[System] polypeptide self-assembly literature information extraction

[User]
Copyright © 2015 Elsevier Ltd. All rights reserved.

a Department of Chemistry, Indian Institute of Technology Delhi (IITD), Hauz Khas, New Delhi 110016, India
b Product Development Cell, National Institute of Immunology (NII), Aruna Asaf Ali Marg, New Delhi 110067, India
A series of tryptophan-based peptides W1a, b–W4a, b, with diverse architectures were designed and
synthesized. These tryptophan containing peptides can self-assemble to spherical particle. This
self-assembled system was demonstrated to encapsulate rhodamine B and penetrate the cell membrane.
(cid:1) 2015 Published by Elsevier Ltd.
CPPs have been shown to interact with the negatively charged
glycosaminoglycans15,16 and lipids17 present on the cell mem-
brane. Membrane hydrophobicity is an important aspect, however
very high hydrophobicity suppresses internalization by promoting
aggregation and strong cell membrane binding. Therefore, a bal-
ance between hydrophobicity and charge is crucial for membrane
penetration.18,19 The application of CPPs range from delivering
low molecular weight drugs to macromolecules such as nucleic
acids and proteins to the speciﬁc site.20 CPPs are widely employed
for delivering chemotherapeutic drugs like doxorubicin21–23, pacli-
taxel24–26, methotrexate27 as well as macromolecules like proteins
and oligonucleotides.28 Though the mechanism of transport across
membrane is still in debate, it is believed that CPPs deliver cargo by
direct penetration of the plasma membrane or by endocytosis.29
The general strategy of CPP design has been the introduction of
Here, we report, self-assembly as a strategy for the design of
spherical particles that can load cargo into its interior and traverse
through the cell membrane with the cargo. We envisioned that
self-assembled hollow sphere is an ideal supramolecular system
for entrapping drugs or biomolecules inside its cavity and such
self-assembled system if garnered with the ability to cross the cell
membrane will turn out to be excellent cell penetrating system.34
The design of molecules with the ability to assemble as spheres
having encapsulation ability coupled with cell penetration is a
challenge. For this purpose, it is necessary to bring both these
aspects, such as self-assembly and cell penetration, into a single
molecule. Amino acid tryptophan (Trp) has a hydrophobic indole
side chain, which can promote p–p interactions, and thus endowed
with dual properties such as membrane afﬁnity and self-
assembly.35
for self-assembly and cellular uptake studies.36 The N-protected
peptide derivatives W1a–W4a were insoluble in aqueous medium,
therefore N-deprotected derivatives W1b–W4b were synthesized
to make them water soluble and suitable for biological studies.
Self-assembling behavior of W1a–W4a and W1b–W4b was
studied by various microscopic techniques. Scanning electron
microscopic (SEM) imaging of W1a–W4a in 1:1 MeOH/CHCl3 (SI,
Fig. S1) and W1b–W4b (Fig. 2(a–d)) in 1:1 MeOH/H2O show that
these peptides assemble into spherical morphology. Transmission
electron microscopic (TEM) (stained with 0.2% phosphotungstic
acid) (SI, Figs. S2 and S3) and atomic force microscopic (AFM) (SI,
Fig. S4) studies revealed that W1a, b–W4a, b form spheres in the
range of (cid:1)0.6–2 lm. The analysis of SEM images of W1b–W4b
show average diameter of vesicles in the range (cid:1)0.3–2 lm (SI,
Fig. S5) which approximately matches from other microscopic
The average sizes obtained from microscopic studies were in
partial agreement with that obtained from the dynamic light scat-
tering studies (SI, Fig. S6). Detailed analysis of SEM images of W1b–
W4b revealed that the average size of these spherical aggregates
increased as the branching increased.
Fluorescence microscopic analysis of W1b–W4b alone and
mixed with rhodamine B revealed the dye entrapment potential
these spherical aggregates. The spheres from W1b–W4b
appeared as blue (kex = 450–490 nm) (Fig. 2(e) and SI, Fig. S7),
while the rhodamine B loaded spheres were red colored
(kex = 510–560 nm) demonstrating encapsulation of the dye by
the spheres (Fig. 2(f) and SI, Fig. S7).
