The crown ether size and stereochemistry affect the self-assembly, hydrogelation, and cellular interactions of crown ether/peptide conjugates

Article abstract of DOI:10.1039/d0tb01913e

The discovery of crown ethers and their unique interactions with ions make them play a key role in supramolecular chemistry. In this study, we have developed a new type of amphiphilic crown ether (DB18C6, DB24C8)-conjugated phenylalanine dipeptides for th

Full text of DOI:10.1039/d0tb01913e

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Materials Chemistry B
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The crown ether size and stereochemistry affect
the self-assembly, hydrogelation, and cellular
interactions of crown ether/peptide conjugates†
Cite this:DOI: 10.1039/d0tb01913e
Abdelreheem Abdelfatah Saddik,
Hsin-Chieh Lin
Mohiuddin Mohammed
and
*
The discovery of crown ethers and their unique interactions with ions make them play a key role in
supramolecular chemistry. In this study, we have developed a new type of amphiphilic crown ether
(DB18C6, DB24C8)-conjugated phenylalanine dipeptides for the gelation of water at physiological pH.
We report here for the first time that the size of the crown ether controlled the morphology of the
self-assembled nanostructures of the hydrogels, as well as their interactions with human mesenchymal
stem cells (hMSCs; 3A6-RFP) and mouse fibroblasts (L929). For example, relative to its D-form and other
crown sizes, DB18C6LFLF exhibited greater cell adhesion and was nontoxic towards hMSCs after
culturing for 72 h. We hypothesize that the steric effect of the crown ether moiety in the assemblies has
substantial influences on the morphology of the nanostructures and the cell–material response. Such
distinct cell responses should be beneficial for the development of supramolecular biomaterials.
Received 8th August 2020,
Accepted 21st September 2020
DOI: 10.1039/d0tb01913e
rsc.li/materials-b
Cell adhesion is necessary for the communication, regulation,
and growth of cells, as well as subsequent tissue formation.52
Introduction
Crown ethers are classical examples of supramolecular systems Many methods have been applied to study cell adhesion toward
exhibiting both hydrophilic and hydrophobic segments. Crown various materials.53 Supramolecular peptide hydrogels are
ethers can act as host molecules for selective complexation materials that mimic the behaviour of the extracellular matrix
with metal ions, and ammonium and primary alkyl ammonium (ECM).54 They can be exploited to encapsulate bioactive
salts.1–4 Accordingly, such molecules have been used in moieties (e.g., epitope sequences) and, hence, can be used as
various applications, including metal–organic frameworks,1–4 (ECM-like) biocompatible scaffolds for tissue engineering.52–61
sensors and ligands for transition elements,5–10 rotaxanes and The nanotopography of material surfaces can modulate the
pseudorotaxanes,11–24 electron transport in solar cells,25,26 morphology, adhesion, and proliferation of cells, as well as the
switchable supramolecular dendronized polymers,27–29 biomi- differentiation of stem cells.45–51 For example, macrophages
metic channels,30 and supramolecular gels.31–37 It is rarely seeded on 10 and 50 nm dot arrays adhered well and displayed
reported that a crown ether is used as a motif for peptide- an extended morphology; cells seeded on 200 nm dot arrays,
based gel materials since supramolecular peptide hydrogels however, displayed a distorted morphology.48
have a wide range of biomedical applications; for example,
Similarly, human mesenchymal stem cells (hMSCs) cultured
in tissue engineering and regenerative medicine.38–42 These peptide on nanotubes of relatively small size (diameter: 30 nm) promoted
biomaterials are generally prepared simply from amino acids that cell adhesion, whereas those seeded on 70 nm nanotubes
exist in enantiomeric forms. The chiral centers in amino acids displayed an elongated morphology and allowed osteogenic
influence the self-assembly of the resulting peptides into nano- differentiation.47 Thus, changes in nanostructures can result
structures (e.g., nanofibers, nanotubes, and nanovesicles)38,39 and in distinctly different cell responses. The three major types of
also influence the morphologies of such nanostructures.39,43–51 For peptide hydrogels are aliphatic peptide amphiphiles, aromatic
instance, changing the chirality of the simple tripeptide PhePheVal peptide amphiphiles, and ionic complimentary peptides.49,62,63
gives several different nanostructure morphologies.43
In this paper, we described the first examples of aliphatic/
aromatic peptide amphiphilic hydrogels (D- and L-forms) featuring
dibenzocrown ether motifs (DBC)50,51 of two ring sizes
(DB18C6LFLF, DB18C6DFDF, DB24C8LFLF, and DB24C8DFDF) at
the N-terminus of the phenylalanine dipeptide (FF) (Scheme 1).
