Supramolecular Hydrogels of Indole-Capped Short Peptides as Vaccine Adjuvants

Article abstract of DOI:10.1002/cjoc.201600813

Many materials as immune adjuvant are researched to help raise immnogenicity of subunit vaccines. Among them, peptide-based hydrogels are gradually coming into notice because of their application in drugs delivery, cancer cell inhibition, vaccine adjuvants and detection of important analytes. In this work, we introduced a novel aromatic capping group based on indole to construct short peptide-based supramolecular hydrogelators Indol-GFFY and Indol-G^DF^DF^DY and demonstrated their potential applications as vaccine adjuvants.

Full text of DOI:10.1002/cjoc.201600813

FULL PAPER
DOI: 10.1002/cjoc.201600813
Supramolecular Hydrogels of Indole-Capped Short Peptides
as Vaccine Adjuvants
a,b
a,b
a,b
,a
,a,b
Zhongyan Wang, Yanbin Cai, Linan Yi, Jie Gao,* and Zhimou Yang*
a
State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China
b
College of Life Sciences, Nankai University, Tianjin 300071, China
Many materials as immune adjuvant are researched to help raise immnogenicity of subunit vaccines. Among
them, peptide-based hydrogels are gradually coming into notice because of their application in drugs delivery, can-
cer cell inhibition, vaccine adjuvants and detection of important analytes. In this work, we introduced a novel aro-
matic capping group based on indole to construct short peptide-based supramolecular hydrogelators Indol-GFFY
D
D D
and Indol-G F F Y and demonstrated their potential applications as vaccine adjuvants.
Keywords self-assembly, hydrogel, vaccine adjuvant
Introduction
ylmethyloxycarbonyl (Fmoc), naphthalene (Nap), phe-
[9]
nothiazine (PTZ) or indole (Indol). We chose to test
whether hydrogels formed by indole-capped short pep-
tides could be potential vaccine adjuvants because in-
dole is commonly found in a wide variety of natural
products, including the amino acid tryptophan, and is
Subunit vaccines are being widely developed owing
to their well-defined structure at the molecular level,
excellent safety properties, and ease of production.
However, relative to live vaccines, subunit vaccines do
not raise strong immune responses. Subunit vaccines
are therefore formulated with adjuvants to stimulate
more potent immune responses. Many materials have
been demonstrated to be promising vaccine adjuvants,
including hydrogels and nanomaterials (e.g., liposomes
[1]
[9l]
highly biocompatible.
Experimental
Materials
[2]
and mesoporous silica nanoparticles). Among these
materials, hydrogels are relatively easy to handle be-
cause antigens can simply be mixed with hydrogels be-
fore use. Recently, Collier et al. conducted pioneering
work whereby supramolecular peptide hydrogels were
2
-Cl-Trityl chloride resin (1.2 mmol/g) was obtain
from Nankai University Resin Co., Ltd. O-Benzotriazol-
-yl-N,N,N',N'-tetramehtyl uronium hexafluorophos-
1
phate (HBTU) and Fmoc-amino acids were bought from
GL Biochem (Shanghai). Chemical regents and solvents
were used as received from commercial sources. Com-
mercially available reagents were used without further
purification, unless noted otherwise. RMPI 1640 me-
dium, fetal bovine serum (FBS) and penicillin/strepto-
mycin were purchased from Gibco Corporation.
EndoFit Ovalbumin (endotoxins＜1 EU/mg) was pur-
chased from InvivoGen (CA, USA). Horseradish pe-
roxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a,
and IgG2b were obtained from Southern Biotechnolo-
gies (AL, USA). Mouse IL-5 and IFN-γ ELISA kits
were obtained from Biolegend (CA, USA). Six- to
eight-week-old female C57BL/6J mice were purchased
from Academy of Military Medical Science (Beijing,
China), and maintained in specific pathogen-free condi-
tions in the animal facility at the Nankai University,
Tianjin, China.
