Self-assembly and hydrogelation from multicomponent coassembly of pentafluorobenzyl-phenylalanine and pentafluorobenzyl-diphenylalanine

Article abstract of DOI:10.1039/c5ra01020a

To screen the possibility of forming self-assembled hydrogels under physiological pH, various molar ratios of the hydrogelators based on pentafluorobenzyl-phenylalanine (PFB-F) and pentafluorobenzyl-diphenylalanine (PFB-FF) were studied. The equimolar ratio of PFB-F and PFB-FF formed coassembled nanofibers and a self-supporting hydrogel at the physiological pH of 7.4. The spectroscopic characterization of the blend gel indicates that π-π interactions and hydrogen-bonding interactions are the major driving forces behind the coassembly. In addition, we also prove that the blend hydrogel is biocompatible, thus making it a scaffolding material for biomedical applications. In this work, the utility of two distinct hydrogelators to promote coassembly under physiological condition expands the repertoire of noncovalent interactions that can be used in the development of sophisticated noncovalent biomaterials. This journal is

Full text of DOI:10.1039/c5ra01020a

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Self-assembly and hydrogelation from
multicomponent coassembly of
Cite this: RSC Adv., 2015, 5, 22943
pentafluorobenzyl-phenylalanine and
pentafluorobenzyl-diphenylalanine†
Received 18th January 2015
Accepted 23rd February 2015
*
Shu-Min Hsu, Fang-Yi Wu, Tsung-Sheng Lai, Yu-Chun Lin and Hsin-Chieh Lin
DOI: 10.1039/c5ra01020a
www.rsc.org/advances
To screen the possibility of forming self-assembled hydrogels under et al. have proven that equimolar mixtures of Fmoc-Phe and
physiological pH, various molar ratios of the hydrogelators based on Fmoc-F5-Phe, which possess side-chain aromatic groups with
pentafluorobenzyl-phenylalanine (PFB-F) and pentafluorobenzyl- complementary quadrupole interactions, readily coassemble to
diphenylalanine (PFB-FF) were studied. The equimolar ratio of PFB-F form two-component hydrogels where Fmoc-Phe alone fails to
and PFB-FF formed coassembled nanofibers and a self-supporting self-assemble.14 In this work, we report that the equimolar ratio
hydrogel at the physiological pH of 7.4. The spectroscopic charac- of PFB-F and PFB-FF efficiently forms coassembled nanobers
terization of the blend gel indicates that p–p interactions and and hydrogels under the physiological pH of 7.4. In addition,
hydrogen-bonding interactions are the major driving forces behind the study of cell survival ratio suggests the blend hydrogel is
the coassembly. In addition, we also prove that the blend hydrogel is biocompatible, thus making it a potentially useful scaffolding
biocompatible, thus making it a scaffolding material for biomedical material for biomedical applications.
applications. In this work, the utility of two distinct hydrogelators to
Molecules of PFB-F and PFB-FF have been synthesized by
promote coassembly under physiological condition expands the solid phase peptide synthesis (SPPS) and their chemical struc-
repertoire of noncovalent interactions that can be used in the devel- tures are shown in Fig. 1.3 The self-assembled hydrogels were
opment of sophisticated noncovalent biomaterials.
prepared by a sequential change in pH. The 1 wt% hydrogels of
PFB-F and PFB-FF efficiently formed at pH 5.0 and 10.0,
respectively. To explore characteristics of PFB-capped hydro-
gels, we have investigated sol–gel transition of PFB-F and PFB-
FF with various pH values (pH ¼ 3–10), concentrations (0.25–
2 wt%) and blend ratios (0 : 1, 3 : 1, 1 : 1, 1 : 3 and 1 : 0) (see
Fig. S1† for details). We found that all the hydrogels with
different blend ratios formed at 1 wt% and their gel images of
PFB-capped hydrogels were shown in Fig. 1. For further study,
Self-assembled hydrogels provide useful nano-sized scaffolds
for biomedical applications.1–5 Amino-acid derivatives and
peptides are attractive in this regard, as they mimic certain
aspects of the natural extracellular matrix, such as their nano-
brous features and high hydration.4 A hydrogel self-assembled
from aromatic peptide amphiphiles is an ideal candidate
material for the development of smart, so and nano-sized
biomaterials due to its synthetic customizability,5 and it has
been used in the applications of tissue scaffolding,6,7 cell
culture,8 drug delivery,9,10 and wound healing.11,12 Recently,
more efforts have been paid to fabricate self-assembled hydro-
gels by mixing two or more aromatic peptide amphiphiles to
adjust their mechanical and morphological properties.13,14 For
example, Ulijn et al. have demonstrated the coassembled
peptide materials combine functional and structural compo-
nents and are potentially useful as tunable matrices for protein
immobilization that can be exploited in biocatalysis.13 Nilsson
Department of Materials Science and Engineering, National Chiao Tung University,
Hsinchu, 300, Taiwan, Republic of China. E-mail: hclin45@nctu.edu.tw; Fax: +886-
3-5724727; Tel: +886-3-5731949
Fig. 1 Upper: the synthetic routes and the chemical structures of
PFB-F and PFB-FF. Lower: the macroscopic appearance in different
blend hydrogels of PFB-F and PFB-FF (1 wt%) (from left to right are
1 : 0, 3 : 1, 1 : 1, 1 : 3 and 0 : 1).