Interestingly, the DB18C6-based peptides self-assembled into
Department of Materials Science and Engineering, National Chiao Tung University,
Hsinchu, 300, Taiwan, Republic of China. E-mail: hclin45@nctu.edu.tw
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb01913e
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Scheme 1 Chemical structures of crown ether/peptide conjugates: (1)
DB18C6LFLF, (2) DB18C6DFDF, (3) DB24C8LFLF, and (4) DB24C8DFDF.
nanofibers at neutral pH, whereas the DB24C8-based peptides
formed twisted nanofibers. Furthermore, the cell adhesion of two
cell lines (3A6-RFP and L929) on the DB18C6-based peptides was
better than that of the DB24C8-based peptides. Thus, we illustrate
that the size of the crown ether ring played a significant role in the
self-assembly and cell adhesion properties of the peptide hydro-
gels, suggesting useful applications in tissue engineering.
Scheme 2 (a) Synthetic scheme of the preparation of crown-ether acids;
and (b) SPPS synthetic scheme of the preparation of crown ether dipep-
tides (i) DIEA, DMF, (ii) 20% piperidine in DMF, (iii) HBTU, DIEA, DMF, and
(iv) TFA : water (95% : 5%).
Results and discussion
We have adopted a convergent synthetic route to prepare the
corresponding crown ether dipeptides. Firstly, the crown ether
acids (DB18C6–COOH and DB24C8–COOH) were synthesized hydrogel nanostructure. The hydrogels of DB18C6LFLF and
according to a literature protocol (see the ESI† for details).64–66 DB18C6DFDF featured nanofibers having a diameter of 11 ꢀ
Briefly, the di-alkylation of catechol resulted in the dihydroxy 1 nm; in contrast, DB24C8LFLF formed left-handed twisted
compounds (3a and 3b). Tosylation of compound 3 was then nanofibers (M-type) having diameter 21
ꢀ
2 nm, and
performed using anhydrous p-toluene sulfonyl chloride (p-TsCl) DB24C8DFDF formed right-handed twisted nanofibers (P-type)
to give compounds 4a and 4b in quantitative yields. The having diameter 19 ꢀ 2 nm (Table 1). The differences in these
corresponding tosylate compounds were then reacted with nanostructures must have resulted from the ring size of
methyl 3,4-dihydroxybenzoate in the presence of KPF6 as a the crown ether moieties, because the dipeptide sequence
templating agent using acetonitrile as a solvent under reflux (phenylalanine) was the same in all compounds. Scanning
for five days to afford the ester intermediates 5a and 5b. electron microscopy (SEM) confirmed the morphologies of the
Alkaline hydrolysis of the esters resulted in the desired nanostructures formed from the DB18C6LFLF, DB18C6DFDF,
crown ether acids DB18C6–COOH (6a) and DB24C8–COOH DB24C8LFLF, and DB24C8DFDF hydrogelators (Fig. S1, ESI†).
(6b). Finally, the crown ether dipeptides that differed in terms To obtain additional information about the geometries and
of their stereochemistry (D- and L-forms) and crown ether ring structural properties of the crown ether dipeptides, we selected
sizes—DB18C6LFLF, DB18C6DFDF, DB24C8LFLF, and DB24C8DFDF the L-forms (DB18C6LFLF and DB24C8LFLF) for computational
(Scheme 2)—were synthesized through standard procedures of studies.
solid-phase peptide synthesis.65 1H-NMR and 13C-NMR spectro-
scopy and high-resolution mass spectrometry confirmed the structures using semi-empirical AM1 calculations and DFT-B3LYP
structures of these newly synthesized dipeptides (ESI†). calculations. The total energies of the optimized structures of
Initially, we optimized the geometry of each of the proposed
We then examined the self-assembly of our crown ether- DB18C6LFLF and DB24C8LFLF were calculated to be 296, and
capped dipeptides in aqueous solutions. Interestingly, all of the 389 kcal molꢁ1, respectively. We found that DB18C6LFLF exhibited
tested compounds self-assembled into hydrogels at a concen- a relatively coplanar structure for its [18]crown-6 motif, between
tration of 2 wt% at physiological pH, suggesting that they might rings A and B, while DB24C8LFLF, presenting larger crown ether
be suitable for tissue engineering.67 The formation of their moieties ([24]crown-8), formed twisted structures when consider-
hydrogels was revealed through the ‘‘invert-vial’’ method ing its A and B rings (Fig. 1e and f). The morphological transition
(insets of Fig. 1). The lower critical gel concentration (cgc) is from coplanar to twisted structures occurred to overcome the
1 wt% for DB18C6FF and 1.25 wt% for DB24C8FF, thus steric effects of the larger crown ether units (Fig. 1g). From this
indicating that increasing the ring size would lead to a higher observation, here we propose a molecular packing model for how
cgc. We then used transmission electron microscopy (TEM) to DB24C8LFLF formed twisted nanofibers while DB18C6LFLF
examine the morphology and size of each self-assembled formed a different morphology (Fig. S5) (see the ESI† for details).
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Fig. 1 (a–d) Negatively stained TEM images of the hydrogels of (a) DB18C6LFLF, (b) DB18C6DFDF, (c) DB24C8LFLF, and (d) DB24C8DFDF at a
concentration of 2 wt% (scale bar: 100 nm; the insets show the optical images of the hydrogels). (e and f) Possible molecular packing in the self-
assembled nanostructures of (e) DB18C6FF and (f) DB24C8FF using the DFT method. (g) Schematic representation of the steric effect of the crown ether
moiety on the self-assembled nanostructures.
