Kinetically Controlled Lifetimes in Redox-Responsive Transient Supramolecular Hydrogels

Article abstract of DOI:10.1021/jacs.7b12198

It remains challenging to program soft materials to show dynamic, tunable time-dependent properties. In this work, we report a strategy to design transient supramolecular hydrogels based on kinetic control of competing reactions. Specifically, the pH-triggered self-assembly of a redox-active supramolecular gelator, N,N′-dibenzoyl-l-cystine (DBC) in the presence of a reducing agent, which acts to disassemble the system. The lifetimes of the transient hydrogels can be tuned simply by pH or reducing agent concentration. We find through kinetic analysis that gel formation hinders the ability of the reducing agent and enables longer transient hydrogel lifetimes than would be predicted. The transient hydrogels undergo clean cycles, with no kinetically trapped aggregates observed. As a result, multiple transient hydrogel cycles are demonstrated and can be predicted. This work contributes to our understanding of designing transient assemblies with tunable temporal control.

Full text of DOI:10.1021/jacs.7b12198

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Article
Kinetically Controlled Lifetimes in Redox-
responsive Transient Supramolecular Hydrogels
Jonathan Wojciechowski, Adam D. Martin, and Pall Thordarson
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12198 • Publication Date (Web): 06 Feb 2018
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Kinetically Controlled Lifetimes in Redox-responsive Transient Supra-
molecular Hydrogels
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Jonathan P. Wojciechowski, Adam D. Martin, Pall Thordarson*
School of Chemistry, the Australian Centre for Nanomedicine and The ARC Centre of Excellence in Convergent Bio-
Nano Science and Technology, the University of New South Wales, Sydney, NSW 2052, Australia
Temporal control, transient assemblies, programmable materials, hydrogels, supramolecular chemistry
ABSTRACT: It remains challenging to program soft materials to show dynamic, tunable time-dependent properties. In this
work, we report a strategy to design transient supramolecular hydrogels based on kinetic control of competing reactions.
Specifically, the pH triggered self-assembly of a redox-active supramolecular gelator, N,N’-dibenzoyl-L-cystine (DBC) in the
presence of a reducing agent, which acts to disassemble the system. The lifetimes of the transient hydrogels can be tuned
simply by pH or reducing agent concentration. We find through kinetic analysis that gel formation hinders the ability of the
reducing agent and enables longer transient hydrogel lifetimes than would be predicted. The transient hydrogels undergo
clean cycles, with no kinetically trapped aggregates observed. As a result, multiple transient hydrogel cycles are demon-
strated and can be predicted. This work contributes to our understanding of designing transient assemblies with tunable
temporal
control.
We proposed using supramolecular gelators, as the rate of
gelation can proceed rapidly (i.e., within seconds) upon
addition of a suitable trigger. In addition, we found reduc-
tion of the disulfide bond in N,N’-dibenzoyl-L-cystine
(DBC), a well-known supramolecular gelator,27 diminished
its aggregation ability. Shown schematically in Figure 1, we
designed a transient hydrogel based on the competitive
self-assembly of DBC in the presence of a disulfide reduc-
ing agent, tris(2-carboxylethyl)phosphine (TCEP).
Introduction
The self-assembly of low-molecular-weight-gelators
(LMWGs) has proven to be an effective strategy to design
highly functional materials based on chemically well-
defined small molecules.1–4 LMWGs have shown excellent
potential in a wide variety of applications such as three-
dimensional cell culture,5 light harvesting,6,7 logic gates,8
and inhibiting cancer cells.9,10 Whilst these materials show
exceptional stimuli responsiveness, they lack the complexi-
ty of natural systems, which show dynamic properties in
space and time.11–13 Key to achieving spatiotemporal con-
trol in these systems is a firm understanding of the self-
assembly process and the kinetics of gelation.14 Exception-
al spatial control has enabled self-sorting of multi-
component LMWGs based on their pKa values,15 which en-
ables hydrogel networks to be selectively removed16 or
asssembled.17 In contrast, strategies to tune the time-
dependent properties of these materials remain limited.
