Nanoscale
PAPER
Ci e his: Nanoscale, 2024, 16, 9887
Recei ed 22nd Janua y 2024,
Accep ed 16 h Ap il 2024
DOI: 10.1039/d4n 00335g
sc.li/nanoscale
Sal -induced Fmoc- ipep ide sup amolecula
hyd ogels: a combined expe imen al and
compu a ional s udy o he sel -assembly†
Mi yam C iado-Gonzalez, *
a,b
Ma io I án Peñas,
a,b
Flo en Ba baul ,
c
Alejand o J. Mülle ,
b,d
Fouzia Boulmedais
e
and Rebeca He nández
a
Del ing in o he mechanism behind he molecula in e ac ions a he a omic le el o sho -sequence
pep ides plays a key ole in he de elopmen o nanoma e ials wi h specific s uc u e–p ope y– unc ion
ela ionships om a bo om-up pe spec i e. Due o hei poo wa e solubili y, he sel -assembly o
Fmoc-bea ing pep ides is usually induced by dissolu ion in an o ganic sol en , ollowed by a dilu ion s ep
in wa e , pH changes, and/o a hea ing–cooling p ocess. He ein, we epo a s aigh o wa d me hod-
ology o he gela ion o Fmoc-FFpY (F: phenylalanine; Y: y osine; and p: PO
42−
), a nega i ely cha ged i-
pep ide, in NaCl solu ion. The elec os a ic in e ac ions be ween Fmoc-FFpY and Na
+
ions gi e ise o
diffe en nanofib illa hyd ogels wi h heological p ope ies and nanofibe sizes modula ed by he NaCl
concen a ion in pu e aqueous media. Ini ia ed by he elec os a ic in e ac ions be ween he pep ide
phospha e g oups and he Na
+
ions, he pep ide sel -assembly is s abilized hanks o hyd ogen bonds
be ween he pep ide backbones and he π–πs acking o a oma ic Fmoc and phenyl uni s. The hyd ogels
showed sel -healing and he mo- esponsi e p ope ies o po en ial biomedical applica ions. Molecula
dynamics simula ions om sys ems de oid o p io aining no only confi m he agg ega ion o pep ides
a a c i ical sal concen a ion and he diffe en in e ac ions in ol ed, bu also co obo a e he seconda y
s uc u e o he hyd ogels a he mic osecond imescale. I is wo h highligh ing he ema kable achie e-
men o ep oducing he mo phological beha io o he hyd ogels using a omis ic simula ions. To ou
knowledge, his s udy is he fi s o epo such a co espondence.
In oduc ion
Sequence-de ined pep ides capable o sel -assembly in o
sup amolecula low-molecula -weigh hyd ogels (LMWH) wi h
con olled nanos uc u es and s imuli- esponsi e p ope ies
ha e a ac ed conside able a en ion in se e al ields, includ-
ing chemis y, physics, biology, ma e ials science, and
nano echnology.
1–5
Pep ide sequences sho e han i e amino
acids a e cu en ly he ocus o esea ch due o hei lowe syn-
hesis cos s compa ed o longe polypep ides and ela i e ease
o modula ion compa ed o la ge biomac omolecules.
6,7
Fo med by non-co alen in e ac ions, i.e., hyd ogen-bonding,
hyd ophobic, a oma ic, and/o elec os a ic in e ac ions,
8
sup amolecula pep ide sel -assemblies a e capable o e ain-
ing wa e wi hin hei s uc u e while possessing shea - hin-
ning and sel -healing p ope ies essen ial o minimally in a-
si e injec abili y ea men s and 3D p in ing.
9
This ype o
sup amolecula hyd ogel wi h physical simila i y o human
issues has excellen p ope ies o be used in he biomedical
ield, i.e., bio-inks, d ug deli e y, ca alysis, o issue enginee -
ing, among o he s.
10,11
N-Fluo enyl-9-me hoxyca bonyl (Fmoc), used as an amine-
p o ec ing g oup du ing he pep ide syn hesis, p o ides in e -
es ing sel -assembly p ope ies o he pep ide chain by p omo -
ing hyd ophobic and π–πs acking in e ac ions o luo enyl
ings.
6,12,13
Among he ple ho a o Fmoc-bea ing pep ides,
hose con aining phenylalanine (F) amino acid in he pep ide
sequence ha e been ex ensi ely s udied.
14–16
Due o hei poo
solubili y in wa e , he sel -assembly o Fmoc-F-de i ed pep-
ides is usually induced by dissolu ion in dime hyl sul oxide
(DMSO), ollowed by a dilu ion s ep in wa e , by pH changes,
and/o by a hea ing–cooling p ocess,
17–20
which make he
sys ems no ully biologically iendly, limi ing hei inal
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d4n 00335g
a
Ins i u o de Ciencia y Tecnología de Políme os (ICTP-CSIC), 28006 Mad id, Spain.
E-mail: [email p o ec ed]
b
POLYMAT and Depa men o Polyme s and Ad anced Ma e ials: Physics, Chemis y
and Technology, Facul y o Chemis y, Uni e si y o he Basque Coun y UPV/EHU,
20018 Donos ia-San Sebas ián, Spain
c
ITODYS, Uni e si é de Pa is, CNRS, F75006 Pa is, F ance
d
Ike basque, Basque Founda ion o Science, Plaza Euskadi 5, 48009 Bilbao, Spain
e
Uni e si é de S asbou g, CNRS, Ins i u Cha les Sad on (UPR 22), 67034
S asbou g, F ance
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applica ions. Gazi and cowo ke s s udied he assembly o
Fmoc-FF in conjunc ion wi h 4,4-bipy idine, esul ing in a con-
o ma ional change om a β-shee o a helix s uc u e o
educe amyloid-associa ed diseases.
21
In addi ion, hey s udied
he assembly o long polypep ide chains wi h mo e han 40
amino acids con aining FF uni s in hei s uc u e, which we e
p e iously solubilized in DMSO in he p esence o diffe en
mono alen , di alen , and i alen me al ion sal s, esul ing
in diffe en sel -assembled seconda y s uc u es, supe helices,
β-shee s, and andom coils, as a unc ion o he coo dina ion
me al ion used.
22
To mimic physiological sel -assembly condi ions, Xu and
cowo ke s designed phospho yla ed Fmoc-pep ides h ough
he inco po a ion o a y osine phospha e (pY) g oup, Fmoc-
pY, which con e ed solubili y p ope ies in a pu e aqueous
solu ion a oom empe a u e and enabled i s con e sion in o
he LMWH (Fmoc-Y) in he p esence o alkaline phospha ase
(AP).