Table 1 Physical properties of crown ether conjugated dipeptides 2 wt% at pH 7.0
Entry
Appr.a
Tgel–sol (1C)
Morphology
Diameter (nm)
G0 and G00 (Pa) at 25 1Cb
G0 and G00 (Pa) at 37 1Cb
DB18C6LFLF
DB18C6DFDF
DB24C8LFLF
DB24C8DFDF
TG
TG
STG
STG
80
80
60
60
Nanofibers
Nanofibers
Left handed twisted fibers
Right handed twisted fibers
11 ꢀ 1.3
11 ꢀ 1.8
21 ꢀ 1.9
21 ꢀ 2.4
1.1 ꢂ 104, 2.2 ꢂ 103
1.7 ꢂ 104, 1.9 ꢂ 103
1.8 ꢂ 105, 4.2 ꢂ 104
1.7 ꢂ 105, 3.6 ꢂ 104
2.3 ꢂ 104, 6.6 ꢂ 103
2.5 ꢂ 104, 3.1 ꢂ 103
4.3 ꢂ 105, 9 ꢂ 104
3 ꢂ 105, 5.7 ꢂ 104
a
b
TG: transparent gel; STG: semi-transparent gel. At 10 rad sꢁ1
.
Further, we used two-dimensional (2D) diffusion-ordered our hydrogels displayed a storage modulus G0 that was higher than
1H-NMR spectroscopy (DOSY) to study the self-assembly of the the loss modulus G00; the storage moduli at 37 1C for DB18C6LFLF,
monomeric crown ether peptides into supramolecular hydrogels DB18C6DFDF, DB24C8LFLF, and DB24C8DFDF were 2.3 ꢂ 104, 2.5 ꢂ
(Fig. 2). As the concentration of monomeric DB18C6LFLF 104, 4.3 ꢂ 105, and 3.0 ꢂ 105 Pa, respectively. In addition, the
increased from 0.1 to 3 mM, the measured weight-average stability of the hydrogels at 25 1C was similar, in terms of
diffusion coefficient decreased significantly, from 4.39 ꢂ 10ꢁ10 viscoelastic properties, to that at 37 1C (Fig. S6, ESI,† and Table 1).
to 3.34 ꢂ 10ꢁ10 m2
s
ꢁ1, indicating the self-assembly of
Thus, all of our hydrogels were elastic in nature—a useful
DB18C6LFLF. In general, a clear solution will have a relatively property for tissue engineering. Furthermore, we measured the
high diffusion coefficient; upon self-assembly, the value decreases transition temperatures from a gel to a solution by heating our
because of the lower molecular mobility of the self-assembled hydrogels in a water bath from 37 to 80 1C. DB18C6FF trans-
system. Our spectra suggested that DB18C6LFLF self-assembled at formed from a gel to a solution at 80 1C, while DB24C8FF
a higher concentration (43 mM) in an aqueous medium. A underwent the transition at 60 1C. Thus, the transition tem-
similar experiment with the DB24C8LFLF hydrogels revealed that peratures of all of our hydrogels were above human body
their critical aggregation concentration was 0.5 mM. Furthermore, temperature (37 1C), again suggesting potential utility of these
the hydrogelation effect of DB18C6FF was evident within 5 min, hydrogels in tissue engineering (Table 1).54
whereas that of DB24C8FF required much longer hydrogelation
times (41 h).
We used circular dichroism (CD) to examine the secondary
structures of self-assembled peptides in water. DB18C6LFLF at a
We used rheological analysis to examine the elastic properties of concentration of 500 mM exhibited a strong negative signal at
the hydrogels such as the storage or elastic modulus (G0) and loss 209 nm, as well as a shoulder peak near 220 nm indicative of a
or viscosity modulus (G00) because they play an important role in superhelical arrangement. Two strong signals in the range from
biomedical applications.68 The minimum storage modulus neces- 260 to 290 nm also appeared, on account of the aromatic group of
sary to support the mass of a cell is approximately 100 Pa.68 Each of an amino acid side chain being perturbed by its dissymmetric
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amide band in DMSO at 1648 cmꢁ1 as a broad peak; upon
hydrogelation in water, this peak shifted to 1628 cmꢁ1
(Fig. S10b, ESI†).71–75 We employed Congo red staining to study
the formation of amyloid fibrils in the hydrogels. The apple-
green birefringence for all of our hydrogels under a polarized
optical microscope suggested b-amyloid-like structures in their
assemblies (Fig. S12, ESI†).76
We used powder X-ray diffraction (PXRD) to study the
molecular packing in the hydrogels of the L-enantiomers
DB18C6LFLF and DB24C8LFLF. Two important peaks appeared
in the pattern of the freeze-dried DB18C6LFLF sample at
9.12 and 16.21, corresponding to d-values of 9.68 and 5.44 Å,
respectively. These signals may have been due to the formation
of Na+ adducts of DB18C6LFLF in the gel state upon adjusting
the pH of the sample, suggesting that it might be useful as an
isoreticular metal–organic framework (IRMOF).78,79 Further-
more, the pattern of DB18C6LFLF featured a peak at 25.11,
corresponding to a d-value of 3.55 Å and indicating the
presence of p–p interactions.4 No such peaks appeared in the
patterns for DB24C8LFLF (Fig. S13, ESI†).77–79
Fig. 2 (a, b, d and e) 2D DOSY plots (at concentrations of 0.3 and 3 mM,
respectively) of (a and b) DB18C6LFLF and (d and e) DB24C8LFLF. (c and f)
Concentration-dependence of the diffusion coefficients (D) for (c)
DB18C6LFLF and (f) DB24C8LFLF (600 MHz, D2O, 293 K).
