Stabilizing self-assembled Fmoc-F5-Phe hydrogels by co-assembly with PEG-functionalized monomers

Article abstract of DOI:10.1039/c0cc02217a

Hydrogels derived from self-assembled Fmoc-F₅-Phe fibrils are stabilized with respect to shear-response by co-assembly with C-terminal PEG-functionalized Fmoc-F₅-Phe.

Full text of DOI:10.1039/c0cc02217a

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Stabilizing self-assembled Fmoc–F5–Phe hydrogels by co-assembly with
PEG-functionalized monomerswz
Derek M. Ryan, Todd M. Doran and Bradley L. Nilsson*
Received 29th June 2010, Accepted 20th September 2010
DOI: 10.1039/c0cc02217a
Hydrogels derived from self-assembled Fmoc–F5–Phe fibrils are
stabilized with respect to shear-response by co-assembly with
C-terminal PEG-functionalized Fmoc–F5–Phe.
and impart stabilizing and shear responsive behavior to these
small molecule gels. The addition of polyethyleneglycol (PEG)
chains has been shown to increase the solution phase stability
of fibrils in the context of amyloid forming peptides.10–12 We
therefore synthesized Fmoc–F5–Phe–PEG (Fig. 1, see ESIz
for details) in order to investigate the influence of PEG
functionality on the self-assembly and stability of the resulting
fibrillar network.
Hydrogels formed from the dynamic self-assembly of peptides
and amino acids have found use in regenerative medicine, drug
delivery, and material fabrication.1,2 While peptides have been
very effective hydrogelators, small molecule systems are attrac-
tive due to cost and synthetic customizability.1 Fluorenyl-
methoxycarbonyl (Fmoc) protected amino acids and short
peptides in particular self-assemble to form hydrogels similar
in rheological strength to those formed from much longer
peptides and covalently cross-linked polymers.3–6
Hydrogels were prepared by dilution of DMSO solutions
of monomer into water. Fmoc–F5–Phe–PEG (Fig. 1) was
dissolved in DMSO (247 mM) and was then diluted in water
to a final concentration of 4.9 mM (2% DMSO/H2O).8 Upon
dilution into water, an opaque suspension was formed which,
when left undisturbed, became optically clear after 3–4 hours.
We previously found that this transition coincides with
the formation of ordered fibrillar assemblies. This transi-
tion time is much longer for Fmoc–F5–Phe–PEG than for
Fmoc–F5–Phe–OH, which requires only minutes to undergo
assembly and hydrogel formation. Heating the opaque
suspension of Fmoc–F5–Phe–PEG to 100 1C accelerated this
transition from hours to minutes. Upon cooling, a self-
supporting hydrogel formed as indicated by vial inver-
sion. In comparison, heating Fmoc–F5–Phe–OH suspensions
resulted in precipitation of the material from solution.
These results suggest that while incorporation of PEG does
in fact increase the water solubility of the assembled network,
it significantly slows the rate of assembly, possibly due to the
steric influence of the C-terminal PEG functionality. To
confirm that the observed optical transition was in fact due
to the self-assembly of Fmoc–F5–Phe–PEG into fibrils, a
circular dichroism (CD) spectrum was obtained (Fig. 2A).