23–25
The inc ease o a oma ic side-chain moie ies could
con e highe sel -assembly yields, he mal s abili y, and elas-
ici y o Fmoc-pep ide-based hyd ogels;
26
he e o e he in o-
duc ion o side-chain phenyl ings was la e conside ed,
leading o he ipep ide Fmoc-FFpY. The localized enzyme-
assis ed sel -assembly (LEASA) o he Fmoc-FFpY ipep ide
ga e ise o sup amolecula Fmoc-FFY hyd ogels in a β-shee
ib illa ne wo k wi h unable mechanical and biological
p ope ies.
27–31
Ne e heless, β-shee - ich assemblies a e
associa ed wi h some amyloid degene a i e diseases such as
ype 2 diabe es, Alzheime ’s disease, and Pa kinson’s disease,
which makes i necessa y o egula e he s uc u al a ange-
men o a o he o ma ion o α-helix o andom coil
con o ma ions.
32,33
This was achie ed by inducing Fmoc-FFpY
sel -assembly h ough elec os a ic in e ac ions wi h posi i ely
cha ged polyme nanopa icles, which led o he appea ance
o an α-helix s uc u e wi h a educ ion in β-shee assem-
blies.
34
The associa ion wi h posi i ely cha ged polyme chains
led only o an inc ease in he andom s uc u e.
35
Knowing ha biological unc ions a e based on molecula
in e ac ions, which in u n a e a consequence o mac omolecu-
la s uc u es, molecula dynamics (MD) simula ions a bio-
logically ele an simula ion imes a e an effec i e ou e o
unde s and LMWH sel -assembly p ocesses and s uc u e–
unc ion ela ionships.
36,37
Thus, del ing in o he mechanism
behind he molecula in e ac ions a he a omic le el allows us
o explo e he dynamic eo ganiza ion and plas ici y wi hin he
sys ems and plays a key ole in he de elopmen o nano-
ma e ials wi h speci ic s uc u e–p ope y– unc ion ela ion-
ships om a bo om-up pe spec i e.
38
While p e ious esea ch
wo ks s udied he pep ide sel -assembly o p e-o ganized con-
igu a ions in he p esence o sal s using densi y unc ional
heo y (DFT) a he nanosecond scale,
18,39–41
and a maximum
pep ide : sal mix u e mola a io o 1 : 3,
22,42,43
unde s anding
wha happens a he a omic scale and highe pep ide : sal con-
cen a ions emains a challenge.
We epo he ein a s aigh o wa d me hodology o
o ming sup amolecula Fmoc-FFpY sel -assembly in pu e
aqueous media by iono opic gela ion wi h sodium chlo ide
(Scheme 1), hus a oiding he use o o ganic sol en s and
polyme nanopa icles and esembling physiological mimick-
ing assembly p ope ies. Ou expe imen al esul s show ha
he pep ide sel -assembly was ini ia ed by he elec os a ic
in e ac ions be ween he pep ide phospha e g oups and Na
+
ions and s abilized hanks o hyd ogen bonds be ween he
pep ide backbones and he π–πs acking o a oma ic Fmoc and
phenyl uni s. To ob ain in o ma ion a he a omic le el, com-
pu a ional s udies we e pe o med om sys ems de oid o
p io aining o e alua e (i) he abili y o he pep ides o spon-
aneously agg ega e, explo ing he ini ial oligome iza ion and
unde lying mechanisms and (ii) he s uc u al a angemen
induced by he agg ega ion o Fmoc-FFpY pep ides a he
mic osecond imescale, o ming nano ibe s and nano ods.
Finally, he he mo- e e sibili y and heological p ope ies o
he sup amolecula pep ide hyd ogels we e assessed as a unc-
ion o he pep ide and NaCl concen a ion. I opens a ou e
o he p edic ion o pep ide sel -assembly a he a omic le el,
which is o pa amoun impo ance o he design and de elop-
men o unc ional nanoma e ials.
Ma e ials and me hods
Ma e ials
Fmoc-FFpY ≥86.0% was p o ided by Pepmic (Suzhou, China)
and cha ac e ized by high-pe o mance liquid ch oma og aphy
(HPLC) and mass spec ome y (MS) (Fig. S1, S2 and Table S1
in he ESI†). The impu i y is an isome ha has he same
chemical o mula. Sodium e abo a e anhyd ous (bo ax) was
pu chased om Sigma-Ald ich and sodium chlo ide was pu -
chased om Fluka. All ma e ials we e used as ecei ed.
Hyd ogel o ma ion induced by Na
+
ca ions
Hyd ogels we e o med by mixing Fmoc-FFpY solu ions (in
25 mM bo ax buffe a pH 9.5) wi h diffe en concen a ions o
NaCl aqueous solu ions a a a io o 1 : 1 ( / ). A ypical hyd o-
gel o med a a concen a ion o 6.4 mM Fmoc-FFpY and
Scheme 1 Schema ic ep esen a ion o Fmoc-FFpY sel -assembly
induced by elec os a ic in e ac ion wi h Na
+
ions, leading o he o -
ma ion o sup amolecula hyd ogels, Fmoc-FFpY/Na
+
.
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50 mM NaCl is named Fmoc-FFpY6.4/Na
+
50. The inal hyd o-
gel olume was 200 µL o in e ed ube es s and 130 µL o
heological es s. The inal pH o he hyd ogels is 8.
Ze a po en ial
The ze a po en ial (ξ) was measu ed by Lase Dopple
Elec opho esis (LDE) using a Mal e n Nanosize NanoZS
ins umen equipped wi h a 4 mW He–Ne lase (λ= 633 nm) a
a sca e ing angle o 173° a 25 °C. Th ee measu emen s o 20
uns we e pe o med o each sample.
Mo phological cha ac e iza ion
The mo phology o he hyd ogels was obse ed by
T ansmission Elec on Mic oscopy (TEM) using a JEOL
JEM-1230 elec on mic oscope equipped wi h a digi al came a
CMOS TVIPS Tem-Cam 16 megapixel. Samples we e obse ed
unde nega i e s aining by incuba ing he Fmoc/Na
+
hyd ogels
wi h a hea y me al sal solu ion, o med by using 1% u anyl
ace a e and 1% phospho ungs ic acid, o 5 min, ollowed by
2 min o washing and b ough in con ac wi h a ca bon-coa ed
coppe g id. Images we e aken a 100 000 V and a magni i-
ca ion o 10 000.