The nanofibrous hydrogels resulting from crown peptides
were tested in cell adhesion with human mesenchymal stem
cells (hMSC; 3A6-RFP cells) which had been genetically mod-
ified to express intracellular red fluorescent proteins (RFPs).
We seeded these cells at a density of 4 ꢂ 104 cells on glass cover
slips pre-coated with hydrogels of the crown ether peptides;80
we used a tissue culture treated plate (TCP) as the control.
The cells were incubated at 37 1C under 5% CO2 for 24 and 72 h
and imaged using fluorescence microscopy to reveal the
morphologies of the adhered cells. The 3A6-RFP cells adhered
well when grown on the DB18C6LFLF hydrogel-coated sub-
strate; they displayed flat and extended conformations and a
cell morphology similar to that of those seeded on the tissue
culture plate during the course of 3 days (Fig. 4). In contrast,
the cell attachment was poor when seeded on the DB18C6DFDF
enantiomer, with a lower cell surface area (Fig. S14, ESI†),
consistent with the chirality of the substrate leading to different
interactions with the cell surface proteins and eventually indu-
cing different cell signals that determined the cellular fate and
function.80 The cells seeded on DB24C8LFLF (Fig. 4) and
DB24C8DFDF (Fig. S14, ESI†) failed to attach to the substrates
and remained spherical. The quantity of adhered live cells on
the DB18C6LFLF hydrogel-coated cover slip was similar to that
of TCP, indicating that improved cell adhesion was the essen-
tial requirement for a high rate of cell proliferation. To further
confirm the cell attachment, the cells grown on the hydrogels
were imaged after 24 h through SEM. The cells grown on
DB18C6LFLF exhibited an extended morphology with a large
number of filopodia (Fig. 5a), indicating the healthy growth of
cultured cells. However, the cells grown on DB24C8LFLF dis-
played apoptosis-like spherical morphologies with a significant
decrease in cell surface area (Fig. 5b). Taken together, the cell
adhesion images obtained using fluorescence microscopy and
SEM images demonstrated that the cells grown on the hydrogel
nanofibers having a random morphology underwent extended
cell adhesion and proliferation, whereas those grown on the
surroundings. DB18C6DFDF exhibited only sign-inverted signals
under the same conditions. The chiral helicity collapsed comple-
tely at higher concentrations (42500 mM), with only weak signals
appearing, owing to precipitation of the monomers in the
CD-cuvette. For DB24C8FF, however, the super helical structures
remained at higher concentrations (Fig. 3 and Fig. S7, ESI†).69,70
This hydrogelator possessed an M-type helical morphology; thus,
its CD signals were evident in the signal range from 215 to
300 nm, due to the chiral environment generated by its helicity.
The D-enantiomers (DB24C8DFDF) exhibited chiral inverted
signals, along with the formation of P-type helical morphologies,
as evident from the TEM data (Fig. 1c and d).
The Fourier transform infrared (FTIR) spectrum of
DB18C6LFLF in DMSO featured amide stretching bands at
1653 and 1603 cmꢁ1. Upon self-assembly in water, these bands
shifted to 1629 and 1593 cmꢁ1, respectively (Fig. S10a, ESI†),
which corresponds to the formation of hydrogen bonds in the
gel state. In contrast, the spectrum of DB24C8LFLF featured the
Fig. 3 CD spectra for DB18C6–COOH and DB24C8–COOH, respectively
(blue), (a) DB18C6LFLF (black) and DB18C6DFDF (red) at 500 mM, and (b)
DB24C8LFLF (black) and DB24C8DFDF (red) at 2500 mM concentration.
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Fig. 6 L929 cells were seeded on a non-plasma treated tissue culture
plate, and DB18C6LFLF and DB18C6DFDF hydrogel coated surfaces for
3 days. (a–c) L929 cells cultured for day 1 on (a) the non-plasma treated
TCP, (b) DB18C6LFLF, and (c) DB18C6DFDF. (d–f) L929 cells cultured for
3 days on (d) the non-plasma treated TCP, (e) DB18C6LFLF, and (f)
DB18C6DFDF. The cell morphology was imaged by optical microscopy
under 10ꢂ magnification (scale bar: 100 mm). (g) The cell densities of L929
cells cultured on DB18C6LFLF and DB18C6DFDF were calculated using
ImageJ software. (The mean ꢀ SD from at least three experiments is
shown).