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra01020a
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Table 1 Physical properties of PFB-capped hydrogels at 1 wt%
PFB-F : PFB-FFa
Appr.b (pH)
G0, G00 (Pa)
Critical strain (%)
Fiber diameter (nm)
1 : 0
3 : 1
1 : 1
1 : 3
0 : 1
SG (5.0)
TG (7.0)
SG (7.4)
TG (9.0)
TG (10.0)
3.0 ꢁ 103, 1.5 ꢁ 102
2.8 ꢁ 103, 5.5 ꢁ 102
3.0 ꢁ 103, 2.0 ꢁ 102
2.6 ꢁ 104, 4.5 ꢁ 103
3.4 ꢁ 104, 8.0 ꢁ 103
0.63
0.57
0.71
0.64
0.89
31.0 ꢂ 5
14.7 ꢂ 3, 6.2 ꢂ 1
8.3 ꢂ 2
7.7 ꢂ 2
5.0 ꢂ 1
PFB-F : PFB-FF: a blend of PFB-F and PFB-FF. b TG: transparent gel; SG: semi-translucent gel.
a
we chose the blend gel in each case with a specic pH value at ratio of PFB-FF (1 : 1 or 1 : 3), the TEM images showed uniform
1 wt% because of its shorter gelation time compared with those nanostructures with a homogeneous diameter of ꢀ8 nm (Fig. 2c
of different pH values (Fig. 1). Notably, the gelation times for and d and Table 1).
different blend ratios were measured which reveal that the
The spectroscopic characterization of the gel states of
co-assembling hydrogelations are much time-consuming than hydrogelators provides relevant evidence regarding the inter-
those of single components (Fig. S2†). The appearances of the molecular interactions in the assemblies.17–19 Therefore, the
hydrogels are transparent for a 3 : 1, 1 : 1, 1 : 3 or 0 : 1 blend of spectroscopic characterization of different blend hydrogels of
PFB-F and PFB-FF, while semi-translucent for pure PFB-F. PFB-F and PFB-FF were then studied. The UV-vis absorption
Interestingly, the increase of the molar ratio of PFB-FF in the spectra of the ve PFB-capped hydrogels displayed absorption
mixture would result in higher pH value for efficient hydro- bands at ca. 260 nm, corresponding to p–p* transitions of the
gelation (Table 1). Importantly, we found that an equimolar typical aromatic rings (Fig. 3a). Notably, a new absorption band
mixture of PFB-F and PFB-FF could efficiently coassemble to at 285 nm (275–305 nm) was observed and its intensity was
form a self-assembled hydrogel under physiological pH.
increased with increasing the ratio of PFB-FF, thus indicating
The microscopic nanostructures of the PFB-capped hydro- the additional Phe in PFB-FF may enhance the p–p interactions
gels were measured by transmission electron microscopy (TEM) in the gel. The emission spectra of the ve materials were
and presented in Fig. 2. The TEM analysis indicated that the obtained with an excitation wavelength of 260 nm under the
PFB-capped hydrogels consisted of a brous network and these gelation conditions, all materials showed red-shied and broad
nanobers entangle to trap water in the gel. The PFB-F gel emissions in gel state (ca. 360 nm) with respect to the corre-
showed a thicker ber diameter (31.0 nm) among the hydrogels, sponding emissions in solution (ꢀ310 nm),3 which is attributed
while the PFB-FF with the relatively thinner ber diameter to aggregate formation in gel state. In addition, we found that
(Table 1). A 3 : 1 blend of PFB-F and PFB-FF leads to the the emission intensity would increase continuously as the ratio
formation of uneven nanobers with the diameters of 14.7 and of the PFB-FF increased (Fig. 3b). The circular dichroism (CD)
6.2 nm. Notably, the presence of two types of brous nano- spectra were shown in Fig. 3c, the hydrogel of PFB-F revealed
structures in the gel of a 3 : 1 blend of PFB-F and PFB-FF may be
attributed to the partial co-assembling behavior of PFB-F and
PFB-FF. For most of the molecules, the nanoscale self-sorting is
possible since multi-gelator gels may independently assemble
their own brous nanostructures in the mixture as reported
recently by Smith et al.15,16 Furthermore, when increasing the
Fig. 3 (a) UV-vis, (b) Fluorescent emission, (c) CD and (d) FT-IR spectra
Fig. 2 TEM images in different blend hydrogels of PFB-F and PFB-FF at in different blend hydrogels of PFB-F and PFB-FF at 1 wt% in water
1 wt%. (a) 1 : 0, (b) 3 : 1, (c) 1 : 1, (d) 1 : 3 and (e) 0 : 1.
under their hydrogelation conditions.