Fibers derived from Fmoc protected aromatic amino acids
have been shown to have characteristic CD signatures
at 270–310 nm, and 200–210 nm corresponding to the
n–p* Fmoc–Fmoc excitation and p–p* phenyl side chain
excitations, respectively.7 The magnitude of the CD signal of
Fmoc–F5–Phe–PEG was significantly weaker than the corres-
ponding signal of assembled Fmoc–F5–Phe–OH at the same
concentration (Fig. 2A). In addition, the CD signals
were inverted relative to Fmoc–F5–Phe–OH, appearing as
maxima rather than minima. This result could indicate that
We recently reported that halogenation of the phenyl side
chain of Fmoc–Phe resulted in rapid self-assembly and hydro-
gelation relative to Fmoc–Phe or Fmoc–Tyr.7,8 Hydrogels
formed from the self-assembly of Fmoc–F5–Phe–OH or
monohalogenated derivatives were mechanically rigid, but
solvolytically unstable. It was found that the fibrillar network
precipitated after extended resting times (1–2 weeks) or
immediately after the application of stress, resulting in the
loss of gel properties; reformation of the gel network could not
be induced once fibrils were desolvated. This phenomenon was
hypothesized to be due to the low water solubility of
Fmoc–F5–Phe–OH-derived fibrils. In order for hydrogels to
be conveniently used in medicinal and biological applications,
they must exhibit shear responsive behavior, spontaneously
recovering, or healing, following the application of stress.9
Thus, even though Fmoc–F5–Phe–OH rapidly self-assembles
and forms rigid hydrogels, it has limited potential in biological
applications since the gel network would not survive the
process of injection that has been shown to be ideal for
delivery of bioactive hydrogels in vivo.9
Existing self-assembled hydrogel systems that exhibit shear-
responsive behavior are derived from fibrils that are highly
water soluble.9 In these systems, the application of stress
temporarily disrupts the entangled fibril network, yet the
fibrils themselves remain solvated, facilitating reformation of
the hydrogel network when stress is removed.9 Based on these
principles we hypothesized that slightly increasing the water
solubility of Fmoc–F5–Phe–OH would solubilize the fibrils
Department of Chemistry, University of Rochester, Rochester, NY,
14627-0216, USA. E-mail: nilsson@chem.rochester.edu;
Fax: +1 585 276-0205; Tel: +1 585 276-3053
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Materials
and methods, synthesis details, digital images of gel, molecular model
of fibrils, TEM images, UV-vis spectra, and fluorescence emission
spectra. See DOI: 10.1039/c0cc02217a
Fig. 1 Structures of Fmoc–F5–Phe–OH and Fmoc–F5–Phe–PEG.
Chem. Commun., 2011, 47, 475–477 475
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disrupt the gel networks. Immediately following the applica-
tion of 100% strain, a second time sweep was performed
(0.2% strain) in order to monitor the ability of the samples
to recover from the stress (Fig. 2D). It was observed that
Fmoc–F5–Phe–OH only recovered 66% of its maximum
G0 and was no longer a rigid hydrogel.13 More importantly,
post strain samples of Fmoc–F5–Phe–OH gels showed the
presence of abundant fibrillar precipitates (Fig. S4, ESIz). In
contrast, the Fmoc–F5–Phe–PEG gels fully recovered after
cessation of 100% strain, demonstrating that while
Fmoc–F5–Phe–PEG fibrils form weak gels, they do display
shear recovery. In fact gels of Fmoc–F5–Phe–PEG were found
to be fully solvated after more than
4 weeks, while
Fmoc–F5–Phe–OH hydrogels were only stable for 1–2 weeks,
after which precipitation was observed.
Fig. 2 (A) CD spectra of assembled Fmoc–F5–Phe–OH (red) and
Fmoc–F5–Phe–PEG (black). (B) TEM image of fibrils formed from the
assembly of Fmoc–F5–Phe–PEG. (C) Dynamic frequency sweep of the
gels formed from Fmoc–F5–Phe–OH (red) and Fmoc–F5–Phe–PEG
(black). (D) Dynamic time sweep of Fmoc–F5–Phe–OH (red) and
Fmoc–F5–Phe–PEG before and after the application of 100% strain
for 30 seconds.
Based on these observations we hypothesized that co-assembly
of Fmoc–F5–Phe–PEG and Fmoc–F5–Phe–OH would result
in rigid hydrogels that exhibit shear recovery ability ideal
for delivery by injection. It is known that the addition of
complementary impurities to a co-assembled gel network
serves to slow precipitation by introducing structural hetero-
geneity that slows crystal growth.14 It was hypothesized that
the statistical distribution of the Fmoc–F5–Phe–PEG through-
out the fibrils, would solubilize the fibrillar network relative to
Fmoc–F5–Phe–OH alone.
Fmoc–F5–Phe–PEG is merely dissolving in water and not
self-assembling into fibrils.