Fig. 4 3A6-RFP cells were seeded onto a tissue culture plate (TCP), and
DB18C6LFLF and DB24C8LFLF hydrogel coated surfaces for 1 day and
3 days. The cell morphology was imaged by fluorescence microscopy
under 10ꢂ magnification (scale bar: 100 mm).
hydrogels featuring twisted nanofibers underwent significantly
lower degrees of cell adhesion and proliferation.
To confirm the cell adhesion properties of the crown ether-
conjugated peptides, we examined their effects towards mouse
fibroblast cells (L929), seeded on 24-well plates at 4 ꢂ 104 cells
in each well pre-coated with the DB18C6LFLF and DB18C6DFDF
hydrogels; a non-plasma treated plate was used as a negative
control, and the cells were cultured for 3 days and adhered cell
images were obtained using optical microscopy. The cells
grown on the hydrogel-coated surfaces displayed excellent
adhesion profiles; in contrast, the cells grown on the non-
treated plate failed to attach and remained spherical (Fig. 6).
The cell density was calculated from the microscopic images of
L929 cells cultured on DB18C6LFLF and DB18C6DFDF hydrogel
surfaces; we found that the proliferation rate of the L929 cells
grown on DB18C6LFLF was higher than that of its corres-
ponding D-enantiomer (Fig. 6g). Similar cell adhesion trends
were observed for the L929 cells grown on the crown ether
dipeptides as seen with 3A6-RFP. Thus, the cell adhesion on
DB18C6LFLF was stronger than that on DB18C6DFDF. On the
contrary, the cells failed to attach when grown on the DB24C8LFLF
and DB24C8DFDF hydrogels and remained spherical.
The different cellular behaviour that arose from the chiral
molecules might be due to the different interactions of the cells
with enantiomorphous surfaces. The L-form nanofibers have
strong affinity with the cells through the stereospecific recogni-
tion between cells and nanofibers; these interactions between
cells and the (ECM) substrate are crucial for the biocompat-
ibility of materials.50,51,81 Also, the different sizes of crown ether or
nanofiber size and morphology could affect the cell behaviour.
The different fibers may induce different signals to the cells and it
ultimately affects the cell–substrate interactions, which influences
the cell adhesion and proliferation behaviour (Fig. 7).82
Next, we studied the biostability of our DB18C6LFLF and
DB18C6DFDF hydrogelators in terms of their incubation with
proteinase K, a powerful protease that hydrolyzes a wide range
Fig. 5 SEM images of 3A6-RFP cells cultured for 24 h on hydrogel-coated
surfaces of (a) DB18C6LFLF and (b) DB24C8LFLF, and (c) the control
(a silicon wafer) [scale bars: (a) 3 mm; (b) and (c) 1 mm].
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due to the oxidative damage which results from H2O2 production
by D-amino acid oxidase (DAO) upon the oxidative deamination of
D-amino acids or from the D-amino acid mis-incorporation in
protein structures, thereby forming functionless proteins and
then showing toxicity.87
Conclusions
In conclusion, we have developed a series of supramolecular
hydrogelators based on crown ether-conjugated dipeptides of
various ring sizes. These hydrogelators self-assembled into
supramolecular hydrogels under aqueous conditions at physio-
logical pH. Hydrogen bonding, electrostatic interactions, and
p–p interactions are the major driving forces responsible for
the self-assembly of these crown ether peptide conjugates. The
cell adhesion properties and cell behaviour were affected by the
size of the crown ether moiety and by the corresponding
morphology of the hydrogelator. The molecular chirality of
the peptide materials also influenced the adhesion and prolif-
eration behaviour of 3A6-RFP and L929 cells. The cell adhesion
and viability on the DB18C6LFLF hydrogel were stronger than
those on its D-enantiomer hydrogel. Thus, the size, shape, and
morphology of small-molecule peptide hydrogelators can mod-
ulate the cell response. To the best of our knowledge, this paper
is the first to report the cell adhesion properties of hydrogels
derived from crown ether peptides. The steric effect of the
crown ether moiety in the assemblies had a substantial influ-
ence on the morphology of the nanostructures as well as the
cell–material responses.
Fig. 7 (a) Positive effect on cell adhesion on DB18C6LFLF nanofibers
derived from L-phenylalanine. (b) Weak effect on cell adhesion on
DB18C6DFDF nanofibers derived from D-phenylalanine. (c and d) Negative
effect on cell adhesion on left- and right-handed twisted nanofibers
DB24C8FF using 3A6-RFP cells.