Small-angle X- ay sca e ing (SAXS) and wide-angle X- ay
sca e ing (WAXS)
SAXS and WAXS measu emen s we e pe o med a BL 11
NCD-SWEET beamline a ALBA Synch o on (Ba celona, Spain)
using an X- ay wa eleng h o λ= 0.1 nm and an acquisi ion
ime o 20 s. Fo SAXS measu emen s, a PILATUS 1 M de ec o
om Dec is was loca ed a 6.70 m, and o WAXS measu e-
men s, a LX255-HS de ec o om Rayonix was loca ed a
0.125 m om he sample posi ion. SAXS and WAXS pa e ns
show he sca e ing in ensi y s. he sca e ing ec o qand
we e analyzed using ATSAS so wa e PRIMUS e sion 3.1 (SAS
da a analysis).
44
The backg ound co esponding o he capil-
la ies con aining he buffe sol en o he hyd ogels was sub-
ac ed om he spec a.
Spec oscopic cha ac e iza ion
Fluo escence spec a we e eco ded be ween 300 and 405 nm
a an exci a ion wa eleng h o 290 nm using a Pe kinElme LS
55 luo escence spec ome e a 25 °C. The sample was placed
be ween wo qua z slides, leading o a pa h leng h o abou
0.1 mm. In a ed (IR) spec a we e eco ded be ween 850 and
1760 cm
−1
in A enua ed To al Re lec ance (ATR) mode using a
Pe kinElme Spec um Two FT-IR spec ome e a 25 °C.
Samples we e p e iously d ied a oom empe a u e o emo e
wa e . To decompose he amide I band, da a p ocessing was
pe o med using OPUS 7.5 so wa e (B uke Op ik GmbH). The
spec a we e smoo hed using a wen y- i e-poin smoo hing
unc ion, cu be ween 1550 and 1710 cm
−1
, and hen no mal-
ized using a no maliza ion “min–max”me hod. The baseline
was hen adjus ed o calcula e he second o he ou h de i a-
i e. The numbe and equencies o diffe en componen s,
o ming he amide I band, and he o he peaks we e de e -
mined using he second de i a i e o he Fou ie smoo hed
spec um using he minimum posi ions. The decomposed
spec um was i ed wi h Gaussian band p o iles using local
leas squa es, ollowed by Le enbe g–Ma qua d ’s me hod,
s a ing wi h in ensi ies o 0.1 and wid hs o 5. The quali y o
i ing was es ima ed by he esidual RMS p o ided by he so -
wa e. The ela i e con ibu ion o each componen o he
amide I band was calcula ed om he a io o he a ea o each
peak o e he a ea o he o al amide I band. Ci cula dich o-
ism (CD) spec a we e eco ded a 25 °C be ween 190 and
320 nm using a Jasco J-815 spec opola ime e wi h a wa e-
leng h da a pi ch o 0.2 nm. Samples we e placed be ween
qua z slides, leading o a pa h leng h o abou 0.1 mm.
Compu a ional de ails
The Fmoc esidue was c ea ed using he Maes o so wa e.
45
The cons uc ion o he Fmoc-FFpY pep ide was gene a ed
using a p e iously published me hod.
46
Sys em assemblies
we e pe o med using he Packmol so wa e.
47
Two se ies o
sys ems we e ini ia ed. In he i s se ies, which aimed o
de e mine he in luence o NaCl concen a ion on agg ega ion
beha io , ou sys ems we e gene a ed whe e nine pep ide
uni s we e andomly placed wi hin a cube wi h an edge leng h
o 130 Å. I was ensu ed ha each pep ide was a a dis ance
g ea e han 20 Å o a oid in luencing he spon anei y o agg e-
ga ion. These ou sys ems we e sol a ed using a TP3P wa e
model,
48
and h ee diffe en NaCl concen a ions o 50, 250,
and 500 mM we e gene a ed. In he second se ies, aimed a
s udying pep ide assembly, ou sys ems consis ing o 40
Fmoc-FFpY uni s we e andomly placed wi hin a simula ion
cube wi h an edge leng h o 70 Å. These dimensions ha e been
chosen o place hem unde condi ions simila o hose o he
wo k o Sasselli e al.,
40
whe e he conside a ion o pe iodic
condi ions makes i possible o ep oduce he s uc u es in he
image boxes and hus o ake in o accoun he possible o -
ma ion o ibe s.
49
These ou sys ems we e sol a ed using he
same wa e model and s udied a wo diffe en concen a ions:
250 and 500 mM.
Molecula dynamics simula ions (MD) we e pe o med wi h
he Ambe so wa e e sion 20.
50,51
All simula ions s a ed
wi h 40 000 s eps o ene gy minimiza ion whe e he pep ides
emain cons ained wi h ha monic es ain s o 5 kcal mol
−1
o he i s 20 000 s eps and elease o he 20 000 o he s eps.
Sys ems we e hen hea ed o 300 K o 100 ps in he NVT
ensemble and hen swi ched o NTP condi ions. MD ajec-
o ies we e engaged o 1 µs, o he i s se ies o sys ems and
1.2 µs o he second se ies. S uc u al analyses we e pe -
o med wi h he cpp aj module o Ambe Tools,
52
while VMD
so wa e
53
was employed o isualize MD ajec o ies and make
igu es. Seconda y s uc u es we e assigned using backbone di-
hed al angle analysis. Two me hods we e used, one using
MolP obi y so wa e
54
and he o he using he de ini ions
gi en by Xiong and cowo ke s.
55
As bo h me hods gi e simila
esul s, we ha e only included he esul s om MolP obi y.
Unless explici ly s a ed o he wise, he p esen ed alues a e
a e aged ac oss he 4 simula ion eplicas.
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Rheological cha ac e iza ion
The heological p ope ies o he hyd ogels we e measu ed
using an AR-G2 heome e (TA Ins umen s) wi h an ac ylic
pla e geome y o 40 mm diame e , a 58 µm gap, and a sol en
ap. Samples we e p epa ed di ec ly on he pla e by mixing
65 µL o Fmoc-FFpY and 65 µL o NaCl solu ion o 30 min a
20 °C un il a pla eau was eached. S ain measu emen s we e
ca ied ou om 0.01% o 1 000% a 1 Hz and equency
sweeps om 100 o 0.01 Hz a 1% s ain. Tempe a u e sweeps
we e pe o med a 1% s ain and 1 Hz. The sel - eco e y p o-
pe ies we e e alua ed h ough dynamic s ep s ain ampli ude
es s by a ying he s ain be ween 1% and 1 000%.