In order to probe this possibility, transmission electron
microscopy (TEM) images of Fmoc–F5–Phe–PEG gels were
obtained. These TEM images clearly indicate the presence
of abundant ordered fibrillar assemblies with diameters of
15 ꢀ 1 nm (Fig. 2B). In some cases helical tapes were observed
(Fig. S1, ESIz), perhaps arising from inhibition of bundling by
the C-terminal PEG groups. Based on our working model for
the packing of Fmoc–F5–Phe–OH (see Fig. S2, ESIz) we
hypothesized that the rather sterically demanding PEG chain
perturbs this mode of assembly, explaining the observed
differences in the respective CD spectra.
The co-assembly behavior of Fmoc–F5–Phe and
Fmoc–F5–Phe–PEG was studied in order to probe this
hypothesis. Stock solutions (DMSO) were prepared with
varying ratios of Fmoc–F5–Phe–OH : Fmoc–F5–Phe–PEG
(1 : 1, 4 : 1, and 9 : 1; 247 mM total monomer concentration).
Each mixture was then diluted to a final concentration of
4.9 mM total monomer in water (2% DMSO/H2O). The 1 : 1
mixture formed an opaque suspension immediately after the
addition of water, and required more than 3 h to become
optically clear, similar to the transition time observed for
the pure Fmoc–F5–Phe–PEG suspension. The 4 : 1 and
9 : 1 mixtures both formed opaque solutions following the
addition of water; however, each became optically clear after
B30 minutes. The resulting hydrogels were found to be stable
to vial inversion, visually indicating the formation of rigid
hydrogels.
Once it was established that Fmoc–F5–Phe–PEG was
assembled into fibrillar networks, the rheological strength of
the resulting hydrogels was measured by an oscillatory
frequency sweep from 0–50 rad sꢁ1 with 0.2% strain
(Fig. 2C). Rheology was collected in the linear viscoelastic
region (Fig. S3, ESIz). Fmoc–F5–Phe–PEG hydrogels have a
G0 and G00 of 130 ꢀ 60 Pa and 30 ꢀ 10 Pa, compared to
Fmoc–F5–Phe–OH (G0 = 3060 ꢀ 70 Pa, G00 = 320 ꢀ 10 Pa).
In addition, the G0 and G00 of Fmoc–F5–Phe–PEG displayed a
large frequency dependence at oscillatory frequencies greater
than 50 rad sꢁ1, consistent with the formation of a viscous
solution, while the G0 and G00 of the Fmoc–F5–Phe–OH
hydrogel was essentially independent of frequency consistent
with a rigid hydrogel.13 (Fig. 2C). These results demonstrate
that the addition of PEG to Fmoc–F5–Phe–OH, while an
efficient method for promoting water solubility of the assembled
fibril network, has a detrimental effect on the rheological
strength of the resulting fibrillar network.
The gels were analyzed by CD spectroscopy in order to
investigate if each possessed the characteristic features associated
with fibrillar assemblies of Fmoc–F5–Phe–OH (Fig. 3A). It
was observed that each of the mixtures had CD signatures
commonly associated with Fmoc–F5–Phe–OH fibrils; how-
ever, there was an apparent change in sign of the signal with
the varying ratios.7 The 4 : 1 mixture possessed a signal with
the same sign as the Fmoc–F5–Phe–OH hydrogels (minima at
215 nm, 270–310 nm), while the 1 : 1 and 9 : 1 mixtures have
the same spectral signatures as maxima. This change in sign
implies there may be a potential change in helicity of the fibrils
relative to Fmoc–F5–Phe–OH.8 Each mixture was then imaged
by TEM in order to see if there were any observable morpho-
logical differences in the samples that could account for the
observed change in CD signal (Fig. 3B–D). All three mixtures
were comprised of densely entangled fibrils with average
diameters of 16 ꢀ 2 nm, 14 ꢀ 1 nm, and 16 ꢀ 2 nm for the
1 : 1, 4 : 1, and 9 : 1 mixtures. While the average diameters of
In order to test the effect of PEG on the mechanical
stability of the hydrogels, a dynamic time sweep (conducted
at 0.2% strain) was performed on freshly prepared gels of
Fmoc–F5–Phe–OH and Fmoc–F5–Phe–PEG. Once the G0 had
plateaued, indicating completion of assembly, the samples
were subjected to 100% strain for 30 seconds in order to
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and 9 : 1 mixtures fully and rapidly (within 1 minute)
recovered their original strength and maintained the order
of magnitude difference between G0 and G00, unlike
Fmoc–F5–Phe–OH-derived hydrogels (Fig. 4B). The 1 : 1
mixture unexpectedly showed a significant increase in rigidity
following the application and cessation of stress. After cessa-
tion of stress, the G0 and G00 increased to 950 ꢀ 20 Pa and
22 ꢀ 5 Pa, respectively, an B40-fold increase in rigidity. This
increase in strength may be due to the forced entanglement of
the PEG side chains on the fibrils from the oscillation of the
rheometer head, imparting gel character. It should also be
noted that while Fmoc–F5–Phe–OH-derived fibril networks
precipitated upon application of stress, the co-assemblies with
Fmoc–F5–Phe–PEG showed no evidence of precipitation,
even after 4 weeks and repeated applications of stress.