[
3]
used as immune adjuvants. They demonstrated that
nanofibres composed of bioconjugates of self-assemb-
ling peptides and peptide/protein antigens could raise
strong humoral immune responses in a long-lasting
[
4]
manner. Our group has also demonstrated that en-
zyme-catalysed short peptide-based hydrogels can raise
strong immune responses when physically mixed with
[5]
HIV DNA or protein (ovalbumin (OVA)) antigens.
These results suggested the great promise of supramo-
lecular hydrogels as vaccine adjuvants.
Peptide-based hydrogels have shown great promise
[
6]
for applications in drug delivery, cancer cell inhibi-
[7]
[8]
tion, and detecting important analytes. Among pep-
tide-based hydrogelators, those based on short peptides
are easier to design and synthesize. To obtain short pep-
tide-based hydrogelators, peptides are typically capped
with an appropriate aromatic group, such as fluoren-9-
*
E-mail: yangzm@nankai.edu.cn, chemgaojie@nankai.edu.cn; Tel.: 0086-022-23502875; Fax: 0086-022-23507760
Received November 21, 2016; accepted January 23, 2017; published online XXXX, 2017.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600813 or from the author.
Chin. J. Chem. 2017, XX, 1—6
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Wang et al.
FULL PAPER
General procedure
liquid chromatography (HPLC).
Characterization of Indol-GFFY H NMR (400
MHz, DMSO) δ: 9.22 (s, 1H), 8.18 (d, J＝24.5 Hz, 3H),
1
1
H NMR (Bruker ARS 400) and HR-MS (Agilent
6
520 Q-TOF LC/MS) were used to characterize the
8
.00 (d, J＝8.5 Hz, 1H), 7.51－7.44 (m, 2H), 7.20 (d,
J＝27.3 Hz, 11H), 7.02 (s, 3H), 6.66 (d, J＝8.0 Hz, 2H),
.37 (s, 1H), 4.52 (d, J＝27.9 Hz, 2H), 4.36 (d, J＝3.7
Hz, 2H), 3.65 (s, 1H), 3.54 (s, 1H), 3.02－2.91 (m, 4H),
.84－2.78 (m, 2H), 2.67－2.59 (m, 4H). MS: calcd M
compounds. TEM (JEM100CXII) was performed at the
Tecnai G2 F20 system, operating at 100 kV. Reology
6
(
4
TA instrument) test was done on an AR 2000ex system,
0 mm parallel plates were used during the experiment
2
at the gap of 500 μm. Fluorescence spectrum was
gained by BioTek (Synergy 4). Circular dichroism (CD)
spectrum was obtained by a BioLogic Synergy 4.
＋
＝
703.78, obsvd. (M＋H) ＝704.3085.
D
D
D
1
Characterization of Indol-G F F Y
H NMR
(
400 MHz, DMSO) δ: 9.22 (s, 1H), 8.19 (dd, J＝29.6,
Synthesis and characterizations
1
2
6
3.3 Hz, 3H), 8.00 (d, J＝8.4 Hz, 1H), 7.52－7.42 (m,
H), 7.23 (t, J＝24.1 Hz, 11H), 7.03 (d, J＝7.8 Hz, 3H),
.66 (d, J＝8.4 Hz, 2H), 6.37 (s, 1H), 4.52 (d, J＝27.9
1
-Indolepropionic acid synthesis As shown in
Scheme S1 (Supporting Information), indole (21.9 mg,
Hz, 2H), 4.36 (d, J＝6.4 Hz, 2H), 3.67 (d, J＝10.2 Hz,
H), 3.54 (s, 1H), 2.98 (dd, J＝32.9, 10.6 Hz, 4H), 2.81
d, J＝18.6 Hz, 2H), 2.70－2.56 (m, 4H). MS: calcd
2
8
.56 mmol), potassium hydroxide (171 mg, 2.61 mmol,
5%, freshly ground), and sodium iodide (39 mg, 0.26
1
(
mmol) were dissolved under argon in 10 mL dry DMF.
After 5 min stirring at room temperature, the ethyl
＋
M＝703.78, obsvd. (M ＋H) ＝704.3077.
3
-bromopropionate (7.7 mmol) was added dropwise.