Conceptually, designing materials with temporal control
is a balancing act between the activation and deactivation
kinetics of the system.12 Strategies to achieve autonomous
temporal control include the use of chemically fuelled as-
semblies,18–21 competing enzyme reactions,22,23 delayed
enzymatic pH cycles24,25 and enzyme coupled programmed
reactions within a polymer.26 Of these systems, however,
examples which do not utilise enzymes are less common.
As such, there is a growing interest in the design of transi-
ent assemblies controlled by chemical reactions, and under
relatively biologically relevant benign conditions.20
Figure 1. Schematic of the sol→gel→sol cycle of the transient
hydrogels. Mixing anionic DBC2- with TCEP in citric acid buff-
er yields hydrogels, triggered from the pH dependent self-
assembly of DBC. The hydrogel formed is transient because
TCEP slowly reduces DBC to N-benzoyl-L-cystine (BC), result-
ing in dissolution of the hydrogel (sol→gel→sol). The lifetimes
of the hydrogels can be chemically controlled by pH or initial
TCEP concentration.
To address this, we envisioned the design of a transient
hydrogel system based upon competing chemical reactions
with orthogonal methods of activation and deactivation.
In this study, we show that we can create transient hy-
drogels with autonomous sol→gel→sol transitions based
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on kinetic control of competing chemical reactions (Figure self-assembly of DBC occurs at pH values below the pKa,
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1). This strategy can be used to tune the lifetimes of the
transient hydrogels based on pH or the concentration of
TCEP. We show through kinetic experiments an under-
standing of the mechanism of the sol→gel→sol cycle of the
transient hydrogels. In addition, we show this system can
achieve multiple transient hydrogel cycles.
and is a well-known mechanism for supramolecular gela-
tors.15 To our knowledge, the pKa value for DBC has not
been reported, only estimated,19 hence we determined the
pKa of DBC by pH titration and found it to be 3.58 (Figure
S1). A pH switch method was used to trigger gelation by
mixing 1:1 (v/v) anionic DBC2- with 100 mM citric ac-
id/trisodium citrate buffer. To make transient hydrogels, a
solution of TCEP in 100 mM citric acid/trisodium citrate
buffer was used. This method of pH switch was chosen
over commonly used lactone hydrolysis34 due to faster
gelation rates of DBC, whilst still yielding consistent hy-
drogels unlike HCl-based methods.
Results and discussion
Transient hydrogel design
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A conceptual framework for designing systems that ex-
hibit autonomous time dependent properties is such that
the rate of activation, νact is much greater than the rate of
The rate of gelation of DBC (k2) is dependent on pH, with
a pH closer to the pKa resulting in a slower gelation rate
(Table S1). As a result of the competing reactions in the
transient hydrogels from the introduction of TCEP, a
boundary condition pH exists that is dictated by the rate of
gelation. Gelation must be significantly faster than the re-
duction rate of TCEP to form transient hydrogels. To de-
termine this pH value, pH dependent time-resolved rheol-
ogy was conducted at pH values below the pKa on 5 mM
DBC hydrogels with an excess of TCEP = 10 mM (Figure
2a). The ratio of citric acid:trisodium citrate was altered to
change the pH of the hydrogels (Figure S2). To measure
the rheological properties of these transient hydrogels, a
constant frequency of 1 Hz and strain of 0.1% was used,
which shows no frequency dependent behavior and lies
within the linear viscoelastic region for these hydrogels
(Figure S3). The lifetime of the transient hydrogels is de-
fined by the time they are no longer self-supporting by the
vial inversion test (G’ ≈ 50 Pa).
12,13
deactivation, νdeact
.
This allows the activated self-
assembled structures to temporarily exist before they are
deactivated by a competing reaction. Redox-active supra-
molecular gelators are ideal for this because they biologi-
cally relevant and impart orthogonal stimuli responsive-
ness. For example the reduction of disulphides28,29 and
oxidation of thiols30 have both been used to trigger gela-
tion. Moreover, the redox activity of disulphides is com-
monly utilised to cross-link polymeric hydrogels.31 Due to
the orthogonal activation and deactivation kinetics, we
reasoned that we could control the kinetics of the compet-
ing reactions to tune the lifetimes of the self-assembled
LMWG hydrogels.