Mic o-diffe en ial scanning calo ime y (mic o DSC)
Mic o-DSC expe imen s we e ca ied ou using a Mic oCal e
VII mic ocalo ime e (Se a am) equipped wi h a double-s age
empe a u e con ol wi h Pel ie coole s. Has elloy C276
essels wi h an elas ome O- ing (NBR) we e employed o he
measu emen s. The sample cell was illed wi h he Fmoc-
FFpY/Na
+
hyd ogel and he e e ence cell wi h a mix u e
(1 : 1% / ) o bo ax buffe , and he co esponding sal concen-
a ion was es ed in each case. The hea ing and cooling scans
om 20 o 75 °C we e eco ded a hea ing and cooling a es o
0.1 °C min
−1
.
Resul s and discussion
Fmoc-FFpY/Na
+
hyd ogel o ma ion and mo phological
cha ac e iza ion
Thanks o he y osine phospha e amino acid (pY), he Fmoc-
FFpY ipep ide can be solubilized in wa e wi hou o ganic
sol en s, such as DMSO. The Fmoc-FFpY solu ion (in 25 mM
bo ax buffe a pH 9) does no o m a gel e en a e se e al
days. The hyd ogela ion o Fmoc-FFpY can be induced wi hou
dephospho yla ion by he addi ion o NaCl. The in e ed ube
es , a commonly accep ed me hod o sc eening whe he a gel
has been o med, was i s pe o med a a ixed concen a ion
o he pep ide (6.4 mM) and diffe en NaCl concen a ions.
The minimum NaCl concen a ion needed o o m a hyd ogel
was 150 mM (mola a io: Fmoc-FFpY : NaCl = 1 : 23) (Fig. 1a).
Fig. 1 (a) In e ed ube es s o sup amolecula Fmoc-FFpY6.4/Na
+
hyd ogels o med a e 24 h a a fixed Fmoc-FFpY concen a ion o 6.4 mM. (b)
Phase diag am o Fmoc-FFpY/Na
+
mix u es a mola concen a ion. (c) Ze a po en ial o Fmoc-FFpY6.4/Na
+
hyd ogels as a unc ion o NaCl concen-
a ion. The dashed lines a e a guide o he eye. (d) TEM mic og aphs o nega i ely s ained Fmoc-FFpY6.4 hyd ogels wi h diffe en NaCl concen-
a ions. The whi e a ows highligh he nanofibe ’s o ganiza ion in o a nano od-like s uc u e.
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A phase diag am was es ablished as a unc ion o pep ide
and sal concen a ions (Fig. 1b). In all cases, he hyd ogels
ob ained we e anspa en and colo less. As he pep ide con-
cen a ion dec eases, a highe sal concen a ion is needed o
o m a gel. Fo 3.2 and 1.3 mM pep ide concen a ions, he
equi ed mola a io o Fmoc-FFpY : NaCl inc eases up o 1 : 78
and 1 : 385, espec i ely (Fig. 1b and Fig. S3†). In he absence
o NaCl, Fmoc-FFpY (in 25 mM bo ax buffe a pH 9) is nega-
i ely cha ged wi h a ze a po en ial o −36.0 ± 2.0 mV (Fig. 1c).
A NaCl concen a ions lowe han 250 mM, he addi ion o
NaCl does no signi ican ly impac he pep ide’s ze a po en ial.
Fo highe NaCl concen a ions, abo e 250 mM, he ze a
po en ial o he hyd ogels dec eases up o −18.6 ± 1.0 mV and
−9.6 ± 1.0 mV o NaCl concen a ions o 375 and 500 mM,
espec i ely. This is indica i e o he elec os a ic in e ac ions
in ol ed in he hyd ogel o ma ion be ween he nega i ely
cha ged g oups o he Fmoc-FFpY pep ide and he posi i ely
cha ged Na
+
ions.
56
The mic os uc u es o Fmoc-FFpY6.4/Na
+
hyd ogels p e-
pa ed a diffe en NaCl concen a ions we e in es iga ed by
TEM. Fmoc-FFpY in solu ion o med some nanod ople s
(Fig. S4†), whe eas in he p esence o e y low NaCl concen-
a ions (50 mM), isola ed nano ibe s we e obse ed (Fig. 1d).
Inc easing he NaCl concen a ion up o 150 mM led o a high
densi y o in e mingled hin nano ibe s wi h an a e age dia-
me e o ∼5 nm. As he NaCl concen a ion inc eases u he ,
he nano ibe s ended o be g ouped and aligned, leading o
he o ma ion o bundles wi h a e age diame e s o ∼29 and
46 nm o 375 and 500 mM NaCl, espec i ely, whe eas he
a e age diame e o e e y single nano ibe emains cons an a
∼5 nm (Fig. S5†). Small-angle X- ay sca e ing (SAXS) cu es
suppo ed he od-like mo phology o he nano ib ils as he
sca e ing cu es o he Fmoc-FFpY6.4/Na
+
hyd ogels o med
in 150, 250, and 500 mM NaCl showed a q
−1
ela ionship a
low q, indica ing elonga ed cylind ical s uc u es a NaCl con-
cen a ions abo e 150 mM (Fig. S6a†).
To del e in o he in e nal s uc u e o he nanoobjec s a
he molecula scale, wide-angle X- ay sca e ing (WAXS)
measu emen s we e pe o med (Fig. S6b†). The WAXS spec a
o Fmoc-FFpY6.4 in solu ion and Fmoc-FFpY6.4/Na
+
hyd ogels
(Fig. S6b†) show h ee maxima a 1.86, 1.53, and 0.98 Å
−1
,
co esponding o dis ances o 3.3, 4.1, and 6.4 Å, espec i ely,
ega dless o he NaCl concen a ion. They can be asc ibed o
he dis ance be ween hyd ogen-bonded backbones in he
β-shee s,
57
as he pep ide molecules ended o be agg ega ed
be ween hem, o ming nanod ople s in solu ion (in bo ax
buffe ),
58
which hen e ol ed o nano ibe o ma ion in he
p esence o Na
+
ions by inc easing he numbe o pep ide
molecules.
Seconda y s uc u e o Fmoc-FFpY/Na
+
sel -assembly
The s acking o he Fmoc moie ies was e i ied a e hyd ogel
o ma ion by luo escence spec oscopy (Fig. 2a and Fig. S7†).
