Temporal regulation of hydrogel stiffness through orthogonal enzymatic reactions
Matthew R. Arkenberg, John C. Bragg, and Chien-Chi Lin
Department of Biomedical Engineering, Purdue School of Engineering and Technology,
Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA
Statement of Purpose: Hydrogels with temporally
controlled mechanical and biochemical properties are
highly useful for studying important cell fate processes. In
particular, two-step crosslinking strategies have been
employed to ‘stiffen’ cell-laden hydrogels[1-3]. Often,
these stiffening strategies involve UV-light and radical
induced polymerization, which may not be ideal for cellbased applications[1,4]. Due to their orthogonality,
efficiency, and predictability, enzymatic reactions have
been increasingly utilized to crosslink hydrogels[4]. Here,
we aimed at exploiting orthogonal enzymatic reactions,
namely Sortase A (SrtA) and mushroom tyrosinase (MT),
to synthesize and subsequently tune the mechanical
properties of the fabricated poly(ethylene glycol) (PEG)peptide hydrogels (Figure 1A). SrtA is a transpeptidase
that covalently ligates an oligoglycine peptide (Gn) to a
short peptide LPXTG, where X is any amino acid[5]. On
the other hand, MT oxidizes tyrosine residues into dityrosine[3]. Due to the orthogonality of the two enzymatic
reactions and the simplicity of the peptide substrates, we
designed PEG-peptide hydrogels that can be crosslinked
by SrtA-mediated transpeptidation and subsequently
stiffened through MT-mediated di-tyrosine formation.
Methods: Macromer 8-arm PEG-norbornene (PEG8NB)
and
photoinitiator
lithium
phenyl-2,4,6trimethylbenzoylphosphinate (LAP) were synthesized per
established protocols[6,7]. All peptides (i.e, GGGGYC,
CYLPRTG) were synthesized by microwave-assisted
solid phase peptide synthesis. PEG-peptide conjugates
were prepared through reacting purified peptides with
PEG8NB via thiol-norbornene photoclick chemistry in the
presence of LAP (5 mM) and light (365 nm, 40 mW/cm2).
Hepta-mutant his-tagged Srt A[8] was expressed in BL21
(DE3) E. Coli. Pellets were lysed and purified by Ni+
Column chromatography. All hydrogels were fabricated
with SrtA and a 1:1 stoichiometric ratio of 8-arm PEGLPRTG and GGGGY-PEG. Gelation kinetics at varying
SrtA concentrations were monitored via in situ oscillatory
rheometry (Bohlin CVO 100). SrtA-crosslinked gels were
incubated in PBS for 24 hours before measuring elastic
modulus (G’). Temporal control of the hydrogel
mechanical properties was achieved through incubating
the gels with MT (1 kU/mL) for 6 hours at 37oC.
Results: The gelation kinetics of PEG-peptide hydrogels
was investigated by in situ rheometry (Figure 1B) using
3wt% PEG-peptide conjugates and varying concentrations
of SrtA (i.e., 150 µM, 300 µM, 600 µM). The gelation
was accelerated at higher SrtA concentrations as indicated
by the rapid increases in G’ (Figure 1B). The effect of
PEG-peptide concentration on the initial elastic modulus
was also investigated (Figure 1C). Formulations
containing 2, 3, and 4 wt% total PEG concentration
yielded gels with elastic moduli of ~800, ~1,200, and
~1,900 Pa, respectively. We also explored the
susceptibility of the SrtA-crosslinked PEG-peptide
hydrogels to MT-mediated secondary crosslinking and
stiffening (Figure 1D). Hydrogels were fabricated
utilizing 3 wt% of PEG-LPRTG and GGGGY-PEG,
followed by stiffening in MT solution for 6 hours. As
shown in Figure 1D, the addition of MT to the gels
increased the gel modulus roughly two-fold,
demonstrating the feasibility of using orthogonal
enzymatic reactions for gel crosslinking and in situ
stiffening.
Figure 1. (A) Schematic of primary SrtA crosslinking and
secondary MT stiffening of PEG-peptide hydrogels. (B)
In situ rheometry of PEG-peptide hydrogels crosslinked
with varying concentrations of SrtA. (C) Effect of PEGpeptide concentration on the elastic modulus of the
hydrogels. (D) MT-mediated in situ stiffening of PEGpeptide hydrogels. (* p < 0.05. Mean ± SEM, n = 3.)
Conclusions: In summary, we have established a purely
enzymatic gelation scheme with tunable initial
crosslinking and temporal regulation of gel stiffness. The
use of sequential enzymatic reactions, namely SrtA and
MT, allows for dynamic tuning of primary and secondary
crosslinking without the necessity of co-initiators and
UV-light. In addition, SrtA and MT reactions yield no
cytotoxic
by-products,
thereby
making
them
advantageous, cytocompatible systems for studying cell
processes. Future work will focus on utilizing this
gelation and stiffening scheme to evaluate the role of
dynamic matrix stiffening on regulating stem cell fate
processes.
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