The kinetics of the transient DBC hydrogels can be de-
fined by the equilibrium constants shown in Figure 1. Gela-
tion is activated when anionic DBC2- is protonated to DBC
(Figure 1). Charge screening of the carboxylate initiates
rapid self-assembly of DBC into a supramolecular hydrogel
driven by non-covalent interactions, namely hydrophobic,
π-π and van der Waal interactions.27 The self-assembly of
DBC competes in the presence of a reducing agent (TCEP)
that reduces the disulfide bond, to give N-benzoyl-L-
cysteine BC, resulting in dissolution of the hydrogel (Fig-
ure 1).
A phosphine based reducing agent (TCEP) was chosen
due to the acidic pH used to trigger the hydrogels (pH 3.00
and below). Other common water-soluble reducing agents
such as dithiothreitol (DTT), 2-mercaptoethanol and dithi-
olbutylamine (DTBA) require higher pH (5.00 and above)
since reduction occurs through thiol-disulfide interchange
by the reducing agent thiolate, with typical pKa values = 8.2
– 9.6.32 In contrast, TCEP proceeds rapidly at low pH due to
the pH independence of the phosphine nucleophile.33
Protonation of DBC (k1) is not rate determining, hence
the lifetimes of the transient hydrogels can be controlled
by understanding the factors which affect the rate of gela-
tion (k2) and the rate of reduction (k3). In addition, the
backwards oxidation reaction (k-3) is negligible since the
systems operates at acidic pH (pH < 3.00). Based on the
unique dynamics of this system, we aimed to fully investi-
gate the kinetics of the transient hydrogels as a function of
pH and initial concentration of TCEP, which allows the
hydrogel lifetimes to be tuned.
Figure 2. a) pH dependent rheology shows an increase in
hydrogel lifetimes and storage modulus (G’) with a decrease
in pH. b) HPLC kinetics of DBC reduction in the hydrogel
shows the rate of reduction of DBC is slower as the pH de-
creases. For both experiments: [DBC]0 = 5 mM, [TCEP]0 = 10
mM. Error bars on uncertainty are the 95% confidence inter-
val (nlparci function in matlab).
The pH dependent rheology of the transient hydrogels
shows a boundary condition pH lower than the pKa of DBC.
Hydrogels triggered at pH = 3.12 only form viscous solu-
tions, as shown by a small increase in G’ below 5 Pa, but
are not self-supporting as demonstrated by the vial inver-
pH dependence on transient hydrogel lifetimes
In aqueous solvents, protonation of DBC2- to DBC (k1) in-
itiates the self-assembly of DBC into a hydrogel (k2). The
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sion test. In contrast, transient hydrogels are formed at pH on the pH dependent results, a 100 mM 85/15 (v/v) citric
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values of 2.98 and below, which indicates the rate of gela-
tion is faster than the rate of reduction of DBC. Unexpect-
edly, the lifetimes of the transient hydrogels increase as
the pH decreases. Transient hydrogels formed at pH = 2.98
have lifetimes of 7 minutes compared with hydrogels
formed at pH = 2.70 which have significantly longer life-
times of 70 minutes. The reduction mechanism of TCEP is
pH independent,33 and hence the increase in lifetimes must
be attributed to the formation of hydrogels. This indicates
once DBC forms supramolecular assemblies, the rate of
reduction by TCEP is reduced. Indeed, the formation of
supramolecular assemblies have shown to reduce reaction
kinetics in a related dissipative supramolecular hydrogel
system.20
acid/trisodium citrate buffer was chosen due to sufficient
rates of gelation, yet also allow for sufficient mixing times
(ca., 20 s). The final pH of the transient hydrogels was in
the range of pH = 2.70 - 3.00.