After discovering the spherical self-assembly and encapsulation
ability of the peptides, these peptides were tested for their poten-
tial for cellular uptake and cargo delivery. Murine macrophage cell
line J774A.1 was chosen for this purpose. Initially the cytotoxicity
Figure 2. SEM images of (a) W1b (2.54 mM), (b) W2b (2.72 mM), (c) W3b (3.3 mM), (d) W4b (2.2 mM). Fluorescence microscopic images of W2b (e) alone (kex =
450–490 nm), (f) with 0.02 equiv of rhodamine B (kex = 510–560 nm) in 1:1 MeOH/H2O.
proﬁle of the peptides was studied by MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide) assay.37 Brieﬂy mur-
ine macrophage cells (J774A.1) were seeded in 96 well plate
(10,000 cells/well) in serum supplemented media at pH 7.4. Serum
free media was used for the dilution of compounds to avoid inter-
actions of peptides with proteins present in serum. Cells were trea-
ted with different concentrations of the peptides (W1b–W4b) for
24 h at 37 (cid:3)C in CO2 incubator followed by incubation with
100 lL of 0.5 mg/mL of MTT in serum free media for 4 h at 37 (cid:3)C
in a CO2 incubator. Formazan crystals formed after reduction of
MTT were dissolved in 100 lL of DMSO and absorbance measured
at 570 nm with reference of 630 nm in a multiwell plate reader.
Cellular viability was calculated as per the formula: (OD of test/
OD of control) (cid:3) 100. Each experiment was performed in tripli-
cates. The Fig. 3(a) shows average percentage viability of cells at
different peptide concentrations revealing that nearly all peptides
(W1b–W4b) exhibit (cid:1)100% viability at a concentration of 60 lg/
mL (45–101 lM) and thus ideal for biological applications. W2b
and W3b showed (cid:1)100% viability even at higher concentrations
(120–500 lg/mL), whereas W1b and W4b showed lesser viability
revealing higher biocompatibility of W2b and W3b. All further
cellular interaction studies were carried out at non toxic concen-
tration of 60 lg/mL for all compounds.
The intrinsic ﬂuorescence of Trp was used for the detection of
these compounds in the cells (SI, Fig. S8).38 Interestingly, we
could observe the spherical aggregates as green (kex = 494 nm)
spots without any additional ﬂuorescent
labeling. Confocal
microscopy of
the cells treated with the peptides for 4 h
showed the presence of peptides in the intracellular region (Fig. 3
(c) and SI, Fig. S9). The nucleus of the cells is stained (blue) with
40,6-diamidino-2-phenylindole (DAPI), actin is stained (red) with
Figure 3. (a) Comparative cytotoxicity proﬁle of W1b–W4b for J774A.1 cells. Cells were incubated with various concentrations of peptides for 24 h. (b) Relative percentage
uptake of W1b–W4b at 37 (cid:3)C, 4 (cid:3)C and with endocytosis inhibitor cytochalasin B (at 37 (cid:3)C) as evaluated by ﬂow cytometry. Peptides were incubated with J774A.1 cells in
serum free medium for 2 h (untreated cells act as control). (c) Confocal microscopy images of J774A.1 cells after incubation with W1b for 4 h. The dyes DAPI (kex = 358 nm;
kem = 461 nm) and rhodamine–phalloidin (red) (kex = 540 nm; kem = 565 nm) were used to stain cell nuclei and actin ﬁlaments, respectively, (Bar = 10 lm). Arrows denote
position of the peptides inside the cell. (d) Relative percentage uptake of rhodamine B by W1b–W4b at 37 (cid:3)C, 4 (cid:3)C and with endocytosis inhibitor cytochalasin B (at 37 (cid:3)C)
(statistical analysis was carried out with Graph Pad Prism V 6.01. Groups were analyzed using ordinary two-way ANOVA. P value less than 0.05 was considered to be
signiﬁcant, ns = not signiﬁcant).
To investigate the uptake mechanism, ﬂow cytometry experi-
ments were performed with W1b–W4b at 4 (cid:3)C. The results (Fig. 3
(b) and SI, Fig. S11) show that the percentage uptake efﬁciency is
reduced at 4 (cid:3)C. Therefore, the plausible mechanism of cellular
uptake of W1b–W4b is largely by energy-dependent pathway. To
conﬁrm the mechanism, we performed cellular uptake study
(Fig. 3b) in the presence of cytochalasin B, an endocytosis inhibitor.
Interestingly in the presence of cytochalasin B, there was no
reduction in the uptake of the peptides, in fact the cellular uptake
was increased slightly for compounds W1b–W4b. This infers that,
these peptides do not enter cells using endocytosis. One possible
explanation could be that as these peptides are hydrophobic in
nature, they may interact with the cell membrane and enters in
to the cells. This is conﬁrmed from the SEM images of the cells
treated with W1b–W4b (SI, Fig. S12) for 24 h which showed gran-
ular appearance on the cell surface which suggests that the spher-
ical aggregates adhere to the cell membrane prior to entry. Thus
the membrane interacting properties of these peptides may be
responsible for its internalization.
