Maintaining homogeneity during a sol-gel transition by an autocatalytic enzyme reaction

Article abstract of DOI:10.1039/c8cc08501c

Kinetic control over supramolecular gelation by increasing the pH can be achieved using an enzymatic reaction. This method allows us to produce homogeneous hydrogels with superior and improved mechanical properties as compared to gels obtained from simple addition of base.

Full text of DOI:10.1039/c8cc08501c

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Maintaining homogeneity during a sol–gel
transition by an autocatalytic enzyme reaction†
Cite this: Chem. Commun., 2019,
5, 47
5
Santanu Panja
and Dave J. Adams
*
Received 24th October 2018,
Accepted 23rd November 2018
DOI: 10.1039/c8cc08501c
rsc.li/chemcomm
1
5
Kinetic control over supramolecular gelation by increasing the pH this is a real issue. Related work showed that the gel properties
1
7
can be achieved using an enzymatic reaction. This method allows us depended heavily on the mixing rates. To improve on this,
to produce homogeneous hydrogels with superior and improved methods that allow a more homogeneous pH decrease were
mechanical properties as compared to gels obtained from simple developed, which lead to significantly more homogeneous
1
5,18
addition of base.
gels.
Similarly, Thornton et al. showed that a slow enzy-
matic trigger at a fixed pH resulted in improved properties over
1
9
Hydrogels derived from the self-assembly of small organic molecules a fast pH change for a Fmoc-amino acid gelator.
have potential in many areas including structuring, sensors, opto-
electronics, catalysis, and cell culture. These gels are formed by gelation, there are more limited options. Beyond the simple
In comparison, for gels where an increase in pH triggers
1–7
non-covalent interactions such as hydrogen bonding, p–p stacking, addition of aliquots of strong base, Stupp’s group have used the
2
0
and van der Waals interactions between the molecules. As the diffusion of gaseous ammonia into a vial. Whilst this is suitable
interactions are individually weak, tuning of the gel properties is for some systems, there are limitations in terms of the volume of
possible by changing the molecular environment, which can be used gel that will be suitable, and there are kinetic issues here since
4–8
to produce a wide variety of materials. However, the final gel gelation will begin from the gas/liquid interface. Similarly, Naka-
properties often depend on the method by which the self- nishi and co-workers introduced a hot aqueous-urea solution to
21
3
aggregation is triggered i.e. upon the kinetics of gelation. In many generate NH in situ to prepare silica aerogels. An alternative
cases, gels which form slowly are more homogeneous, and often approach is to generate ammonia locally by the enzymatic
exhibit superior and improved mechanical properties compared hydrolysis of urea by urease. The urease-catalysed hydrolysis of
6
–10
to the gels obtained under kinetic trapping.
urea produces NH , which in turn results in an increase in the pH
3
22
Gels can be formed by applying a trigger to a solution or of the medium. The rate of the enzymatic reaction depends
suspension of the gelator molecules which result in a significant upon the initial pH of the solution and the nature of the acid.
decrease in their solubility. The most common triggers that are This method has been used to prepare temporally-controlled
used for hydrogelation include temperature, pH, light, the use of polymer gels by Jee et al. but not exploited in low molecular
a co-solvent, and an increase in ionic strength.
2
2
1
,3–5,10–16
The weight gels. Here, we utilise this method to form hydrogels by
choice of gelator determines the trigger that is appropriate. The increasing the pH. We show that the kinetics of gelation affects
desired application will also determine the appropriateness of the microstructure of the gels and the mechanical properties.
5
the trigger. pH-Triggered gels are very common. In many cases,
For the gelators, we investigated a small library of Fmoc-
the pH changes are driven by the addition of a mineral acid or a derivatives. Among the many classes of gelators, Fmoc-derivatives
strong base, resulting in rapid gelation. This can result in gels have gained attention because of their propensity towards hydro-
9
,15,23–28
with irreproducible properties. Since the change in pH results in gel formation.
a decrease in the gelator’s solubility, addition of a strong acid as a protecting group during peptide synthesis. It is extremely
The Fmoc group is of course widely used
2
9
or base means that mixing competes with gelation, meaning stable under acidic conditions, but unstable at higher pH
2
4
that preparing homogeneous gels can be difficult in many cases. (typically 4 pH 10.5). Unsurprisingly, when Fmoc-derivatives
For example, in the case of acid-triggered Fmoc-dipeptide gels, are used as gelators, these tend to form gels at low pH, with the
time at higher pH minimised.