The luo escence spec um o 6.4 mM Fmoc-FFpY solu ion
showed a peak a 315 nm, which is assigned o non-assembled
luo enyl moie ies. The same peak appea ed in he case o
Fmoc-FFpY6.4/Na
+
50, whe e no en angled nano ibe ne wo k
was obse ed by TEM (Fig. 1d). Inc easing he NaCl concen-
a ion esul ed in a ed shi o he luo escence band up o
332 nm o Fmoc-FFpY6.4/Na
+
500 due o luo enyl excime o -
ma ion a e he pep ide assembly, as con i med by he ib il-
la en angled ne wo k isualized by TEM. These esul s a e
Fig. 2 (a) Fluo escence spec a no malized o he peak a 315 nm, (b)
ATR-FTIR spec a, and (c) CD spec a o Fmoc-FFpY in solu ion in he
absence (dashed black cu e) and in he p esence o diffe en NaCl con-
cen a ions: Fmoc-FFpY6.4/Na
+
50 (pu ple cu e), Fmoc-FFpY6.4/
Na
+
150 (g een cu e), Fmoc-FFpY6.4/Na
+
250 ( ed cu e), Fmoc-
FFpY6.4/Na
+
375 (blue cu e), and Fmoc-FFpY6.4/Na
+
500 (pink cu e).
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consis en wi h p e ious wo k on he sel -assembly o Fmoc-
FFpY h ough elec os a ic in e ac ions wi h posi i ely cha ged
polyme s.
35,59
To u he in es iga e he assembly mechanism
o Fmoc-FFpY/Na
+
, he seconda y s uc u e was i s s udied by
ATR-FTIR spec oscopy (Fig. 2b). The ATR-FTIR spec um o
6.4 mM Fmoc-FFpY in solu ion shows a peak a 1688 cm
−1
co esponding o ca bama es,
60
he amide I band wi h wo
peaks a 1649 and 1640 cm
−1
assigned o uns acked and
β-shee s uc u es, and wo peaks a 980 and 872 cm
−1
assigned o phospha e g oups.
61
In he case o he Fmoc-
FFpY6.4/Na
+
500 hyd ogel, he in ensi y o he amide I band is
loca ed a 1644 cm
−1
assigned o β-shee s uc u es. The p es-
ence o he phospha e peaks indica es ha he pep ide is no
dephospho yla ed. The appea ance o a peak a 910 cm
−1
,
cha ac e is ic o C–OH bending, could indica e elec os a ic
in e ac ions be ween he ca boxylic acids and he sodium
ca ions.
62
To gain u he s uc u al insigh , he amide I band was
decomposed o iden i y he con ibu ions o he seconda y
s uc u es adop ed by he pep ides in he hyd ogels (Fig. S8†).
The ela i e con ibu ions o diffe en seconda y s uc u es o
he amide I band o he Fmoc-FFpY6.4/Na
+
500 hyd ogel a e
shown in Fig. S9†and summa ized in Table S2.†The Fmoc-
FFpY6.4/Na
+
500 hyd ogel con ains 41% β-shee s (wi h 20% o
an ipa allel β-shee s), 30% andom s uc u es, and 29%
α-helices. The s uc u al a angemen was u he cha ac e -
ized by ci cula dich oism (Fig. 2c and Fig. S10†). The CD
spec a o Fmoc-FFpY in pu e wa e o a low concen a ion o
NaCl (50 mM) show no signal, which is consis en wi h he
non-gel s a e (Fig. 1). In he p esence o 150 o 500 mM NaCl,
Fmoc-FFpY showed a posi i e peak a 194 nm oge he wi h
wo nega i e bands a 204 and 215 nm, which is he signa u e
o α-helix con o ma ions. The helical con o ma ion o he i-
pep ide/me al ion could be a ibu ed o me al coo dina ion
and in e molecula non-co alen in e ac ions. The posi i e
peak a 227 nm is a ibu ed o he s acking in e ac ions o he
a oma ic uni s o Fmoc-FFpY, while he nega i e band a
252 nm is a ea u e o offse ace- o- ace s acking o he Fmoc
moie ies. These esul s a e in ag eemen wi h o he wo ks on
he sel -assembly o Fmoc-FF pep ides induced by elec os a ic
in e ac ions and me al ions.
22,34
We can no ice ha he FTIR
decomposi ion ga e simila con ibu ions om he β-shee ,
α-helix, and andom coil, which could explain he diffe ence in
he esul wi h CD. I is no ewo hy ha highe concen a ions
o NaCl a e equi ed o ob ain he α-helix signa u e when
using lowe Fmoc-FFpY concen a ions, 250 mM and 500 mM
NaCl o 3.2 and 1.3 mM Fmoc-FFpY, espec i ely (Fig. S11†).
Molecula dynamics simula ions o Fmoc-FFpY/Na
+
assembly
Compu a ional s udies we e pe o med o ob ain in o ma ion
a he a omic le el ha allowed he s udy o complex bio-
molecula sys ems wi h high p ecision and accu acy. Two dis-
inc s udies we e pe o med, each add essing he speci ic
aspec s o pep ide agg ega ion. The i s s udy ocused on he
pep ides’abili y o spon aneously agg ega e and explo ed he
unde lying mechanisms ha d i e his p ocess. To s udy his
beha io , nine Fmoc-FFpY pep ides we e andomly dis ibu ed
wi hin la ge wa e boxes, ensu ing ha he ini ial dis ance
be ween any wo pep ides was a leas 20 Å o a oid any po en-
ial associa ion due o p oximi y. Molecula dynamics (MD)
simula ions we e pe o med a h ee diffe en sal concen-
a ions (50, 250, and 500 mM) o 1 µs and eplica ed ou
imes o ensu e s a is ical signi icance.
MD simula ions show ha he Fmoc-FFpY pep ide ends o
sel -assemble and a ies signi ican ly wi h he NaCl concen-
a ion (Fig. 3a). A 500 mM NaCl, pep ide agg ega es a e
dense and consis o all nine pep ide uni s. A 250 mM NaCl,
agg ega es a e loose and eplica es show agg ega es consis ing
o 7, 8, o 9 uni s. A 50 mM NaCl, agg ega es include only a
ew pep ides, while o he s emain isola ed in he solu ion. To
Fig. 3 MD simula ions o Fmoc-FFpY in he p esence o diffe en NaCl concen a ions: (a) he p o ocol used and (b) he numbe o non-na i e con-
ac s (dis ance less han 7 Å), along he MD ajec o y, be ween a oms om diffe en pep ides. An in e pola ion using Bézie cu es allows us o
obse e he end.