Having established the cellular uptake ability of these peptides,
we evaluated whether these peptides enter into the cell in the form
of spheres. For investigating this, J774A.1 cells were treated with
the rhodamine B alone and rhodamine B encapsulated peptide
spheres of W1b–W4b for 2 h. Flow cytometry experiments
carried out with cells treated with rhodamine B encapsulated
peptide spheres showed higher uptake of rhodamine B into the
cells as compared to the cells treated with rhodamine B alone
underscoring that the self-assembling peptides act as carrier
systems (Fig. 3(d)). The dot plot based on FACS data clearly sup-
ported this ﬁnding (SI, Fig. S13). The confocal microscopic images
showed presence of rhodamine B (kex = 510 nm) in the cell interior
(Fig. 4(a, b) and SI, Fig. S14), providing a compelling evidence of
rhodamine transport by the self-assembled spherical aggregates.
Fig. 4(b) showing very faint spot corresponding to W3b indicates
lowest uptake efﬁciency in agreement with the FACS data (Fig. 3
(b and d)). Amongst all the compounds, W1b showed highest
efﬁciency in delivering cargo rhodamine B into the cells. The quan-
tiﬁcation of uptake of peptides (W1b–W4b) with and without dye
by ﬂow cytometry data along with the results obtained from con-
focal microscopic studies suggest that W1b (Figs. 3(b–d) and 4(a))
showed highest uptake efﬁciency while W3b (Figs. 3(b, d) and 4
(b)) has relatively less efﬁciency for cellular uptake as compared
to other peptides.
In summary, we have designed and synthesized linear and
dendrimeric tryptophan-based self-assembling peptides. These
peptides self-assemble to spheres that encapsulate rhodamine B
and traverse the cell membrane by energy-dependent process.
The studies presented here demonstrate that self-assembly
provides the encapsulation ability, while Trp units provide cellular
uptake properties. These topologically and chemically diverse class
of self-assembling peptides would be of tremendous interest to
chemical, biological, medical and nanotechnological applications.
The enormous chemical space available for peptide design further
expands the scope of this work.
12. Pooga, M.; Hallbrink, M.; Zorko, M.; Langel, U. FASEB J. 1998, 12, 68.
13. Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Chem. Soc. Rev. 2011, 40, 3638.
14. Stewart, K. M.; Horton, K. L.; Kelley, S. O. Org. Biomol. Chem. 2008, 6, 2242.
15. Bechara, C.; Pallerla, M.; Zaltsman, Y.; Burlina, F.; Alves, I. D.; Lequin, O.; Sagan,
17. Koren, E.; Torchilin, V. P. Trends Mol. Med. 2012, 18, 385.
18. Copolovici, D. M.; Langel, K.; Eriste, E.; Langel, U. ACS Nano 2014, 8, 1972.
19. Fillon, Y. A.; Anderson, J. P.; Chmielewski, J. J. Am. Chem. Soc. 2005, 127, 11798.
20. Fonseca, S. B.; Pereira, M. P.; Kelley, S. O. Adv. Drug Deliv. Rev. 2009, 61, 953.
21. Mandal, D.; Shirazi, A. N.; Parang, K. Angew. Chem., Int. Ed. 2011, 50, 9633.
28. Mae, M.; Langel, U. Curr. Opin. Pharmacol. 2006, 6, 509.
29. Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.;

[Assistant] [{"peptide_sequence": "WWW", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/Chloroform", "peptide_concentration(mg/ml)": "2.22", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "WWW", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "1.46", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficacious Anticancer Drug Delivery Mediated by a pH-Sensitive Self-
Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase
NGF Receptor**
Parikshit Moitra, Krishan Kumar, Paturu Kondaiah, and Santanu Bhattacharya*
Abstract: We present herein a short tripeptide sequence (Lys–
Phe–Gly or KFG) that is situated in the juxtamembrane region
the tyrosine kinase nerve growth factor (Trk NGF)
receptors. KFG self-assembles in water and shows a reversible
and concentration-dependent switching of nanostructures from
nanospheres (vesicles) to nanotubes, as evidenced by dynamic
light scattering, transmission electron microscopy, and atomic
force microscopy. The morphology change was associated with
a transition in the secondary structure. The tripeptide vesicles
have inner aqueous compartments and are stable at pH 7.4 but
rupture rapidly at pH (cid:2) 6. The pH-sensitive response of the
vesicles was exploited for the delivery of a chemotherapeutic
anticancer drug, doxorubicin, which resulted in enhanced
cytotoxicity for both drug-sensitive and drug-resistant cells.