Presumably because of the lack of stability at high pH,
Fmoc-based gelators that form gels at high pH are rare.
E-mail: dave.adams@glasgow.ac.uk
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc08501c Rajbhandary et al. have recently reported hydrogelation of
School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK.
2
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†
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Fmoc-derivatised cationic gelators but gelation was triggered by
27
NaCl. Mandal and co-workers reported gelation of some acid-
28
functionalized Fmoc-derivatives in DMSO/H O at basic pH,
2
although the stability in the assembled state at high pH was only
explored by FTIR and it is known that DMSO can facilitate Fmoc
29
cleavage even under base-free conditions. Whilst there are
potential issues in using Fmoc-derivatives for gels at higher pH,
we will be targeting a pH lower than 10.5. Aggregation is also known
30
to improve the stability of Fmoc-amino acids to deprotection.
Initially for screening, the gelation ability of a small library
Fig. 1) was examined by addition of an aliquot of a solution of
(
sodium hydroxide (1 molar equivalent) to aqueous solutions
of the compounds (Table S1, ESI†). This resulted in sudden
switching of the local pH from acidic (pH 3–5) to basic (pH 9–10.3)
that led to the formation of kinetically trapped self-assembled
systems. Only Fmoc-2 and Fmoc-3 formed gels (Fig. 1). The
resulting gels were turbid and inhomogeneous, presumably due
to the kinetics of gelation being faster than the kinetics of mixing
as described above. Under identical conditions, the other com-
pounds remained either insoluble in water at low pH (Fmoc-HZ) or
Fig. 2 Change in pH with time from the urease–urea reaction in (a) the
absence and (b–d) the presence of Fmoc-2 under different conditions at
2
5 1C. In (b), we compare solutions where the pH has either not been
adjusted, or adjusted with either HCl or AcOH. For (c) and (d), the initial pH
resulted in precipitation upon addition of base (Fmoc-4, Fmoc-5 of the solutions was adjusted to pH 3.9 by using HCl and AcOH, respec-
ꢀ
1
tively. For (a) and (b) concentration of urease is 0.1 mg mL . For (c) and
and Fmoc-6).
ꢀ1
ꢀ1
(
(
d) concentration of urease is 0.1 mg mL (black data) and 0.03 mg mL
In developing a method for triggering gelation at high pH, the
ꢀ1
red data). In all cases, concentration of Fmoc-2 is 2 mg mL , initial
apparent pK
the changed pH in the medium should exceed the apparent pK
the gelator to allow the formation of the corresponding conjugate
a
of the gelator needs to be considered. The value of
concentration of urea is 0.01 M.
a
of
base that undergoes self-aggregation. Hence, the apparent pK of and reached a plateau at pH 9.2–9.3 within a few minutes (Fig. 2a).
a
Fmoc-2 and Fmoc-3 were determined and were found to fall in the However, when the initial pH of the solution was adjusted to pH 3.9
range of 8.4–8.7 (Fig. S1, ESI†). We then used the urease-catalysed using dilute HCl (0.1 M) or AcOH (0.1 M), the rate of increase in pH
3
hydrolysis reaction of urea to produce NH which results in an was significantly lower (Fig. 2a) and produced a sigmoidal curve for
22
increase in the pH of the medium above pH 9 (Fig. 2a). The rate the pH–time profile in both cases. There are slight differences in
of the reaction depends upon the initial pH of the solution and the the profile of pH change depending on the acid used as expected
22
22
acid used to lower the pH. Initially, we used dilute HCl (0.1 M) to from the effect of in situ generated buffer.
adjust the pH of the urease solution. In the absence of gelator, for When adding Fmoc-2 or Fmoc-3, the pH–time profiles were no
solutions initially at pH 5.0 and pH 6.0, the increase in pH upon longer sigmoidal curves. In the presence of Fmoc-2, irrespective
addition of urea was similar to that of the solution of urease only of the nature of the acid used to adjust the initial pH of the
solutions, a slow but steady increase in pH with time was observed
with the propagation of the enzyme-catalyzed reaction (Fig. 2b).