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quan i y his phenomenon, he numbe o non-na i e con ac s
was de e mined along he MD ajec o ies and plo ed o each
concen a ion (Fig. 3b). The numbe o non-na i e con ac s
along he ajec o y quan i ies he numbe o dis ances
be ween a oms o less han 7 Å (one con ac ) om diffe en
pep ides. A high alue indica es s ong and dense agg ega ion,
while a low alue indica es a less dense agg ega e o one wi h
ewe Fmoc-FFpY uni s. A 500 mM NaCl, agg ega es o m
wi hin he i s 200 nanoseconds and emain s able. This is
hus a spon aneous and apid assembly. A 250 mM NaCl, pep-
ides also sel -assemble, bu he agg ega es ake a much longe
ime, abou 700 nanoseconds, and p o ide less dense agg e-
ga es. A 50 mM NaCl, he pep ide assembly is incomple e and
emains s able, demons a ing he inabili y o Fmoc-FFpY o
ully agg ega e a his sal concen a ion. This phenomenon is
ema kably consis en wi h expe imen al obse a ions.
Sufficien ly high ionic s eng h sc eens he nega i e cha ges o
each Fmoc-FFpY pep ide, he eby educing elec os a ic epul-
sion and allowing o he o ma ion o new in e ac ions a
sho e dis ances.
An al e na i e simula ion s a egy was employed o s udy
he s uc u al a angemen induced by he agg ega ion o
Fmoc-FFpY pep ides. To achie e his, mo e compac sys ems
wi h a highe numbe o pep ides we e cons uc ed.
Speci ically, 40 pep ides we e andomly placed in a con ined
space o p omo e hei associa ion, as shown in Fig. 4a. MD
simula ions we e hen pe o med o a du a ion o 1.2 µs a
wo diffe en concen a ions o NaCl, namely 250 and 500 mM.
As be o e, he simula ions we e epea ed ou imes o ensu e
he ep oducibili y o he esul s.
As expec ed, spon aneous and comple e agg ega ion occu s
du ing he i s 400 ns and e ol es sligh ly, as shown by he
RMSD cu e (Fig. S12†). Two diffe en agg ega ion beha io s
we e obse ed a 250 and 500 mM NaCl. A 250 mM NaCl,
h ee ou o ou simula ions showed he collapse o all 40
Fmoc-FFpY uni s in o a globula s uc u e, while a single
simula ion showed a nano od-like s uc u e. In his las simu-
la ion, he pep ides ook ad an age o pe iodic bounda y con-
di ions in one di ec ion o o ganize in o a nano od-like con o -
ma ion p oducing a 2D ma e ial. These s uc u al o ganiz-
a ions a e shown in Fig. S13.†In con as , a 500 mM NaCl, all
simula ions showed a nano od-like o ganiza ion by epea ing
he s uc u al o ganiza ion in a pe iodic di ec ion. Fig. S14†
shows hese o ganiza ions o he 4 simula ions. The a ia ion
obse ed in he concen a ion dependence is o conside able
in e es , as i effec i ely mi o s he esul s obse ed in he
expe imen al TEM mic og aphs. Speci ically, Fig. 1d shows
ha indi idual nano ibe s a e isola ed a a concen a ion o
250 mM NaCl, whe eas comple e nano ibe o ma ion is
obse ed a 500 mM. I is wo h highligh ing he ema kable
achie emen o ep oducing he mo phological beha io o he
hyd ogel using a omis ic simula ions. To he bes o ou
knowledge, his s udy is he i s o epo such a
co espondence.
Fig. 4 (a) MD simula ions, whe e 40 Fmoc-FFpY uni s we e andomly a anged in he p esence o 500 mM NaCl a = 0 (le ) and a e 1.2 µs
dynamics ( igh ), whe e he 40 pep ide uni s a e assembled, leading o pep ide fibe s wi h his eplica ed cubic uni . Na
+
ions a e shown in ligh blue
and H
2
O molecules a e hidden o be e cla i y o he assembly isualiza ion. (b) π–πs acking o a oma ic Fmoc g oups in he hyd ophobic nucleus
o he assembly. (c) Elec os a ic in e ac ion be ween he phospha e g oups o 3 Fmoc-FFpY uni s and he Na
+
ions in he ou e pa . (d)
Ramachand an map o one Fmoc-FFpY assembled s uc u e. (e) Con o ma ional ee ene gy landscape o Fmoc-FFpY/Na
+
assemblies. ( ) TEM
mic og aph o a nega i ely s ained Fmoc-FFpY6.4/Na
+
500 hyd ogel, whe e he a ows ma ked a helical wis ing. The inse shows a zoom-in image
o isualize he a angemen o fibe s o ming a nano od and he helical s uc u e.
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Since he s uc u al incidence a 500 mM NaCl is he mos
in e es ing, comple e s uc u al analyses we e pe o med a
his concen a ion, ocusing on he las 200 ns o he ou
simula ions o p o ide meaning ul in o ma ion a e he s abi-
liza ion o he assembly. Th ee dis inc s uc u ally a o able
in e ac ions we e highligh ed: (i) π–πs acking o a oma ic
g oups p esen in he pep ide s uc u e. The e a e many s acks
in each s uc u e, almos exclusi ely ela ed o Fmoc and Phe
g oups. Phospho yla ed y osine has a olume o 197 Å
3
ins ead o 157 Å
3
, co esponding o 20% o he whole amino
acid olume. The phospha e moie y is hus qui e massi e and
no plana , dis a o ing his way o app oach o ano he a o-
ma ic nucleus. S acks o wo, h ee, and up o ou pep ide
uni s a e obse ed (Fig. 4b and c). These s acks, which occu a
dis ances be ween 4 and 5 Å, a e obse ed in he cen e o he
clus e , highligh ing he hyd ophobic co e o he clus e . (ii)
Many elec os a ic in e ac ions in ol ing sodium ions a e
iden i ied. A he end o he simula ion, 89% o he Na
+
ions
in e ac wi h he pep ide (i.e., a a dis ance o less han 3 Å). In
con as o he a oma ic s acks, he elec os a ic in e ac ions
a e localized on he su ace o he a ay, as shown in Fig. 4d
and Fig. S15.†Na
+
ions s ongly shield he nega i e cha ges o
Fmoc-FFpY, allowing he app oach o se e al pep ide ag-
men s on he side o he phospho yla ed y osine. They a e,
he e o e, an essen ial elemen in he o ganiza ion o he
assembly and occu a dis ances be ween 4.5 and 6.4 Å. (iii)
Many hyd ogen bonds a e also p esen du ing he molecula
dynamics, bu only hose wi h an a endance ime o a leas
20%, i.e., H-bonds ha a e obse ed o a leas 40 ns wi hin
he 200 ns o he analyzed ajec o y, we e conside ed o a dis-
ance be ween 1.8 and 2 Å. The only hyd ogen bond dono
a oms a e he NHs o he amides o he pep ide backbone
( h ee o each Fmoc-FFpY molecule). I is obse ed ha
H-bonds almos exclusi ely in ol e in e ac ions wi h he
pep ide backbone. The e o e, H-bonds a e he sign o “second-
a y s uc u e”elemen s and a e classi ied in o wo g oups,
in e molecula and in amolecula . In e molecula H-bonds
ai ly iden i y β-shee o ganiza ions (Fig. S15b, le †). Fo hese
β-shee s, up o 3 H-bonds a e obse ed, which co esponds o
he maximum o his pep ide size, implying ha he pep ide
backbone is shee -like, i.e., mo e o less la . On he o he
hand, i we ocus on he in amolecula H-bonds, we obse ed
ha he pep ide backbone shows w inkling simila o ha o
an α-helix (Fig. S15b, igh †). The single H-bond b ings he
y osine close o he Fmoc agmen , o cing he pep ide back-
bone o adop a helical s uc u e.