Efficient intracellular release of the drug was confirmed by
fluorescence-activated cell sorting analysis, fluorescence mi-
croscopy, and confocal microscopy.
mimicking natural processes is among the leading approaches
in nanobiotechnology.[1] Toward therapeutic goals, one of the
emerging interests is the development of biocompatible
nanocarriers for efficient drug delivery.[2] The main aim is to
overcome barriers that stand against the therapeutic potential
of drugs, like overload side-effects,[3] specific cell targeting,[4]
the development of multidrug resistance (MDR),[5] and the
blood–brain barrier in gliomas.[6] In order to solve such
outstanding problems, “decorated” liposomes,[7] multifunc-
tional micelles,[8] polymeric hydrogels,[9]
functionalized
organic[10] and inorganic nanomaterials,[11] etc. have been
employed. Among these, building blocks that are organized
from short peptide sequences have become promising tools
for biomedical applications.[12, 13]
Prof. P. Kondaiah
Department of Molecular Reproduction, Development and Genetics
Indian Institute of Science, Bangalore 560 012 (India)
Recently, stimuli-responsive morphological transforma-
tions of short peptide sequences in water have also been
reported.[12, 14] We considered a short tripeptide sequence
(Lys–Phe–Gly or KFG) that is situated in the juxtamembrane
region of the tyrosine kinase nerve growth factor (Trk NGF)
receptors, which are involved in neuronal growth and differ-
entiation. The absence of the KFG sequence in the receptor
polypeptide chain seriously affects the activation of signaling
cascades including the Ras superfamily, phosphoinositide 3-
kinase, and the Suc1-associated neurotrophic factor target
(SNT) protein.[15] This also results in impaired neurite out-
growth and sometic hypertrophy. This small but critically
conserved entity, which plays an important role in the growth
factor signal transduction process, was chosen for the present
study. We investigated this biologically important tripeptide,
KFG (Figure 1), which spontaneously self-assembled into
defined nanostructures in aqueous media. The tripeptide
showed an exciting phenomenon of reversible and concen-
tration-dependent switching of nanostructures between nano-
vesicles and nanotubes. This change in morphology was
associated with a transition in the secondary structure at the
peptide level. The vesicles that resulted from the self-
assembly of KFG were extremely sensitive toward the pH
value of the medium. They were stable at pH 7.4 but ruptured
rapidly at pH (cid:2) 6. This property is often exploited for
improved cellular internalizations in anticancer drug deliv-
ery.[11c, 16] The pH-sensitive nature of the vesicles was accord-
ingly tested for the delivery of one of the most widely used
drugs for many types of cancer, doxorubicin (DOX). Cellular
uptake of DOX dramatically increased after the drug was
loaded into the vesicles. Overall, we report for the first time
a smart stimulus-responsive (pH-induced) tripeptide-based
self-assembled nanocarrier that induces significant therapeu-
tic delivery of a chemotherapeutic drug, DOX, to cancer cells.
To discern the nature of the supramolecular structures
that were formed upon self-assembly of Lys–Phe–Gly,
dynamic light scattering (DLS) measurements were per-
formed. At
tripeptide
low concentration of
(0.5 mg mL(cid:3)1), nanostructures of approximately 50–70 nm
were observed, whereas aggregates of about 190–200 nm were
formed at a higher concentration (5 mg mL(cid:3)1; Figure S5 in the
Supporting Information). Interestingly, we found that this
transition in the sizes of the nanostructures is reversible in
nature (Figure S6). These observations prompted us to
investigate the KFG aggregates at various concentrations by
using transmission electron microscopy (TEM). The tripep-
tide formed spherical structures with diameters of (50 (cid:4)
10) nm at the low concentration, whereas it formed tubular
aggregates with diameters of (190 (cid:4) 10) nm at the higher
concentration (Figure S7). Atomic force microscopy (AFM;
Figure 2) showed morphological features of the tripeptide
aggregates similar to those observed in the TEM observa-
Figure 2. AFM images showing the topography of the KFG tripeptide
at different concentrations: a) 0.5 mg mL(cid:3)1 and b) 5 mg mL(cid:3)1. The
double arrow between (a) and (b) indicates that the transition from
nanovesicles to nanotubes is reversible as a function of concentration.