of peptides.83–86 Over 90% of our DB18C6FF samples (both the
D- and L-forms) remained after incubation with proteinase K,
with the degradation rate monitored using HPLC over a period
of 72 h. Thus, our hydrogelators are biostable toward protei-
nase K and are likely suitable for in vivo studies (Fig. S15,
ESI†).83,84 Encouraged by the results of the bio-stability study,
we examined the biocompatibility of our crown ether dipeptide
hydrogels (2 wt%) with the gel extraction medium using MTT
assay.84 The gel extraction medium was exposed to 3A6-RFP
cells over a period of 2 days. The toxicity of the L-enantiomer
was lower than that of the D-enantiomer, suggesting that the
former can be considered as a biocompatible material. The
DB18C6LFLF hydrogel provided cell survival ratios of greater
than 87% on day 2, and the DB24C8LFLF hydrogel displayed a
cell survival ratio of approximately 90% at day 2, compared with
the DB24C8DFDF hydrogel, which had a cell survival ratio of
approximately 70% (Fig. 8). The toxicity of the D-form might be
Experimental
Materials and methods
Catechol, 2-(2-chloroethoxy)ethanol and potassium hexafluor-
ophosphate were purchased from Alfa Aesar, and 2-(2-(2-
chloroethoxy)ethoxy)ethanol and 3,4-dihydroxymethyl benzoate
from Tokyo Chemical Industry. Anhydrous grade solvents were
purchased from Sigma-Aldrich and Acros Organics. The synth-
esis of DB18C6FF, DB21C7FF, and DB24C8FF was performed by
solid phase peptide synthesis (SPPS) using the classical Fmoc
protocol. The purity of the final compounds was confirmed
1
using H NMR, 13C NMR, and MS techniques. NMR spectra of
the crown ether compounds were recorded at 300 MHz using a
Bruker NMR instrument in deuterated solvents. Coupling con-
stants (J) are denoted in Hz and chemical shifts (d) in ppm.
Multiplicities are denoted as follows: s = singlet, d = doublet,
t = triplet, m = multiplet. High resolution mass spectra were
recorded on a Micromass Q-TOF MS spectrometer.
General procedure for the synthesis of crown ether-
phenylalanine-phenylalanine
Fig. 8 Cell viability of 3A6-RFP cells exposed to the gel extraction media
of the L- and D-enantiomers for 2 days: (a) DB18C6LFLF and DB24C8LFLF;
and (b) DB18C6DFDF and DB24C8DFDF (for each data point at least six
experimental measurements were used to calculate the standard
deviation.)
Crown ether/peptide derivatives were prepared through solid
phase peptide synthesis (SPPS) using 2-chlorotrityl chloride
resin. The resin (1.2 g) was pre-swollen in anhydrous THF for
1 h under N2 conditions. Then Fmoc-L-Phe-OH or Fmoc-D-Phe-OH
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(0.75 g, 2 mmol) in anhydrous N,N0-dimethylformamide (DMF) 37.32, 53.99, 54.95, 69.16, 69.34, 69.43, 69.50, 69.62, 70.85,
was added along with N,N-diisopropylethylamine (DIEA) 70.89, 112.87, 113.30, 114.57, 121.61, 126.63, 126.89, 126.95,
(0.85 mL, 5 mmol) and the mixture was allowed to react for 128.62, 129.62, 137.82, 138.89, 147.99, 148.93, 151.43, 166.14,
1 h. The block solution was added (DCM : MeOH : DIEA) and 172.03, 173.24; HRMS [ESI+]: m/z (%): calcd 786.3364, obsvd
stirred for 30 min. Then 20% piperidine in DMF was used to 809.3247 [M + Na]+.
treat the resin for 30 min to de-protect the Fmoc group; this
Dibenzo[24]crown[8]ether-D-phenylalanine-phenylalanine
procedure was repeated twice (each for 5 min). Then the (DB24C8DFDF). Yield (0.62 g). 1H NMR (300 MHz, DMSO-d6,
coupling of Fmoc-L-Phe-OH or Fmoc-D-Phe-OH (0.75 g, 2 mmol) 25 1C): d = 2.85–3.00 (m, 2H; CH2), 3.05–3.15 (m, 2H; CH2),
amino acid was performed using O-(benzotriazol-1-yl)- 3.64–3.65 (d, J = 1.8 Hz, 8H; 4CH2), 3.74–3.77 (t, J = 4.2 Hz, 8H;
N,N,N0,N0-tetramethyluroniumhexa fluorophosphate (HBTU) 4CH2), 4.03–4.10 (m, 8H; 4CH2), 4.40–4.50 (m, 1H; CH), 4.65–
(0.75 g,
2 mmol) and N,N-diisopropylethylamine (DIEA) 4.75 (m, 1H; CH), 6.84–6.87 (q, J = 4.13 Hz, 2H; 2CH), 6.91–6.99
(0.85 mL, 5 mmol) in DMF for 1 h. Then 20% piperidine in (m, 3H; 3CH), 7.13–7.24 (m, 8H; 8CH), 7.30–7.38 (m, 4H; 4CH);
DMF was used to treat the resin for 30 min to de-protect the 13C NMR (75 MHz, DMSO-d6, 25 1C): d = 37.11, 37.34, 53.99,
Fmoc group; this procedure was repeated twice (each for 54.95, 69.18, 69.35, 69.51, 69.64, 70.86, 112.88, 113.31, 114.57,
5 min). Finally, crown ether acid (2 mmol) was added to the 121.63, 126.64, 126.91, 126.96, 128.47, 128.64, 129.63, 137.81,
free amino group in the presence of O-(benzotriazol-1-yl)- 148.00, 148.95, 151.43, 166.14, 172.06, 173.17; HRMS [ESI+]:
N,N,N0,N0-tetramethyluroniumhexa fluorophosphate (HBTU) m/z (%): calcd 786.3364, obsvd 787.3452 [M + H]+.