Hydrogel formation and characterizations
The reaction mixture was heated at 50 ℃ for 42 h. Af-
ter the reaction mixture was cooled back to room tem-
perature for 30 min, it was diluted with DCM (15 mL)
and washed with water (10 mL×3). The combined or-
ganic layers were dried over magnesium sulfate, filtered,
and rotary-evaporated to give a yellow liquid. The crude
product was purified on a flash silica column with
EtOAc/hexanes (4% to 8% to 15%) as eluents to give
the indolepropionic ethyl ester, which was then hydro-
lyzed in EtOH/MeOH/KOH (25∶25∶8, 116 mL) at
Preparation of peptide hydrogels Compound 1
or 2 was dispersed in sterile endotoxin-free PBS buffer
(
pH＝7.4) at a final concentration of 0.2 wt% with so-
dium carbonate to adjust the final pH at 7.4. The sus-
pension was then heated to 80 ℃ in order to dissolve
the compound completely, then the hot solution was
cooled back to room temperature (25 ℃). The hydrogel
formed after about 60 min at room temperature.
Rheology Rheology test was carried out on an AR
2
000ex (TA instrument) system, 40 mm parallel plates
6
0 ℃ for 30 min. The reaction mixture was poured
into 100 mL water and acidified by slow addition of
0% citric acid until the salt was converted completely
were used during the experiment at the gap of 500 μm.
For the dynamic time sweep, the solution of compounds
was directly transferred to the rheometer and it was
conducted at the frequency of 1 rad/s and the strain of
1
to the free acid. The resulting crystal was filtered and
washed with cold water. The crude acid was recrysal-
lized from ethanol to afford the final product.
1
% immediately. The gels were also characterized by
Synthesis of peptides Both Indol-Gly-Phe-Phe-
Tyr-OH (Indol-GFFY, 1) and Indol-Gly-D-Phe-D-Phe-
the mode of dynamic frequency sweep in the region of
0.1－100 rad/s at the strain of 1%. All samples were
tested at temperature of 37 ℃.
D
D D
D-Tyr-OH (Indol-G F F Y, 2) were prepared by
[
10]
standard solid phase peptide synthesis (SPPS) by us-
ing 2-chlorotrityl chloride resin and the corresponding
N-Fmoc protected amino acids with side chains properly
protected. Firstly, the C-terminal of the first amino acid
was conjugated on the resin. Anhydrous N,N'-dimethyl
formamide (DMF) containing 20% piperidine was used
to remove Fmoc protected group. To couple the next
amino acid to the free amino group, HBTU was used as
coupling reagent. Peptides chain was extended accord-
ing to the standard SPPS protocol. After the 1-indole-
propionic acid coupling the glycine, 5 times DMF wash
followed by 5 times DCM wash were used to remove
excessive reagents. Lastly, 95% TFA containing 2.5%
TEM sample preparation
Negative staining
technique was used to observe the TEM images of mi-
cro structure in hydrogels. Firstly, 10 μL hydrogels were
loaded on the 400 mesh copper grids coated with con-
tinuous thick carbon film, dd-water was then used to
rinse grid twice. After rinsing, the grids containing sam-
ples were stained with 2 % uranyl acetate. Finally the
grids were allowed to dry in air.
Preparation of peptide/OVA gels 20 L OVA
stock solution (10 mg/mL) was added and mixed with
980 L hydrogel with pipette at room temperature
(25 ℃). Then the OVA was dispersed in the hydrogel
evenly by vortex and the mixture became a sticky liquid.
The final concentration of OVA in hydrogel was 200
g/mL. After about 60 min, the sticky liquid formed
hydrogel again and became stable. When conducting
immunization, first shakes the OVA/hydrogel and then
aspirates the hydrogel with a syringe.