Figure 3. a) CD spectra of KFG at two different concentrations.
b) Schematic illustration of the concentration-dependent self-assembly
process of the tripeptide.
The thermotropic properties of the tripeptide self-assem-
blies at both concentrations in aqueous media were inves-
tigated by high-sensitivity differential scanning calorimetry
(DSC).[17] The suspension of KFG at low concentration
(vesicles) did not show any transition in the temperature
range 20–70 8C, but at the higher concentration (nanotubes),
the tripeptide showed a quasireversible transition during
heating and cooling scans. Two peaks appeared at approx-
imately 47 and 60 8C (Figure S8). The DSC results were
corroborated by using temperature-dependent AFM studies
at low and high concentrations (Figure S9). There was no
change in the morphology of the vesicles even after the
sample had been heated up to 85 8C, but the nanotubes
changed topology upon heating. The transition from tubular
to spherical nanostructures (vesicles) started occurring from
45 8C and ended at 60 8C. Above 90 8C, the vesicular structures
collapsed, probably due to water evaporation. Interestingly,
such a temperature-induced transition from tubular-to-vesic-
ular morphology is quite unprecedented.
the tripeptide
exhibited a maximum at 196 nm and a minimum at 218 nm
in the CD spectrum (Figure 3). This signature in the CD
spectrum is analogous to those of b sheets in polypeptides.
After dilution of this suspension to 0.5 mg mL(cid:3)1, the CD
spectrum showed a negative band at 195 nm together with
a positive band at 212 nm, which suggests the formation of
a (cid:2)random-coil(cid:3) structure.[18] This change in the secondary
structure from a b sheet to a random coil occurs upon dilution
of the tripeptide, which is also associated with the morpho-
logical transition. Again, such a reversible transition from
vesicles to nanotubes along with changes in the secondary
structure is rarely observed, to the best of our knowledge.
Furthermore, we examined the self-assembly by molecular
modeling studies (Figure S10), which evidenced that the
peptide assemblies are formed mainly due to the interplay of
electrostatic, p-stacking, and H-bonding interactions.
Even if the particles appear to be nearly spherical, it may
not necessarily signify that they are vesicles and not solid
nanoparticles. It is important to know whether the nano-
spheres derived from the tripeptide assemblies possess inner
aqueous compartments. Therefore, we attempted to encap-
sulate a water-soluble solute or a drug in such particles.
Toward this end, we employed a cationic dye, methylene blue
(MB),[19] to check the entrapment capacities of the spherical
aggregates in aqueous media. Indeed, the vesicles could
encapsulate approximately 6.8 % of the total dye inside their
inner aqueous compartments (Figure S11). In another experi-
ment, we found that these tripeptide vesicles were also able to
encapsulate the neutral, impermeant, water-soluble solute
glucose[20] (Figure S12), which has no p-stacking unit, unlike
the commonly available aromatic dyes.
Furthermore, because the tripeptide formed nanovesicles
spontaneously at physiological pH values and the same
vesicles could be disrupted at pH 6, we thought of exploiting
this property for drug delivery.[16, 21] Accordingly, we loaded
Figure 4. Fluorescence microscopy images of a) DOX-loaded vesicles
at pH 7.4 and b) the disruption of the vesicles and drug release at
pH 6. Scale bar: 20 mm.
the vesicles with DOX at pH 7.4 (Figure 4 a) and followed the
release of the drug at pH 6 upon rupture of the vesicles
(Figure 4 b). The percentage of DOX encapsulation was
approximately 25.7 %, and importantly, the resulting DOX-
loaded vesicles were also quite stable in buffer containing
serum (Figure S14). The kinetic profile obtained for the
system showed a zero-order drug release at pH 6, which could
be explained on the basis of a non-Fickian diffusion mech-
anism,[22] as fitted from the Peppas model (Figure S15). Also,
the drug release profile of the DOX-loaded vesicles showed
a bimodal release profile, with an initial burst release
followed by a sustained release (Figure S14). A plausible
reason for this burst release could be the disruption of the
vesicular self-assembly and the increased hydrophilicity of
DOX molecules at this lowered pH value. The remaining
amount of DOX, which followed a sustained release profile
thereafter, could be attributed to the partial adsorption
effects between the tripeptide assembly and the DOX
molecules, as reported in other nanocarrier systems.[11a]
The toxicity of the KFG nanovesicles was assayed in cell
lines of different origins, namely HeLa (human cervical
carcinoma), HEK 293T (human embryonic kidney, trans-
formed), U251 (glioblastoma), and A549 (human lung
adenocarcinoma) cells. The toxic effect of the empty vesicles
appeared to be insignificant toward each cell line, with > 95 %
cells found to be viable (Figure 5 a). The toxicity of the DOX-
loaded vesicles was then assessed in the same set of cell lines
and compared with that of free DOX. Notably, the cytotox-
icity due to the DOX-loaded vesicles was significantly greater
than that of free DOX at each of the examined concentrations
(10, 20, 30, and 40 mg mL(cid:3)1) for the same period of incubation
in all of the cells studied. The differences in cell viabilities
between the treatment with DOX alone and the DOX-loaded
vesicles at a representative 20 mg mL(cid:3)1 drug concentration in
the above cells are shown in Figure 5 b.