(1.14 g,
3
mmol) and N,N-diisopropylethylamine (DIEA)
Hydrogel preparation. Hydrogels were prepared by weighing
(1.3 mL, 7.5 mmol) in DMF. The reaction mixture was stirred 4.0 mg of compound in a screw-capped 2 mL vial (diameter
overnight; excessive reagents were removed by subsequently 10 mm). Aqueous NaOH solution was added and the resulting
washing with various solvents. The desired compounds were solution was vortexed and sonicated for a few minutes to get a
achieved by treating the resin with 90% trifluoroacetic acid clear solution. Then the pH and concentration of the solution were
(TFA) in DI water for 3 h. The resulting solution was air-dried, carefully adjusted using HCl solution to trigger hydrogelation.
and then diethyl ether was added to precipitate the target
Transmission electron microscopy (TEM). Images were
product. The solid was dried under a vacuum to remove obtained with a Hitachi HT7700 transmission electron micro-
residual solvents.
scope at an accelerating voltage of 100 kV. The hydrogels were
applied directly onto 200 mesh carbon-coated copper grids. The
Dibenzo[18]crown[6]ether-L-phenylalanine-phenylalanine
(DB18C6LFLF). Yield (0.44 g). 1H NMR (300 MHz, DMSO-d6, excess amount of the hydrogel was carefully removed by capil-
25 1C): d = 2.85–3.00 (m, 2H; CH2), 3.05–3.15 (m, 2H; CH2), 3.84 lary action (filter paper), and the grids were then immediately
(s, 8H; 4CH2), 4.04 (s, 4H; 2CH2), 4.05–4.10 (m, 4H; 2CH2), 4.40– stained with uranyl acetate for 30 s. Excess stain was removed
4.50 (m, 1H; CH), 4.60–4.80 (m, 1H; CH), 6.80–7.00 (m, 5H; by capillary action, and the grids were allowed to air dry.
5CH), 7.10–7.25 (m, 8H; 8CH), 7.30–7.40 (m, 4H; 4CH); 13C
Scanning electron microscopy (SEM). Samples were visua-
NMR (75 MHz, DMSO-d6, 25 1C): d = 37.11, 37.35, 53.94, 54.97, lized with a JEOL JSM-6700F scanning electron microscope at
67.87, 68.15, 68.26, 69.21, 69.40, 111.69, 112.74, 121.16, 126.63, an accelerating voltage of 5 kV and a working distance of
126.72, 126.91, 128.46, 128.64, 129.62, 137.82, 138.91, 147.58, 6.3 mm. The hydrogels were applied directly onto silicon wafers
148.26, 150.81, 166.20, 172.06, 173.17; HRMS [ESI+]: m/z (%): and the samples were allowed to air dry.
calcd 698.2839, obsvd 721.2724 [M + Na]+.
Rheological tests. Rheological tests were conducted on a TA
Dibenzo[18]crown[6]ether-D-phenylalanine-phenylalanine
Discovery. A 20 mm parallel plate was used during the experi-
(DB18C6DFDF). Yield (0.54 g). 1H NMR (300 MHz, DMSO-d6, ment. 100 mL of the hydrogel sample (2 wt%) was placed on the
25 1C): d = 2.85–3.00 (m, 2H; CH2), 3.05–3.15 (m, 2H; CH2), 3.84 parallel plate. Angular frequency sweep test: test range (0.1 to
(s, 8H; 4CH2), 4.05–4.06 (d, J = 3 Hz, 4H; 2CH2), 4.05–4.10 100 rad sꢁ1 frequency, strain = 0.8%), 13 points per decade. The
(m, 4H; 2CH2), 4.40–4.50 (m, 1H; CH), 4.65–4.75 (m, 1H; CH), sweep mode is ‘‘log’’ and the temperature was 25 1C and 37 1C.
6.84–6.89 (m, 2H; 2CH), 6.90–6.99 (m, 3H; 3CH), 7.11–7.25 (m,
Circular dichroism (CD) spectra. CD spectra of the hydrogels
8H; 8CH), 7.32–7.39 (m, 4H; 4CH); 13C NMR (75 MHz, DMSO- were recorded in the UV vis region (190–600 nm) using a
d6, 25 1C): d = 37.11, 37.35, 53.99, 54.98, 67.94, 68.19, 68.28, 0.1 mm quartz cuvette with the total gelator concentration at
69.22, 69.41, 111.73, 112.79, 121.13, 126.64, 126.68, 126.92, 1000 mM (0.07 wt%) for DB18C6FF, and 2500 mM (0.095 wt%)
128.47, 128.64, 129.63, 137.81, 138.92, 147.65, 148.35, 150.90, for DB24C8FF.
166.23, 172.08, 173.18; HRMS [ESI+]: m/z (%): calcd 698.2839,
obsvd 721.2725 [M + Na]+.