All hese indings a e suppo ed by he Ramachand an
map, which shows he phi and psi angles o he pep ide back-
bone wi hou aking in o accoun hyd ogen bonds (Fig. 4d).
The phi/psi pai s con i m he p esence o bo h β-shee (57 ±
4%) and α-helix (43 ± 3%) s uc u es wi hin he assemblies. In
compa ison, he FTIR decomposi ion o he amide I band ga e
he β-shee (∼41%) and α-helix (∼29%) o he o al seconda y
s uc u es. When he a io is calcula ed on he o al amoun o
β-shee and α-helix s uc u es only, we ob ain he β-shee
(∼59%) and α-helix (∼41%), which is e y close o he simu-
la ion esul s. The ac ha we obse ed an almos iden ical
a io o seconda y s uc u es be ween he calcula ions and he
expe imen al da a s ongly alida es he MD simula ions.
Simila ly, i can be obse ed ha in e ac ions p omo ing
pep ide assembly occu a h ee ypical dis ance anges: 1.8 o
2 Å o hyd ogen bonds, 4 o 5 Å o he s acking o a oma ic
co es, and 4.5 o 6.4 Å o ionic bonds. These alues co es-
pond o he h ee alues ob ained om he WAXS spec a (3.3,
4.1, and 6.4 Å), which exhibi a simila end.
To in es iga e he s uc u al s abili y o he sys ems, an
ene gy mapping was pe o med using Bol zmann s a is ics
and by conside ing he elonga ion dis ance o he pep ide as
well as i s o e all dihed al angle as coo dina es (Fig. 4e). On
his ene gy map, wo ene gy basins a e highligh ed, co es-
ponding o he o ma ion o α-helices and β-shee s. Ano he
in e es ing inding is ha he wo minima ( amed in Fig. 4e),
which a e a signa u e o he wo seconda y s uc u es, a e no
isola ed in he map, meaning ha i is ene ge ically economi-
cal o swi ch om he β-shee o α-helix and ice e sa. This is
con i med by he TEM mic og aph (Fig. 4 ), which shows he
p esence o bo h non- wis ed nano ibe s, which can be a ibu-
ed o β-shee s and wis ed nano ibe s, which can be a ea u e
o α-helix seconda y s uc u es, in he Fmoc-FFpY6.4/
Na
+
500 hyd ogel.
Mac oscopic p ope ies o Fmoc-FFpY/Na
+
hyd ogels: a s udy
o heological and he mo- esponsi e p ope ies
Oscilla o y heological measu emen s we e pe o med o de e -
mine he mechanical p ope ies o he Fmoc-FFpY/Na
+
hyd o-
gels. Fig. 5a shows he e olu ion o he elas ic modulus (G′)
and he loss modulus (G″) o e ime o he Fmoc-FFpY6.4/Na
+
hyd ogels o med a diffe en NaCl concen a ions. Time
sweep expe imen s we e pe o med wi hin he iscoelas ic
egime (1% s ain) a 1 Hz o p o ide in o ma ion abou he
ime needed o o m a s able gel. Fmoc-FFpY6.4/Na
+
hyd ogel
o ma ion (G′>G″) is ins an aneous o NaCl concen a ions
highe han 250 mM, which exhibi ed G′and G″ alues ha
emained s able o e he ime in less han 300 s, whe eas a
longe gela ion ime (∼900 s) was equi ed a 150 mM NaCl o
each a pla eau o bo h G′and G″. The equency sweep expe i-
men s con i med ha in all cases, G′was highe han G″and
he ma e ials beha ed as iscoelas ic solids. G′( espec o G″)
inc eases wi h he NaCl concen a ion eaching alues o 2.7 ±
1.2 Pa (G″= 1.4 ± 0.5 Pa), 4.5 ± 2.2 Pa (G″= 2.1 ± 0.2 Pa), 24.2 ±
11.0 Pa (G″= 5.4 ± 2.4 Pa), and 53.3 ± 14.9 Pa (G″= 7.7 ± 1.3
Pa) o he hyd ogels o med wi h 150, 250, 375, and 500 mM
NaCl, espec i ely (Fig. 5b). Wi h inc easing NaCl concen-
a ion, G′and G″become mo e independen o he equency,
indica ing he s eng hening o he hyd ogel ne wo k. This is
p obably due o he inc ease in he numbe o ibe s and he
en anglemen s be ween hem, as shown in he TEM images
(Fig. 1d). By keeping he NaCl concen a ion ixed a 500 mM,
he G′o he Fmoc-FFpY/Na
+
hyd ogels inc eases wi h he
pep ide concen a ion om 3.9 ± 0.9 Pa (G″= 0.6 ± 0.1 Pa) o
17.9 ± 1.1 Pa (G″= 2.6 ± 0.4 Pa) and 53.3 ± 14.9 Pa (G″= 7.7 ±
1.3 Pa) when p epa ed wi h 1.3, 3.2, and 6.4 mM Fmoc-FFpY
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(Fig. S16†). The elas ic modulus alues o Fmoc-FFpY/Na
+
500
p epa ed a 6.4 mM in he pep ide a e in he ange o hose
ound in o he Fmoc-based hyd ogels such as Fmoc-FF (G′∼
80 Pa)
63
o Fmoc-GFFRGD (G′∼100 Pa).