To explore the possible reasons for the enhanced toxicity,
the internalization of DOX before and after being loaded into
the KFG vesicles was examined, both by fluorescence-
activated cell sorting (FACS) analysis and fluorescence
microscopy. Based on the FACS data, the geometric mean
of fluorescence intensity (GMFI) values for cells treated with
DOX-loaded KFG vesicles were significantly higher than
those of the free-DOX-treated cells. This effect was more
prominent with increased exposure to and concentrations of
DOX (Figure 6 a) and remained consistent among all of the
cell lines studied. Representative flow cytometry dot plots
and histograms for DOX internalization are shown in Fig-
Figure 5. a) Cytotoxicity of the tripeptide vesicles at different concen-
trations. b) Cytotoxicity of DOX-loaded vesicles in comparison with
free DOX at a concentration of 20 mg mL(cid:3)1.
ure S16 and S17a, respectively. The fluorescence microscopy
results substantiated the observation that the fluorescence
intensity of the DOX was significantly higher for cells treated
with DOX-loaded peptide vesicles than for the free-DOX-
treated cells after 1 h of incubation (Figure S17). We also
assessed the intracellular localization of the drug by confocal
microscopy. Interestingly, cells treated with the DOX-loaded
vesicles for 4 h showed nuclear localization of the drug with
Figure 6. a) GMFI of DOX internalizations in HeLa cells at two differ-
ent concentrations based on FACS analysis. The first two stacked bars
show the results after 1 h and the second two stacked bars show the
results after 4 h. b) Cytotoxicity of DOX-loaded vesicles in HeLa-DOXR
cells relative to the cytotoxicity with free DOX (20 mg mL(cid:3)1), and GMFI
values in HeLa-DOXR cells after a 4 h incubation (10 mg mL(cid:3)1).
Figure 7. Representative confocal microscopy images after DOX treat-
ment of HeLa cells at 5 mg mL(cid:3)1 for 4 h (a,b) and HeLa-DOXR cells at
20 mg mL(cid:3)1 for 24 h (c,d). Panels (a) and (c) represent treatment with
free DOX and panels b and d represent treatment with DOX-loaded
vesicles. Each panel, from left to right, shows the bright-field image,
DOX fluorescence (red), 4’,6-diamidino-2-phenylindole (DAPI) stained
nuclei (blue), and an overlay of the last two.
This interesting phenomenon of accelerated drug release
and, thereby, increased cytotoxicity selectively toward the
cancer cells encouraged us to deal with drug-resistant cell
lines. We, therefore, performed cell viability experiments and
drug internalization studies in DOX-resistant HeLa cells
(HeLa-DOXR). In this case also, the DOX-loaded vesicles
were found to be more efficient in delivering a larger amount
of the drug at fixed concentrations and time points, which
imparted greater toxicity than the free DOX (Figure 6 b).
Confocal microscopy was performed for HeLa-DOXR at
relatively longer time points and higher drug concentrations
because of the drug-efflux-related issues in drug-resistant cell
lines. At an optimized concentration and time point, cells
treated with DOX-loaded vesicles showed much higher DOX
fluorescence than those treated with free DOX, although the
drug was mainly localized in the cytoplasm this time (Fig-
ure 7 c,d). Thus, the increased internalization of the drug
could be attributed to the pH-sensitive response of the
vesicles.