2
H O and 2.5% TIS was used to cleave peptides deriva-
tive from resin and the mixture was filtered. Ice cold
diethylether was poured into filtrate performed by rotary
evaporation. The precipitate was centrifuged for 5 min
at 5000 r/min speed. The solid was dried by vacuum
pump to gain resulting compounds. We then obtained
the pure compounds by reverse phase high performance
Immunizations For in vivo immune evaluation,
female C57BL/6J mice were randomly divided into 4
2
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Chin. J. Chem. 2017, XX, 1—6

Supramolecular Hydrogels of Indole-Capped Short Peptides as Vaccine Adjuvants
groups and each group contained 6 mice. 4 groups mice
were each given 100 μL subcutaneous injections (0.2
wt% supramolecular hydrogels composed of 20 μg OVA
vaccine, 100 μL PBS with 20 μg OVA and 20 μg OVA
with 25 times alum, respectively). All immunizations
were given 14 days apart. Serum was collected at 7 d
after the second immunization for the antibody detec-
tion.
tide FF or tripeptide FFY had excellent self-assembling
and gelation properties.
[11]
Scheme 1 The chemical structures of possible hydrogelators,
D
D D
Indol-GFFY (1) and Indol-G F F Y (2)
O
O
O
H
H
N
N
Determination of antibody titers OVA-specific
antibody responses in mice were examined by ELISA.
N
N
N
OH
H
H
O
O
9
6-well ELISA plates were coated with 10 μg/mL OVA
antigen and stored at 4 ℃ overnight. After three wash-
es with PBST (PBS buffer containing 0.05% tween 20),
the plates were blocked by 1% BSA buffer (1% BSA in
PBST solution) for 1 h at room temperature. Individual
antisera were serially diluted in the 1% BSA buffer and
incubated in the wells for 2 h at 37 ℃. After five wash-
es with PBST, the wells were incubated with goat an-
ti-mouse IgG horseradish peroxidase for 1 h at 37 ℃.
After washing, antibody binding was assessed by adding
OH
1
O
O
O
H
H
N
N
N
N
N
OH
H
H
O
O
OH
2
1
00 μL 3,3',5,5'-tetramethyl-benzidine peroxidase sub-
strate to each well. The substrate reaction was terminat-
－
1
After synthesis and purification by HPLC, the gela-
tin properties of these two compounds were tested. Like
our previous results of Nap-GFFY, Fmoc-GFFY and
ed by adding 50 μL of 2 mol•L
2 4
H SO . Antibody iso-
types were determined similarly using goat anti-mouse
IgG1, IgG2a and IgG2b horseradish peroxidase. The
plates were then read by an ELISA reader at an optical
density of 450 nm. Antibody titers were calculated as
the reciprocal serum dilution giving O.D. readings＞0.1
standard deviations above background levels as calcu-
lated using PBS at the same dilutions.
Splenocytes cytokine production At 7 days after
the second immunization, splenocytes (5×106 cells/ml)
were collected and seeded in 24-well plates, and then
were re-treated with soluble OVA (50 μg/mL) at 37 ℃
for 96 h. The production of IFN-γ and IL-5 in cell cul-
ture supernatants was detected by using ELISA kit (Bi-
ologend, San Diego, CA, USA).
[12]
PTZ-GFFY could gel aqueous solutions efficiently,
compound 1 and its D-enantiomer, compound 2 could
also form transparent hydrogels in phosphate buffer sa-
line (PBS, pH 7.4) solutions via a heating-cooling pro-
cess (Figure 1A, L-gel and D-gel, respectively) in 60
min. The minimum gelation concentration (MGC) for
both compounds 1 and 2 was about 1.2 mg/mL.
We then characterized L-gel, D-gel, L-gel/OVA and
D-gel/OVA by a rheometer and transmission electron
microscopy (TEM). As shown in Figure 1E, both gels
exhibited weak frequency dependences at the range of
0
.1－100 rad/s. The elasticity (G' or storage modulus)
value of both gels was about 10 times bigger than their
corresponding viscosity (G'' or loss modulus) value,
which means they were all true hydrogels. The G' value
of all gels was more than 1000 Pa. These observations
implied medium mechanical properties of all gels.
However, the incorporation of OVA in gels made the
gels slightly stronger. We observed flexible and uniform
nanofibers in L-gel, D-gel, L-gel/OVA and D-gel/OVA
Cytotoxicity assays Biocompatibility of L-gel
D-gel was evaluated by an MTT assay. The RAW 264.7
cells were seeded in 96-well plates at a density of 5000
cells per well with medium volume of 100 μL. After
incubation for 24 h, 100 μL fresh medium containing a
serial of concentrations of two gels was added. 72 h lat-
er, 10 μL MTT (5 mg/mL) was added into cells. After 4
h, DMSO was added to dissolve the formazan crystals.