Congo red staining. To freshly prepared saturated sodium
chloride ethanol solution (80%) Congo Red dye was added and
the solution was vortexed vigorously and filtered through a
Dibenzo[24]crown[8]ether-L-phenylalanine-phenylalanine
(DB24C8LFLF). Yield (0.6 g). 1H NMR (300 MHz, DMSO-d6, 0.45 micron filter. 50 mL Congo Red solution was placed onto
25 1C): d = 2.85–3.00 (m, 2H; CH2), 3.05–3.15 (m, 2H; CH2), the glass slide and then 25 mL of hydrogel was placed onto the
3.66 (d, J = 1.8 Hz, 8H; 4CH2), 3.75–3.95 (m, 8H; 4CH2), 4.00– Congo Red solution and the hydrogel was then stained for
4.15 (m, 8H; 4CH2), 4.40–4.50 (m, 1H; CH), 4.65–4.75 (m, 1H; about 1 to 2 minutes. After the Congo Red dye evaporated,
CH), 6.80–7.00 (m, 5H; 5CH), 7.10–7.25 (m, 8H; 8CH), 7.30–7.40 the hydrogel was then covered with a cover slip and the excess
(m, 4H; 4CH); 13C NMR (75 MHz, DMSO-d6, 25 1C): d = 37.12, amount of dye was removed by blotting. The hydrogel containing
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slide was then placed between crossed polarizers and images were added to dissolve the formazan crystals, and then 100 mL of
obtained using a Zeiss (Axio Vert.A1) microscope. each 24 well plate was transferred to a 96-well plate and the
Scanning electron microscopy for cell adhesion. The har- optical density of the resulting solution was measured at
vested cells were washed with PBS and then fixed with 2.5% 595 nm using a TECAN Infinite F50 microplate reader. Cells
glutaraldehyde in PBS at 4 1C overnight, followed by post- without the treatment with the gel extract were used as the
fixation in 1% osmium tetroxide for 30 min. Dehydration was control.
performed through a series of ethanol concentrations (10 min
Biostability test with proteinase K. 1 mg of each compound
incubation each in 50%, 60%, 70%, 80%, 90%, 95%, and 100% was dissolved in 5 mL HEPES buffer at pH = 7.5. Subsequently,
ethanol) and the samples were air-dried. The specimen was proteinase K was added at a concentration of 3.2 units per mL
sputter-coated with platinum and examined using a Zeiss-SEM and incubated at 37 1C for 24 h. After that, 100 mL of sample was
at an accelerating voltage of 5 keV.
taken out each time and analyzed by HPLC.
Computational study. All the computations were carried out
using the semi-empirical AM1 method as implemented within
the Gaussian 16 program package. The geometries of all Conflicts of interest
compounds were fully optimized to elucidate the effect of the
The authors declare no conflict of interest.
large-size crown ethers, i.e. 18-crown-6 (DB18C6) and 24-crown-
8 (DB24C8), in the structure of the synthesized compounds.
Powder XRD. The PXRD study was carried out to understand
the periodic arrangement of molecules during the self-
assembly process. DB18C6LFLF and DB24C8LFLF hydrogels
were prepared based on the procedure discussed above and
the sample was frozen and dried using a lyophilizer (FDM-2,
UNISS) to get the xerogel. The xerogel sample was analysed
by PXRD. The measurements were performed on a Bragg–
Brentano-type powder diffractometer (Bruker D8 Advance
diffractometer, operated at 40 kV and 40 mA, with Cu Ka
radiation, l = 1.5418 Å). The data were collected in a 2y range
of 5–301 using a step size of 0.0161.
Acknowledgements
We would like to thank Dr Rajan Deepan Chakravarthy and
Dr Satish Kumar Talloj for proofreading of this paper. This study
was supported by the Ministry of Science and Technology of the
Republic of China, Taiwan (grants MOST 109-2113-M-009-025
and MOST 108-2113-M-009-024); the ‘‘Aim for the Top Univer-
sity’’ program of National Chiao Tung University, Taiwan; and
the Ministry of Education, Taiwan, Republic of China.
2D cell culture. Prior to cell seeding, glass coverslips
(15 mm) were coated with 2 wt% hydrogels (40 mL of each
DB18C6LFLF and DB24C8LFLF), and allowed for drying. 3A6-
RFP cells at 4 ꢂ 104 cells per well were seeded on the hydrogel
coated coverslips in a 24 well plate with DMEM low glucose
containing 10% fetal bovine serum (FBS), supplemented with
100 IU mLꢁ1 streptomycin and penicillin, and the cells were
then incubated at 37 1C for 24 h and 72 h. L929 cells were
cultured on 2 wt% hydrogel (DB18C6LFLF and DB18C6DFDF)
coated on non-plasma treated 24 well plate with EMEM con-
taining 10% horse serum (HS), supplemented with 100 IU mLꢁ1
streptomycin and penicillin, and then the cells were incubated
at 37 1C for 24 h and 72 h. The cultured cells were imaged using
a Zeiss (Axio Vert.A1) fluorescence microscope.
Notes and references
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