When AcOH was used to adjust the pH, the rate of pH change was
slower than when HCl was used within the pH range 3.9–8.3 but it
was slightly higher from pH 8.3 to pH 9.1. This is probably due to
ꢀ
ꢀ
the higher basicity of the conjugate base AcO compared to Cl ,
which was generated within the reaction medium because of the
ꢀ
neutralization of the AcOH. The AcO ions could play the role of a
supportive base in solution and along with NH
4
3
result in the
acceleration of the pH of the medium slightly faster than the HCl
case. In all cases, we obtained gels that were more transluscent in
comparison to the NaOH-triggered gel (Fig. 1). Interestingly,
Fmoc-3 which formed gels when NaOH was used to change the
pH did not form gels when using the enzymatic reaction. We
Fig. 1 (top) Library of Fmoc-derivatives used here. (bottom) Photographs
of hydrogels of Fmoc-2 formed by (a) NaOH and (b)–(f) urea–urease ascribe this to the fact that the final pH of the medium (pH 8.6)
ꢀ
1
reaction. (b) No acid, [urease] = 0.1 mg mL ; (c) pH 3.9 (HCl, [urease] =
did not exceed the apparent pK of Fmoc-3 (8.6–8.7) even after 24
hours (Fig. S2, ESI†).
To further control the rate of gelation, we decreased the
concentration of urease keeping other parameters unchanged.
A significant decrease in the rate of pH change was recorded
a
ꢀ
1
ꢀ
1
0
.1 mg mL ); (d) pH 3.9 (AcOH, [urease] = 0.1 mg mL ); (e) pH 3.9, (HCl,
ꢀ
1
ꢀ
1
[
urease] = 0.03 mg mL ); (f) pH 3.9 (AcOH, [urease] = 0.03 mg mL ). In all
ꢀ1
cases, final concentration of Fmoc-2 is 2 mg mL , initial urea concentra-
tion is 0.01 M. (g) Photograph of hydrogel of Fmoc-3 (5 mg mL ) formed
ꢀ1
by addition of NaOH.
4
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with time for both the solutions containing either HCl or AcOH
initially (Fig. 2c and d). Here also the pH–time profiles show the
existence of two different rate zones below and above the apparent
pK of Fmoc-2, and the rate of increase of pH with time was slow
a
within the pH range 8.3–9.0. This was more pronounced for the
solution where HCl was used to adjust the initial pH as compared
to AcOH. This indicates the persistence of a new acid–base
equilibrium at pH 8.4, where the gelator becomes the free amine
and allows gelation (Scheme S1, ESI†). These gels showed no
significant change in their visual appearance as compared to the
gels obtained at high urease concentrations (Fig. 1). In the absence
of any acid, turbidity appeared within 1–2 min, and gelation took
Fig. 3 Strain sweep experiments of the enzyme catalyzed hydrogels of
Fmoc-2 under different conditions. Initial pH of the solutions adjusted to
3
.9 by using (a) HCl and (b) AcOH. In all cases, the closed symbols represent
0
00
ꢀ1
G , the open symbols G . Concentration of urease is 0.1 mg mL (black
ꢀ1
ꢀ1
place for around 15 minutes (as confirmed by the inversion of vial data) and 0.03 mg mL (red data). Concentration of Fmoc-2 is 2 mg mL
method). When we decreased the initial pH using HCl or AcOH,
,
initial concentration of urea is 0.01 M.
gelation occurred for 75 and 50 minutes respectively. A decrease in
the concentration of urease further reduced the gelation rate
and extended the overall time until gelation more than 4 times. conditions (Fig. 3 and Fig. S5, ESI†). At high enzyme concen-
Table S2 (ESI†) summarises the pH–time data for Fmoc-2 under trations while using HCl to lower the pH produced gels with
0
different conditions.