The optical density of the solutions at 597 nm was
measured by a microplate reader. Meanwhile, cells
without any treatment were used as the control. The
percent of cell viability was calculated according to the
following formula:
(
Figures 2A, 2B, 2C and 2D) and these flexible nano-
fibers entangled with each other to form three dimen-
sional networks for hydrogelation. The diameter of nan-
ofibers in L-gel and D-gel was similar and it was about
1
5－25 nm, and the incorporation of OVA in gels made
the size of nanofibers slightly bigger (30－35 nm),
which was likely the reason for the stronger mechanical
properties of gels containing OVA.
The percent of cell viability＝ODsample/ODcontrol
×
1
00%.
In order to understand the molecular arrangement of
peptides in nanofibers, we collected circular dichroism
(CD) and emission spectra of solutions and gels. As
shown in Figure 3A, we observed a positive peak near
194 nm (* transition) and negative bands at 208 nm
Results and Discussion
We designed two possible gelators (Indol-GFFY (1)
D
D D
and Indol-G F F Y (2) in Scheme 1) based on short
peptide of GFFY, because many compounds with dipep-
Chin. J. Chem. 2017, XX, 1—6
© 2017 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Wang et al.
FULL PAPER
Figure 1 Optical images of (A) L-gel containing 0.2 wt% of 1,
(
(
B) D-gel containing 0.2 wt% of 2, (C) L-gel containing OVA
L-gel/OVA) and (D) D-gel containing OVA (D-gel/OVA). (E)
Dynamic frequency sweep of L-gel, D-gel, L-gel/OVA and
D-gel/OVA at 37 ℃. (For rheological characterization, three
samples were tested for each group, one of the results was shown
in this figure and the rest were shown in Supporting Information.)
Figure 3 (A) The circular dichroism (CD) spectra of the hy-
drogels and (B) the emission spectra (excitation wavelength＝
260 nm) of 1 and 2 in solution and in hydrogel. (C) Possible mo-
lecular arrangement of the peptide in nanofibers.
wt%), while PTZ-GFFY was a super-gelator with an
MGC value of 0.01 wt%. These results were consistent
with the conclusion in our previously published work
that PTZ-GFFY with an additional glycine could form
more extensive hydrogen bonds than peptides with cap-
[12a]
ping groups such as Fmoc, Nap or Indol.
We found a
near mirror CD spectrum from D-gel, compared with
that from L-gel, suggesting that compound 2 also
adopted -sheet conformation in the gel. Figure 3B
showed the emission spectra of both solutions and gels.
Both solutions of compounds 1 and 2 exhibited a peak
centered at 349 nm. This peak showed a red-shift to
about 368 nm in both gels, suggesting that the packing
of indole groups was more efficient in gel stage than
that in solutions. Based on these information, we pro-
posed a possible molecular arrangement of the peptide
in nanofibers (Figure 3C). Assisted by the -interac-
tions between aromatic capping group of indole and
hydrogen bond interactions between peptides (shown as
purple ribbons with arrows), compound 1 or 2 adopted
an anti-parallel packing mode with each other, and
therefore helped to extend the supramolecular chain to
form nanofibers.
Figure 2 TEM image of (A) L-gel, (B) D-gel, (C) L-gel/OVA
and (D) D-gel/OVA.