We performed time-resolved rheology on 5 mM DBC
transient hydrogels with varying concentrations of TCEP
(5-20 mM). As shown in Figure 3a, the storage modulus
(G’) of the DBC hydrogels decrease faster in the presence
of higher concentrations of TCEP, and show reduced hy-
drogel lifetimes. All hydrogels reach a maximum storage
modulus of 600-700 Pa, before decreasing with time. As
expected, higher equivalents of TCEP results in a shorter
hydrogel lifetime, due to the faster rate of reduction on
DBC. The lifetimes of the transient hydrogels are as short
as 14 minutes for 5 mM DBC with 20 mM TCEP (4 equiv.)
or as long as 82 minutes when 5 mM TCEP (1 equiv.) is
used, showing a clear dependence on initial concentration
of TCEP. Interestingly, the lifetimes of the transient hydro-
gels with [TCEP]0 = 5 mM are significantly longer than the
higher initial concentrations of TCEP, which show an al-
most linear relationship. To understand the process be-
hind this increase in the hydrogel lifetimes, HPLC kinetics
were performed on the hydrogels to investigate the de-
pendence on [TCEP]0. A constant concentration of DBC = 5
mM was used, with the concentration of TCEP varied be-
tween 5-20 mM.
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We sought to measure the kinetics of DBC reduction (k3
– we assume k-3 = 0) in a hydrogel and developed a HPLC
method to achieve this (See SI for details). In brief, transi-
ent hydrogels are formed, dissolved in 14-fold volume-
excess of methanol at various kinetic time points and in-
jected into the HPLC. The concentrations of DBC were de-
termined using a HPLC calibration curve of the peak areas
(Figure S4). The concentration of DBC with respect to time
were fit to pseudo-first order kinetic equations to deter-
mine the reaction rate constant, k. The HPLC kinetic data
on the transient hydrogels shown in Figure 2b shows
slower reduction kinetics of DBC as the pH is decreased
and agrees with the rheology data. The reaction rate con-
stant at pH 3.12, where a gel is not formed, is 2.06 ± 0.12 x
10-3 s-1 and is almost five times larger than the reaction
rate constant at pH 2.70, k = 0.443 ± 0.016 x 10-3 s-1, which
shows the longest hydrogel lifetimes.
Figure 3b shows the concentration of DBC in a hydrogel
at various time points with increasing [TCEP]0. The HPLC
data was fit to a pseudo-first order kinetic equation with
respect to DBC degradation, which is found for TCEP in
solution.33
Taken together, the pH dependent rheology and HPLC
kinetics of the hydrogels show that upon assembly of DBC
into a transient hydrogel, the kinetics of DBC reduction is
reduced, and therefore the lifetimes of the hydrogels in-
crease. These results indicate the mechanism of transient
hydrogel reduction likely occurs through dissociation of
DBC from the transient hydrogel, which is then rapidly
reduced by TCEP to BC. Once DBC is reduced, it is unable
to exchange with the supramolecular fibres and hence the
hydrogel is slowly dissolved. The dissociation of gelator
monomers from self-assembled fibres is a well-known
mechanism and has been observed in related one-
dimensional fibres.35
This is an interesting property, since control of the dis-
sociation constant of the hydrogel (k-2) affects the lifetime
of the supramolecular assemblies. In summary, there is a
clear pH dependence on the lifetimes of the transient hy-
drogels. Above pH ≈ 3.00, transient hydrogels will not form
because the rate of gelation does not outcompete the rate
of reduction. Below pH ≈ 3.00, transient hydrogels can be
formed due to a higher rate of gelation. These results show
the lifetimes of the transient hydrogels can be tuned by pH.
Figure 3. a) Time-resolved rheology of 5 mM DBC hydrogels
shows a decrease in the hydrogel lifetimes with increasing
concentrations of TCEP. b) Kinetic analysis of the rate of re-
duction of 5 mM DBC transient hydrogels by TCEP fits to a
pseudo-first order kinetic equation. Error bars on uncertainty
are the 95% confidence interval (nlparci function in matlab).