higher stiffness (high G ), use of AcOH resulted in substantial
To probe the development of the gels, we carried out rheo- increases in gel strength with higher crossover points, as com-
logical time sweep experiments and the data were correlated with pared to the gel formed without using any acids (Table S3,
pH–time profiles. First, the NaOH-triggered gels were analysed. ESI†). As the urease concentration was decreased, gelation was
0
For Fmoc-2 and Fmoc-3, gelation begins immediately after the slower and there was a significant increase (42 times) in G
0
0
addition of NaOH (pH 4 9.3) and the gel stiffness (as measured and G (Fig. 3). These gels also showed an increased resistance to
by the storage modulus, G ) gradually increases with time (Fig. S3, strain. The gels were essentially frequency independent (Fig. S6,
0
00
0
00
ESI†). However, analysis of tan d(G /G ) (G is the loss modulus) ESI†). Hence, in comparison to the NaOH-triggered gel, hydro-
suggests that no plateau was reached even after 16 hours. In gelation by the urea–urease reaction with low enzyme concen-
comparison, the hydrogelation of Fmoc-2 by the urea-urease tration (irrespective of the acid used) resulted in gels with
reaction showed a different behaviour (Fig. S4, ESI†). In all cases, superior mechanical properties in terms of both gel stiffness and
gelation begins (shown by G 4 G ) at BpH 8.4, i.e. when the pH gel strength (Table S3, ESI†). We also prepared gels of Fmoc-2 by
had reached the apparent pK (8.4–8.5) of Fmoc-2. Noticeably, the adding NaOH and NH OH slowly over 1 hour (Fig. S7, ESI†). These
0
00
a
4
0
00
time required for the initialization of gelation (G 4 G ) depends gels were found to be less stiff and less strong compared to those
upon the initial reaction conditions. As observed, the appearance obtained by the single addition of NaOH. Comparison of the
0
00
of G 4 G was delayed proportionally with the decrease in the rheological data (Table S3, ESI†) emphasises therefore that a slow
0
rate of pH change. With time, both the storage modulus (G ) and and homogeneous pH change is necessary to prepare gels with
00
the loss modulus (G ) increased and the tan d values reached a improved mechanical properties. The data also show that the
plateau after a time span that is comparable with the pH–time viscoelastic properties of the gels can be tuned by using different
profiles (Table S2, ESI†).
triggers for gelation.
The final mechanical properties of the gels were affected by the
The microstructure of the respective gels was characterised
kinetics of hydrogel formation. Gels formed using the different by confocal microscopy imaging. In all cases, fibres are formed
0
00
triggers exhibited significant differences in G and G values (Fig. 3 (Fig. 4 and Fig. S8, ESI†). The fibres in the network are generally
0
and Fig. S5, ESI†). For all gels, the storage modulus (G ) was arranged in spherulitic domains. Interestingly, when the gela-
00
significantly higher than the corresponding loss modulus (G ) as tion rate is high, the gels contained more spherulitic domains,
expected for a gel. At the minimum gelation concentration (mgc), which are less interlinked (Fig. 4a–d). Gels which were formed
0
the NaOH-triggered gel of Fmoc-3 exhibited a higher G than the more slowly exhibit a higher density of long fibres (Fig. 4e and f).
0
gel formed from Fmoc-2. In the strain sweeps for all gels, both G
This difference in the network structure correlates with the lower
00
0
and G were essentially constant at low strain, but deviated from stiffness (G ) of the gel as mentioned above.
linearity after a certain applied stress. Deformation starts (critical
strain) at a slightly lower strain for the NaOH-triggered gels molecular packing. The absorption and emission spectra of
The rate of assembly is likely to affect the quality of the
3
1
(
(
B0.5% strain) compared to the enzyme-triggered hydrogels Fmoc-2 and Fmoc-3 were compared in their respective solution
0.7–0.9% strain). The enzyme-triggered gels could withstand a and gel states. In the case of Fmoc-2, on moving from solution
higher strain with higher crossover points (yield point), where to gel states, the absorption bands at 264 nm and 299 nm were
00
0
G 4 G (4390% strain), than the NaOH-triggered hydrogels slightly red shifted (1–4 nm) (Fig. S9, ESI†). By fluorescence,
o100% strain) (Table S3, ESI†). gelation resulted in around a 10 nm red shift in the monomer
The enzyme-triggered gels of Fmoc-2 exhibited different emission at 327 nm along with appearance of the excimer bands in
rheological properties depending on the exact formation the 400–500 nm region (Fig. S9, ESI†). The excimer peaks can be
(
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for help with rheometers and confocal microscope and Annette
Taylor (Sheffield) for helpful discussions.
Conflicts of interest
There are no conflicts to declare.
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3
1
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29
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0 | Chem. Commun., 2019, 55, 47--50
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