(
n* transition) from L-gel, suggesting that compound 1
[13]
adopted the -sheet conformation in the gel. We did
not observe distinct peaks at wavelength longer than
2
50 nm, suggesting that the indole moiety failed to
adopt helical arrangement. The CD spectrum pattern
from L-gel was similar to those from gels of Nap-Gly-
Phe-Phe-Tyr-OH (Nap-GFFY) and Fmoc-Gly-Phe-
Phe-Tyr-OH (Fmoc-GFFY), but slightly different from
that from gel of PTZ-Gly-Phe-Phe-Tyr-OH (PTZ-GFFY)
that possessed a negative peak at about 254 nm. The
MGC value of compound 1 was therefore similar to
those of both Nap-GFFY and Fmoc-GFFY (0.08－0.1
4
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© 2017 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2017, XX, 1—6

Supramolecular Hydrogels of Indole-Capped Short Peptides as Vaccine Adjuvants
We then tested the potency of both gels as vaccine
adjuvants. The protein ovalbumin (OVA) was chosen as
a model antigen to be physically mixed with the gel at a
weight ratio of (peptide∶OVA＝10∶1). Four groups
of C57BL/6J mice were injected with OVA, aluminium
hydroxide containing OVA (Alum∶OVA＝25∶1),
L-gel containing OVA (L-gel), D-gel containing OVA
IgG antibody and its isotope productions with FDA ap-
proved Alum adjuvant. Even though both gels promoted
the production of IFN-γ and IL-5 more than free OVA,
cytokines did not reach the desired levels like Alum
(Figure 5). These observations suggested that our hy-
drogels
(D-gel), respectively at day 0. A secondary immuniza-
tion was performed at day 14. The bleed of all mice
were collected at day 21 and an ELISA assay was per-
formed to determine the anti-OVA IgG titer. As shown
in Figure 4A, soluble OVA group exhibited poor im-
mune responses, while both hydrogel groups showed
significantly higher anti-OVA IgG antibody productions.
D-gel group was more potent to increase IgG production
than L-gel group, which was similar to our recent results
that the other two D-peptide-based hydrogels could in-
crease anti-OVA titer more significantly than their
Figure 5 The production of IFN-γ and IL-5 in splenocytes cul-
ture supernatants (**: p＜0.01, ***: p＜0.001).
[
5b,5c]
L-counterparts.
For IgG isotopes, both gels could
significantly increase the production of IgG1, IgG2a,
and IgG2b than free OVA, and D-gel was more potent
than L-gel (Figures 4B, 4C and 4D). We believe the
reason for D-gel showing more efficacy than L-gel is the
same with that proposed in our recently published work,
that the release of OVA from D-gel was faster than that
from L-gel in lysosome because the binding between
protein OVA and L-nanofiber was stronger than that
between OVA and D-nanofiber, resulting in more anti-
gen escape from lysosome and release into cytosol,
which were important for stronger immune responses.
might be used as vaccine adjuvants to raise humoral
immune response, which was similar to Alum adjuvant.
Moreover, we also evaluated the cytotoxicity of both
gels against immune cells of RAW 264.7 cells by an
MTT assay (Figure 6), and demonstrated that both gels
have high biocompatibility, which confers them great
potential in applications of biomedicine.
Figure 6 Cytotoxicity assay of L-gel and D-gel.
Conclusions
Our study provides a novel candidate of aromatic
capping group (Indole) to construct short peptide-based
hydrogelators. Our hydrogels, especially the D-peptide
gel shares a similar potency with Alum as a vaccine ad-
juvant to raise humoral immune response. The good
biocompatibility, ease of synthesis, and ease of vaccine
formulation suggest its good promise for vaccine pro-
duction. One shortcoming of the gels in this study is
their poor potency to raise cellular immune response,
which is believed to be very important for the develop-
ment of vaccines to protect against or treat cancers, HIV,
and malaria. We will opt to develop more gels and test
their potency to raise both humoral and cellular immune
responses.
Figure 4 The production of anti-OVA IgG antibodies and its
isotopes in plasma on day 21 determined by ELISA (*: p＜0.05,
*
*: p＜0.01).
Our D-gel showed a comparable potency to evoke
Chin. J. Chem. 2017, XX, 1—6
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5

Wang et al.
FULL PAPER
Acknowledgement
Xu, B. Angew. Chem., Int. Ed. 2014, 53, 8104; (c) Li, J.; Kuang, Y.;
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This work is supported by National Natural Science
Foundation of China (31370964, 51403105), Science
and Technology Guiding Project of Guangdong Prov-
ince (2013B091500071), and Program for Changjiang
Scholars and Innovative Research Team in University
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