead staining kit (Invitrogen),
which differentiates viable cells from dead cells based on membrane
integrity. Stained hydrogels were imaged on a Zeiss LSM NLO laser
scanning confocal microscope, and percent viability was determined by
analysis in Image J.
EXPERIMENTAL SECTION
Hydrogel Precursors. A clickable PEG-tetrazine (PEG-Tz)
macromer was synthesized by coupling 5-(4-(1,2,4,5-tetrazin-3-yl)-
benzylamino)-5-oxopentanoic acid (Tz-COOH; see Scheme 1 for
■
Scheme 1. Synthesis of Tz-COOH
Protein Photopatterning. To demonstrate orthogonality with
photochemical patterning techniques, hydrogels were prepared using
7.5 wt % PEG-Tz, 4.75 mM norb-KGPQGIWGQKK-norb, and 1 mM
norb-GGKGGC. After gelation, the gels were immersed in a solution
of 0.1 mg/mL norbornene-functionalized fluorescein−bovine serum
albumin (norb/FL-BSA; see SI for functionalization protocol) and 2.2
mM 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1- propa-
none (trade name I2959; Ciba) photoinitiator in PBS, incubated at
room temperature on an orbital shaker for 2 h, and then irradiated
with collimated UV light (365 nm, 10 mW/cm2, Omnicure lamp)
through a chrome on a quartz photomask (100 μm lines with 100 μm
spacing) for 10 min. The patterned gel was transferred to fresh PBS,
incubated for 1 h at room temperature on an orbital shaker, and the
patterning process was repeated with norbornene-functionalized
tetrametheylrhodamine-BSA (norb/TAMRA-BSA). In the second
patterning step, the chrome photomask was rotated approximately
90° to generate a grid pattern. Single and dual protein patterned
hydrogels were imaged at 10X magnification through a water
immersion lense on a Ziess widefield fluorescence microscope.
Quantitative measurements of the patterned lines were made to
confirm pattern fidelity.
(i) 1.1 equiv Et3N, CH3CN, reflux, 15 h. (ii) 18 equiv NH2−NH2, 4
equiv HNCHNH2·CH3COOH, 1 equiv S, room temp, 20 h. (iii)
CH3COOH, 5 equiv NaNO2, 0 °C, ∼ 1 h.
summary of synthetic route) to a multifunctional PEG-NH2 (four-arm,
Mn ≈ 20 000 Da; JenKem Technologies USA) using standard acid-
amine conjugation techniques. The PEG-Tz was estimated to be 75%
functionalized, which should lead to a statistical distribution of
polymer functionalization with an average functionality of 3. A
dinorbornene cell degradable cross-linker peptide norb-
KGPQGIWGQKK-norb and mononorbornene pendant peptides
norb-AhxRGDS and norb-GGKGGC were synthesized using standard
Fmoc solid phase peptide synthesis protocols and purified by reverse
B
dx.doi.org/10.1021/bm4000508 | Biomacromolecules XXXX, XXX, XXX−XXX