In summary, we have synthesized a natural tripeptide
(KFG), which self-assembles to produce vesicles at lower
concentration (0.5 mg mL(cid:3)1) and hollow nanotubes at higher
concentration (5 mg mL(cid:3)1). The secondary structure of the
tripeptide changes from random-coil to b-sheet-like assem-
blies as the concentration is increased. The transition scheme
in the secondary structure is corroborated well with a molec-
ular modeling study. The vesicles rupture and release their
contents at pH (cid:2) 6. A continuous drug release profile
consistent with zero-order kinetics is maintained so that the
concentration of drug remains constant
delivery period. The pH-targeted drug delivery results in an
enhanced cytotoxicity to both drug-sensitive and drug-resist-
ant cells in vitro. The efficient intracellular release of the drug
after loading into vesicles was confirmed by FACS analysis,
fluorescence microscopy, and confocal microscopy. These
tripeptide vesicles show the distinct advantage of pH-driven
translocation even in serum, which may be important because
it may bypass factors involved in drug resistance.
Such a system is therefore highly promising as a stimulus-
responsive biocompatible nanovehicle that translocates into
cells once exposed to the tumor pH regime. The fact that this
tripeptide could be either synthesized readily or obtained
commercially makes it particularly attractive for the delivery
of anticancer drugs selectively to the cancer site with relevant
functionalizations. We are now extending the utility of this by
exploring its therapeutic potential toward other MDR cell
lines with specific recognition sites in vivo. With the aim of
eradicating the root of cancer, the specialized cancer stem
cells that are responsible for the resistance to cancer therapies
and the recurrence of cancer, work is underway to achieve
targeting by a combination chemotherapeutic approach with
the design of appropriate aptamer-loaded tripeptide nano-
carriers.

[Assistant] [{"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "20.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "30.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "40.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "50.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "60.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "70.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "20.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "30.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "40.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "60.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "70.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "KFG", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "7.4", "temperature(celsius)": "80.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
In this study, we examined the self-assembly of four dipeptides conjugated with
the electroactive dye naphthalenediimide (NDI). The presence of the NDI group at
the N-terminus of Phe-Phe and Phe-Gly promoted the formation of one-dimensional
(1-D) nanostructures and three-dimensional (3-D) colored hydrogels under both
acidic and physiological conditions. The 1-D nanostructures of these gels were
stabilized through intermolecular π-π interactions of the conjugated systems and
extended hydrogen bonding of the dipeptide units.

Electroactive Organic Dye Incorporating Dipeptides in the Formation
of Self-Assembled Nanoﬁbrous Hydrogels
Yu-Hao Liu, Shu-Min Hsu, Fang-Yi Wu, Hsun Cheng, Mei-Yu Yeh, and Hsin-Chieh Lin*
ABSTRACT: In this study, we examined the self-assembly of
four dipeptides conjugated with the electroactive dye
naphthalenediimide (NDI). The presence of the NDI group
at the N-terminus of Phe-Phe and Phe-Gly promoted the
formation of one-dimensional (1-D) nanostructures and three-
dimensional (3-D) colored hydrogels under both acidic and
physiological conditions. The 1-D nanostructures of these gels
were stabilized through intermolecular π−π interactions of the
conjugated systems and extended hydrogen bonding of the dipeptide units.
Supramolecular hydrogels, which feature a solid-like gel state
arising from the self-assembly of small molecules in water, are
potentially useful candidate materials in the rapidly expanding
ﬁeld of soft biomaterials.1 Because the morphology of a
supramolecular hydrogel is similar to that of the extracellular
matrix in a biological tissue, supramolecular hydrogels form a
particularly interesting class of biomaterials. Three major
building blocks of supramolecular peptide hydrogels have
been established previously: ionic complementary peptides,2,3
peptide amphiphiles,4,5 and low-molecular-weight hydrogels.6,7
The latter have been explored for a variety of applications,
including scaﬀolds for tissue engineering,8−13 carriers for drug
delivery,6,14−19 matrices for enzyme assays,7,20,21 ﬂuorophores
for biosensing,22−24 and as
for wound
healing.25,26 Recently,
functional naphthalenediimide (NDI)
dyes have been studied as key components in several systems,
including polymeric solar cells,27,28 polymer
light-emitting
transistors,30−37 and ion
diodes,29 organic ﬁeld eﬀect
sensors,38,39 as well as in photodynamic therapy40 and cell
imaging.38,41,42 In addition, NDI motifs have been employed as
components
in various (supra)molecular nanostructures,
including catenanes, rotaxanes, barrels, and vesicles,43−45 as
well as one-dimensional (1-D) nanostructures (e.g., nanorib-
bons, nanoﬁbers),46,47 which can be used to construct more
complex three-dimensional (3-D) materials (e.g., organogels,48
hydrogels47,49−51).