The effect of TCEP on transient hydrogel lifetimes
Next, we sought to investigate the rate of reduction in
the transient hydrogels with increasing concentrations of
TCEP. We expected higher concentrations of TCEP would
increase the rate of DBC reduction and allow another pa-
rameter to control the transient hydrogel lifetimes. Based
The plot of k3 versus initial concentration of TCEP shows
a linear relationship, which is not seen in the hydrogel life-
times by rheology. Based on the previous results showing
the pH dependence of k3, it is known that formation of the
hydrogel hinders reduction of DBC. Therefore, the reduc-
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tion of DBC to BC likely occurs when it is dissociated from some viscoelastic properties at two hours, however, do not
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the hydrogel. In the case of the pH dependent results, an
excess of TCEP (2 equiv.) was used to study these kinetics,
and it can be assumed once DBC is dissociated, it would
likely be reduced to BC. In the case of [TCEP]0 = 5 mM, the
ratio is stoichiometric, and it is possible the rate of DBC
reduction is slower than association of DBC to the hydro-
gel. This suggests that DBC is not being constantly reduced
when dissociated, which would increase the lifetimes of
the hydrogel. This is further exemplified when the pH of
the hydrogel is decreased, giving hydrogel lifetimes of 6.7
hours in the case of a 95/5 citric acid/trisodium citrate
buffer (Figure S5). This result further supports the hy-
pothesis that once DBC self-assembles into a hydrogel, the
rate of reduction by TCEP is limited by the dissociation of
DBC. As a result, the dissociation constant of the hydrogel
(k-2) is potentially rate limiting the reduction of DBC to BC.
form self-supporting hydrogels. Thus, the time-resolved
AFM measurements suggests the transient hydrogel net-
work initially starts as a population of large, cross-linked
fibers which over time lessen in both fiber size and fiber
length.
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Morphology and secondary structure changes in the transi-
ent hydrogel cycle
Clearly, the lifetime of the hydrogels can be tuned by
changing the pH or initial concentration of TCEP. We were
interested to visualize the self-assembled structures which
form in the cycle, since in some cases kinetically trapped
Figure 4. a-d) Time-resolved atomic force microscopy of a 5
mM DBC transient hydrogel with 5 mM TCEP; a) 10 min, b) 30
min, c) 1 h (60 min) and d) 2 h (120 min). e) Time-resolved
FTIR on a 5 mM DBC transient hydrogel with 5 mM TCEP in
D2O shows a decrease in the amide C=O stretch at 1627 cm-1
with respect to time. Scale bars represent 1 ꢀm.
products
are
formed
upon
assembly
and
disassembly.14,16,22 These kinetically trapped products are
undesirable, reducing the efficiency of multiple transient
cycles. We firstly investigated the nanostructures using
atomic force microscopy of the pre-gel solutions (DBC2-),
hydrogels (DBC) and reduced gel solutions (BC) (Figure
S6).
As AFM had revealed a change in the density and length
of the fibrous network for the transient hydrogels over
time, we then used FTIR to probe whether a change in sec-
ondary structure was also observed. The DBC hydrogels
display a peak at 1627 cm-1, which represents the C=O
stretching vibrations of the amide bond (Figure S9). This
stretching frequency is not observed in the anionic DBC2-
solution, BC solution or TCEP solution, and hence can be
used to monitor the disassembly of the transient hydrogels
(Figure S9).
Initially, no structures are observed in the anionic DBC2-
solution, suggesting monomeric solvation of the solution.
Upon acidification of this solution, fibrous networks are
observed by AFM for the DBC hydrogels, which is con-
sistent with previously reported network structures for
these hydrogels.27 Upon reduction to BC, these fibrous
structures are no longer detected by AFM. In addition,
small aggregate structures are also not observed which is
assumed to be the result of high solubility of the reduced
BC, and hence it does not form any aggregates.
To follow the kinetics of transient hydrogel disassembly,
5 mM DBC hydrogels with 5 mM TCEP were used (Figure
4e). Initially, the transient hydrogels show the characteris-
tic C=O stretching frequency at 1627 cm-1 (10 minutes)
found in the DBC hydrogels. This stretching frequency de-
cays over four hours as the assemblies are reduced by
TCEP. No increase in other peaks in the Amide I region
(1700 – 1600 cm-1) are observed. This result suggests the
transient hydrogel assemblies do not undergo any changes
to their secondary structure upon dissolution.
The monomeric solvation of DBC2- and BC is supported
by 1H NMR and viscosity measurements which shows
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sharp, resolved peaks in H NMR (Figure S7) and that vis-
cosity is independent of shear rate (Figure S8). Combined,
these results show that the DBC hydrogels do not form
kinetically trapped aggregates upon reduction, demon-
strating the clean assembly and disassembly of the transi-
ent hydrogels.