In conclusion, these results indicate that co-fibrils of
Fmoc-F5–Phe–OH and Fmoc–F5–Phe–PEG exhibit the strong
gel rigidity of Fmoc–F5–Phe–OH fibrils and the solvolytic
stability of Fmoc–F5–Phe–PEG fibrils. The combination of
these desirable attributes results in a hydrogel that exhibits
ideal stress-responsive behaviors. These significant discoveries
will enable the development of small molecule derived hydrogels
that can be further functionalized for application to problems of
biological significance.
Fig. 3 (A) CD spectra of Fmoc–F5–Phe–OH : Fmoc–F5–Phe–PEG
co-fibrils (1 : 1, blue; 4 : 1, green; 9 : 1, orange). (B) TEM image of
fibrils observed in 1 : 1 Fmoc–F5–Phe–OH : Fmoc–F5–Phe–PEG
mixture. (C) TEM image of fibrils observed in 4 : 1
Fmoc–F5–Phe–OH : Fmoc–F5–Phe–PEG mixture. (D) TEM image
of fibrils observed in 9 : 1 Fmoc–F5–Phe–OH : Fmoc–F5–Phe–PEG
mixture.
We gratefully acknowledge Chris Willoughby and TA
Instruments for use of an AR-G2 rheometer. This work was
supported in part by DuPont (Young Professor Award to
BLN), Alzheimer’s Association (NIRG-08-90797), and the
ACS PRF (48922-DNI).
Fig.
4
(A) Dynamic frequency sweeps of Fmoc–F5–Phe–OH :
Notes and references
Fmoc–F5–Phe–PEG co-fibrils (1 : 1, blue; 4 : 1, green; 9 : 1, orange).
(B) Dynamic time sweeps of each mixture before and after the
application of 100% strain (G00 omitted for clarity).
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the bundles were similar in dimension, the handedness of
individual helical fibrils was not clearly distinguishable and
further structural characterization is needed in order to address
the observed changes in CD spectra. These spectra are inter-
preted as evidence of Fmoc–Fmoc, phenyl–phenyl transitions,
supporting the structural model shown in Fig. S2, ESIz.7,8
The rheological strength of each of the hydrogels was measured
using a dynamic frequency sweep from 0–50 rad sꢁ1 with 0.2%
strain (Fig. 4A). The G0 and G00 of the 1 : 1 co-fibrils displayed
significant frequency dependence and the sample was essen-
tially liquid like at frequencies greater than 20 rad sꢁ1
(Fig. 4A). The G0 and G00 of the 4 : 1 mixture were
3000 ꢀ 200 Pa and 400 ꢀ 100 Pa, respectively, and the
G0 and G00 of the 9 : 1 mixture was 2900 ꢀ 400 Pa and
320 ꢀ 50 Pa. The G0 and G00 of both the 4 : 1 and 9 : 1
mixtures were an order of magnitude apart within error and
were essentially independent of the applied frequency, consis-
tent with the formation of rigid hydrogels.13 The gels of these
co-fibrils had nearly identical rigidity to Fmoc–F5–Phe–OH
gels. The response of the mixtures to 100% strain was
tested as described above for Fmoc–F5–Phe–OH and
Fmoc–F5–Phe–PEG (Fig. 4B). Interestingly both the 4 : 1
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Chem. Commun., 2011, 47, 475–477 477