64
S ain sweeps we e pe o med o de e mine he linea isco-
elas ic egime and he gel- o-sol ansi ion o sel -healing
es s (Fig. 5c). A 1% s ain, all Fmoc-FFpY6.4/Na
+
hyd ogels
(G′>G″) p epa ed a diffe en NaCl concen a ions a e in he
linea iscoelas ic egion. As s ain inc eases, a gel- o-sol an-
si ion occu s, and a 1000% s ain, he Fmoc-FFpY6.4/Na
+
gels
a e in he solu ion s a e (G″>G′). The sel -healing hyd ogels
exhibi a eco e y o hei iscoelas ic p ope ies a e being
subjec ed o a shea o ce. To e alua e his p ope y, dynamic
s ep s ain ampli ude es s we e pe o med by a ying he
s ains be ween 1 and 1000% o sho imes, 200 s (Fig. 5d).
A s age I (γ= 1%), he Fmoc-FFpY6.4/Na
+
hyd ogels exhibi a
solid-like beha io (G′>G″). As he s ain is inc eased in s age
II (γ= 1000%), he elas ic modulus apidly dec eases wo
o de s o magni ude and well below ha o G″, eaching a
luid-like s a e (G″>G′) h ough he b eaking o he weak
physical in e ac ions (elec os a ic in e ac ions, π–πs acking,
and H-bonds) ha o m he gel s uc u e.
65
In e es ingly, when
he hyd ogels a e no longe subjec ed o la ge de o ma ions
(s age III, γ= 1%), he ini ial mechanical p ope ies a e eco -
e ed almos ins an aneously, showing a solid-like beha io
again (G′>G″), which p o ed he apid sel -healing beha io o
he gels, which migh be acili a ed by he sol en ha
enhances he mobili y and ea angemen o he molecules.
66
No ably, his sel -healing beha io is main ained a e a
second de o ma ion (s age IV, γ= 1000%) and eco e y (s age
V, γ= 1%) cycle. Howe e , sligh ly lowe G′ alues we e
obse ed a e he i s high–low s ain cycle o he hyd ogels
o med wi h NaCl concen a ions highe han 150 mM, which
indica es ha he g adual build-up o he gel s uc u e o he
equilib ium s a e would equi e longe pe iods o “ es ”(low
s ain), a p ope y ypically obse ed o se e al low molecula
weigh gela o s.
67,68
The he mo- esponsi e p ope ies o he Fmoc-FFpY/Na
+
hyd ogels we e i s e alua ed by in e ed ube es s unde con-
olled he mal condi ions in an o en (Fig. 6a). The pep ide
Fmoc-FFpY in solu ion (6.4 mM in bo ax buffe ) has no gelling
abili y. The Fmoc-FFpY/Na
+
150 hyd ogels exhibi a gel- o-sol
ansi ion (T
g–s
) when hea ed up o 60 °C and a he mo- e e s-
ible sol- o-gel ansi ion when cooled o 20 °C.
A mo e de ailed in es iga ion was pe o med by mic o-DSC,
whe e he hyd ogels we e hea ed om 20 o 75 °C (Fig. 6b).
Fmoc-FFpY in solu ion (6.4 mM in bo ax buffe ) showed an
endo he mic peak a 62 °C, which can be a ibu ed o he gel–
sol ansi ion o he nanod ople s o he pep ide (Fig. S3†).
This was con i med by he X- ay diff ac og am o Fmoc-FFpY
in powde , which does no show any c ys alline s uc u e
(Fig. S17†). A single b oad diff ac ion band a 2θ=18–20°
emains s able du ing hea ing and subsequen cooling p o-
cesses. This co esponds o a dis ance o 4.4 Å a ibu ed o
he in e -s and dis ance o Fmoc-F pep ides.
69
The hyd ogels
o med wi h a lowe NaCl concen a ion, Fmoc-FFpY6.4/
Na
+
150 and Fmoc-FFpY6.4/Na
+
250, show a single endo he mic
peak a 62 °C co esponding o he gel–sol ansi ion empe a-
u e (T
g–s
) o he hyd ogels as de e mined by he in e ed es s.
In e es ingly, a he in e media e NaCl concen a ion, Fmoc-
FFpY6.4/Na
+
375, he endo he mic signal is weak and shi ed
o lowe empe a u es wi h he appea ance o wo bands a 53
and 57 °C, which can be a ibu ed o a ansi ion s a e in gel
o ma ion om small nano ibe s andomly dis ibu ed in he
hyd ogel ne wo k o a long bundle o nano ibe s as obse ed
by TEM (Fig. 1d). Howe e , a a highe NaCl concen a ion,
Fmoc-FFpY6.4/Na
+
500, he endo he mic peak a 62 °C eap-
pea ed wi h highe in ensi y. This phenomenon can be
explained as an effec o cha ge compensa ion by compa ing
he en halpy (ΔH
g–s
) o each sample (Table S3†).
ΔH
g–s
dec eases om 16.0 J g
−1
o 5.9 J g
−1
o Fmoc-FFpY
and Fmoc-FFpY6.4/Na
+
250, espec i ely, and emains cons an o
Fmoc-FFpY6.4/Na
+
375. This may e lec he lowes ene gy s a e o
his hyd ogel as a esul o he mo phological change. Then,
ΔH
g–s
inc eases up o 35.4 J g
−1
o Fmoc-FFpY6.4/Na
+
500, whe e
mo e s able nano ods a e o med. These Fmoc-FFpY6.4/Na
+
hyd ogels show a eco e y o he gel p ope ies a e cooling hem
back o 20 °C (Fig. 6c), wi h he p esence o a single exo he mic
peak a 47 °C, a ibu ed o he sol- o-gel ansi ion (T
s–g
). The
en halpy alues (ΔH
s–g
) a e simila o hose ob ained in he
hea ing s ep (ΔH
g–s
). Thus, i demons a es he he mo- e e sible
beha io o hese sup amolecula pep ide hyd ogels, which is
main ained e en a e wo epea ed hea ing and cooling cycles
(Fig.6dande),showing hesameΔH
g–s
and ΔH
s–g
alues as in
he i s cycle (Table S3†).
Fig. 5 Rheological p ope ies o Fmoc-FFpY6.4/Na
+
hyd ogels p e-
pa ed a 150 mM, 250 mM, 375 mM, and 500 mM NaCl. S o age
modulus (G’–solid symbols) and loss modulus (G’’ – hollow symbols) as
a unc ion o (a) ime (1 Hz, 1% s ain), (b) equency (0.01–10 Hz, 1%
s ain), (c) s ain (0.01–1000%, 1 Hz), and (d) dynamic s ep s ain ampli-
ude es s (1% o 1 000% s ain) a 20 °C.
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