To gain insights into the morphology changes during
transient hydrogel disassembly we conducted a time-
resolved AFM study on a 5 mM DBC transient hydrogel
with 5 mM TCEP (Figure 4a-d). Initially, 10 and 30 minutes
after hydrogel formation (Figure 4a-b), the transient hy-
drogels show a thick, fibrous network similar to the DBC
hydrogels in Figure S6b. One hour after hydrogel for-
mation (Figure 4c), the fibrous network is noticeably less
dense compared with the 10 and 30 minutes images. After
two hours (Figure 4d), only short fibers of randomly dis-
tributed lengths are observed. This agrees with the rheolo-
gy data (Figure 3a) which shows the hydrogels display
Multiple pre-programmed transient hydrogel cycles.
A significant challenge in transient assemblies which
show temporal control is the ability to complete multiple
reaction cycles without degradation of the system compo-
nents.23,26,36 Encouraged by the clean assembly and disas-
sembly of the hydrogels, we sought to benchmark this ap-
proach to achieve multiple transient hydrogel cycles (i.e.,
sol→gel→sol after addition of a trigger). Multiple cycles of
transient assemblies are particularly interesting because
they may be used in continuous stirred tank reactors or
microfluidics to achieve out-of-equilibrium steady
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states.37,38 In addition, they could be applied in soft robot-
ics or used in microfluidics as temporary fluid guides.
TCEP has the capacity for three, 5 mM DBC transient hy-
drogel cycles.
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To achieve multiple transient hydrogel cycles, we used
an excess initial concentration of TCEP (Figure 5a). TCEP
reacts stoichiometrically with DBC, hence we can pre-
program the number of transient hydrogel cycles. Addition
of DBC2- to the solution with TCEP causes formation of a
transient hydrogel which is then autonomously converted
over time to a solution (one cycle). Consequent addition of
DBC2- to this solution enables another transient hydrogel
cycle. The transient cycles can be repeated as long as the
concentration of TCEP is sufficient to reduce the DBC2-
added. We used UV-vis scattering at 350 nm to monitor the
transient cycles since only the DBC hydrogels absorb at
this wavelength (Figure S10). A typical scattering profile of
the transient hydrogel cycle is shown in Figure 5b.
Three transient hydrogel cycles can be seen in Figure 5d,
monitored using UV-vis. The increase in scattering upon
addition of DBC2- is due to transient hydrogel formation
(Figure S11) The lifetimes of the hydrogels increase as
TCEP is consumed, since higher equivalents of TCEP result
in faster dissolution of the hydrogel which is shown by
HPLC and rheology data. However, on the fourth cycle,
there is not enough TCEP to reduce DBC and deactivate the
hydrogel, hence the hydrogels do not dissolute. There is a
small decrease in scattering as the remaining TCEP is con-
sumed. The resulting hydrogel after the fourth cycle is ki-
netically stable, and remains self-supporting after 24 hours
(Figure S11).
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Conclusions
We have outlined a general approach to enable transient
lifetimes in redox-active materials based on orthogonal
reaction kinetics. In this work, we show competing kinetics
of supramolecular gelation (activation) and disulfide re-
duction (deactivation) can be kinetically controlled to cre-
ate transient hydrogels with show autonomous temporal
control (i.e., gel-sol reversibility without input). We study
the mechanism of this system using rheology and HPLC
and show the deactivation of the transient assemblies oc-
curs through a dissociative pathway. Moreover, this study
highlights the delicate balance between solubility and self-
assembly. We show this approach can be used to achieve
multiple transient hydrogel cycles, which can be predicted
based on the concentrations of starting materials.