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bisignate Cotton effects, with a negative part at 260 nm and a
positive part at 285 nm. With increasing the ratio of PFB-FF, the
bisignate signal would disappear, and instead of a strong
positive CD signal at 260 nm. As shown in Fig. 3d, the FT-IR
spectra of a pure PFB-F and a 3 : 1 blend of PFB-F and PFB-FF
hydrogels showed that there are no secondary structures
formed in the hydrogels, suggesting the exibility of PFB-F
hydrogelator does not allow the single amino acid to interlock
in a b-sheet-like conformation.3 With increasing the ratio of
PFB-FF, the spectra of the 1 : 1 and the 1 : 3 blend of PFB-F and
PFB-FF as well as pure PFB-FF gels showed two peaks located
around 1656 cmꢃ1, 1630 cmꢃ1 and a less intense peak around
1545 cmꢃ1, indicating the formation of b-sheet-like structure in
the assemblies.20
The mechanical properties of the PFB-capped hydrogels were
obtained by oscillatory rheometry. In comparison of blend
hydrogels with different molar ratios, the viscoelastic properties
of PFB-capped hydrogels are determined at the concentration of
1 wt% and the results are collected in Fig. 4a and b and Table 1.
The storage moduli of all the hydrogels are higher than their
loss moduli, thus indicating these materials formed viscoelastic
gels. The average storage modulus of the 1 : 1 blend was 3.0 kPa
which supports the mass of a cell (ꢀ0.1 kPa). Therefore, it is
possibly to be used as scaffolding material for tissue engi-
neering.21,22 Moreover, the strain sweeps results of the gels are
shown in Fig. 4c and d and their critical strains of the blend gels
are collected in Table 1.
Fig. 5 Viability data of (a) CTX TNA2 and (b) MCF-7 cells incubated
with 10–500 mM of a 1 : 1 blend of PFB-F and PFB-FF after 24, 48 and
72 h.
experiments revealed that aer being incubated the MCF-7 cells
with the blend material (10–500 mM) for 72 h, cells that were
grown in liquid medium showed the proliferation capacities
with high survival ratio (above 80% at 500 mM).24 These obser-
vations illustrate the potentially useful of this material in the
applications of 3D cell culturing and drug delivery.
In summary, we have studied various molar ratios of PFB-F
and PFB-FF to screen the possibility of forming self-
assembled hydrogel under physiological pH. We found that
equimolar ratio of PFB-F and PFB-FF efficiently formed coas-
sembled nanobers and the hydrogel at the pH of 7.4. The
spectroscopic characterization of the blend gel indicates the
p–p interactions and hydrogen-bonding interactions are the
major driving forces behind the coassembly. Additionally, the
results of cell survival ratio of CTX TNA2 and MCF-7 cells
suggest the blend hydrogel is biocompatible, thus making it a
potentially useful scaffolding material for biomedical applica-
tions such as drug delivery and tissue engineering. In this study,
the successful usage of two distinct hydrogelators to promote
the coassembly under physiological pH expands the repertoire
of noncovalent interactions that can be used in the develop-
ment of sophisticated noncovalent biomaterials.
Based on the success of the blend hydrogel formed under
physiological pH, we further examined the biocompatibility of
the 1 : 1 blend material. The biocompatibility of a 1 : 1 blend of
PFB-F and PFB-FF was examined using colorimetric MTT
assay.23 In Fig. 5a, we examined the viability of CTX TNA2 cells,
the results indicate the 1 : 1 blend gel of PFB-F and PFB-FF was
biocompatible because the 50% inhibition (IC50) are higher
than 500 mM aer 72 h, and its survival ratio is higher than 80%.
As a potential drug carrier, we used MCF-7 cell line as a model to
evaluate the cell compatibility of the material. In Fig. 5b, the
Acknowledgements
This study was supported nancially by the National Science
Council of the Republic of China, Taiwan (grant NSC 102-2113-
M-009-006-MY2); the “Aim for the Top University” program of
National Chiao Tung University; and the Ministry of Education,
Taiwan, R.O.C. We thank Jhong-Hua Lin and Yu-Tang Huang
for help with gelation tests.
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