The insights gained from the kinetic analysis on this ap-
proach contributes to our understanding of spatiotemporal
control in self-assembled structures. The general concept
of competing chemical reactions outlined in this work is
applicable not only to redox-active systems, but also stimu-
li responsive systems. We envision this concept will con-
tribute to the strategies used in the next generation of spa-
tiotemporal hydrogel material39 and could potentially be
used to create out-of-equilibrium systems using microflu-
idics or continuously stirred tank reactors.37
Figure 5. a) Schematic of the transient hydrogel cycles. The
number of cycles can be programmed based on the starting
concentration of TCEP. b) Typical time-resolved UV-vis scat-
tering of the transient hydrogel cycle. c) Amount of TCEP re-
maining, DBC2- added and volume change after each cycle for
[TCEP]0 = 50 mM. d) UV-vis scattering showing three transi-
ent hydrogel cycles with a starting concentration of TCEP = 50
mM.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures, details of kinetic analysis, HPLC
method for transient hydrogels, pH titration, rheological char-
acterization, NMR characterization, AFM images, FTIR spectra,
viscosity and UV-Vis characterization are available free of
charge on the ACS Publications website as a PDF.
Initially, the solution of TCEP exhibits no scattering at
350 nm. Transient hydrogels are triggered upon the addi-
tion of DBC2-, resulting in a rapid increase in the scattering
which can be attributed to the formation of a hydrogel. The
scattering then slowly decreases over time as DBC is re-
duced to BC, which does not absorb at 350 nm. On each
cycle, the transient hydrogel concentration is always 5 mM.
Due to the volume increases on each cycle, the concentra-
tion of DBC2- added increases on each cycle to maintain a
hydrogel concentration = 5 mM and is shown in Figure 5c.
Based on stoichiometry, an initial concentration of 50 mM
AUTHOR INFORMATION
Corresponding Author
*p.thordarson@unsw.edu.au
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
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4825–4828.
We would like to thank the Mark Wainwright Analytical Cen-
tre (UNSW) for access to instruments. We acknowledge the
Australian Research Council for an ARC Centre of Excellence
grant (CE140100036) to P.T and National Health and Medical
Research Council for a Dementia Development Research Fel-
lowship (APP1106751) to ADM. We would also like to thank
the Australian Government for an APA to J.P.W.
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Figure 1. Schematic of the sol→gel→sol cycle of the transient hydrogels. Mixing anionic DBC2- with TCEP in
citric acid bufer yields hydrogels, triggered from the pH dependent self-assembly of DBC. The hydrogel
formed is transient because TCEP slowly reduces DBC to N-benzoyl-L-cystine (BC), resulting in dissolution of
the hydrogel (sol→gel→sol). The life-times of the hydrogels can be chemically controlled by pH or initial
TCEP concentration.
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Figure 2. a) pH dependent rheology shows an increase in hydrogel lifetimes and storage modulus (G’) with
a decrease in pH. b) HPLC kinetics of DBC reduction in the hydrogel shows the rate of reduction of DBC is
slower as the pH decreases. For both experiments: [DBC]0 = 5 mM, [TCEP]0 = 10 mM. Error bars on
uncertainty are the 95% confidence interval (nlparci function in matlab).
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Figure 3. a) Time-resolved rheology of 5 mM DBC hydrogels shows a decrease in the hydrogel lifetimes
with increasing concentrations of TCEP. b) Kinetic analysis of the rate of reduction of 5 mM DBC transient
hydrogels by TCEP fits to a pseudo-first order kinetic equation. Error bars on uncertainty are the 95%
confidence interval (nlparci function in matlab).
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Figure 4. a-d) Time-resolved atomic force microscopy of a 5 mM DBC transient hydrogel with 5 mM TCEP;
a) 10 min, b) 30 min, c) 1 h (60 min) and d) 2 h (120 min). e) Time-resolved FTIR on a 5 mM DBC
transient hydrogel with 5 mM TCEP in D2O shows a decrease in the amide C=O stretch at 1627 cm-1 with
respect to time. Scale bars represent 1 µm.
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Figure 5. a) Schematic of the transient hydrogel cycles. The number of cycles can be programmed based on
the starting concentration of TCEP. b) Typical time-resolved UV-vis scattering of the transient hydrogel
cycle. c) Amount of TCEP remaining, DBC2- added and volume change after each cycle for [TCEP]0 = 50 mM.
d) UV-vis scattering showing three transient hydrogel cycles with a starting concentration of TCEP = 50 mM.
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