In a microscopic sense, a 3-D supramolecular hydrogel
constructed through physical cross-linking and entanglement of
self-assembled nanoﬁbers, which themselves comprise units of
molecular hydrogelators. An eﬃcient strategy toward realizing a
low-molecular-weight hydrogelator is to covalently bind a π-
conjugated hydrophobic group to a short peptide.52,53 In
contrast to the symmetric NDI-peptide hydrogelators that have
been investigated previously,49 in this paper we report
asymmetric NDI-capped hydrogelators formed from a hydro-
phobic octyl-capped NDI dye linked to dipeptides at the N-
terminus. With this design, we constructed hydrogelators that
could gel water under physiological conditions; indeed, our
peptide/dye conjugates featuring the peptide sequences Phe-
Phe and Phe-Gly underwent eﬃcient self-assembly to form
nanoﬁbers and hydrogels in aqueous media at pH 7.4. In
addition, we could use these water-soluble molecules for cell
imaging at concentrations below their minimum gel concen-
tration (mgc).

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "4.8", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "4.7", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "GF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "4.3", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "GG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "3.8", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "GF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "GG", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Mixture", "conjugate_partner": "NDI : 1,4,5,8-naphthalenetetracarboxylic acid diimide", "solution": "100 H2O", "peptide_concentration(mg/ml)": "20.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
romatic amino acid phenylalanine,
with a double bond between Ca and Cb atoms.
The introduction of DPhe induces conforma-
tional constraints in the peptide backbone [19,20]
and  also  imparts  proteolytic  stability  to  the
peptide  [21–23].  All  the  dipeptides  tested  have
C-terminal DPhe whereas they differed in their
N-terminal amino acids, which dictated their
assembly behavior. The dipeptides formed stable
self-assembled nanostructures that could encap-
sulate drug-like molecules. They also showed
high in vivo stability and some could even evade
uptake by reticuloendothelial systems (RES), a
desirable property for polymeric nanosystems.
Materials & methods
  Materials
The following materials were used: tetrahydrofu-
ran; N-methyl morpholine; dimethylformamide;
piperidine;  N,N’-disopropylcarbodimide;
isobutyl  chloroformate;  tri-isopropylsilane;
trifluoroacetic  acid  (TFA);  1,1,1,3,3,3-hexa-
fluoro-isopropanol  (HFIP);  phenol  (Sigma);
dl-threo-b-phenylserine;  stannous  tartar-
ate  (Sigma-Aldrich);  t-butoxycarbonyl(Boc)–
Phe-OH; Boc–Arg(Pmc)–OH; Boc–Leu-OH;
Boc–Glu(OtBu)-OH (Novabiochem); sodium
acetate; ethyl acetate; acetonitrile (Spectrochem);
anhydrous sodium sulfate; citric acid (Merck);
RPMI–1640 (Invitrogen); and 3-(4,5-dimeth-
ylthiazolyl-2)-2,5-diphenyltetrazolium bromide
(MTT; Sigma).
  Peptide synthesis
Dipeptides were synthesized (at a scale ranging
from 10 to 30 mM) as described below.
Synthesis of H-Phe–DPhe-OH
The peptide was synthesized by using a method
described previously  [24]. Briefly, Boc–Phe-OH
(5 mM) was dissolved in dry tetrahydrofuran,
chilled to -20°C and stirred in an ice–salt bath.
Equivalent moles of N-methyl morpholine and
isobutyl chloroformate were added to the solu-
tion. After 10 min, a precooled aqueous solution
of dl-threo-b-phenylserine (5.5 mM) and sodium
hydroxide (5.5 mM), were added, stirred over-
night and concentrated in vacuo. The residue was
acidified with citric acid and extracted with ethyl
acetate. The ethyl acetate layer was washed three
times with water and saturated sodium chloride,
dried over anhydrous sodium sulfate, and evapo-
rated to get Boc–Phe–dl-threo-b-phenylserine,
which was then mixed with anhydrous sodium
acetate (6.5 mM) in freshly distilled acetic anhy-
dride (50 ml) and stirred for 36 h. The thick slurry
obtained was poured over crushed ice and the pre-
cipitate was filtered, washed with 5% NaHCO3,
cold  water  and  dried  in  vacuo.  The  resulting
compound  (Boc–Phe–DPhe–azalactone)  was
dissolved in methanol, treated with 1.5 equiv of
1 M NaOH solution for 3–4 h, and concentrated
in vacuo. Acidification with solid citric acid lib-
erated the peptide which was extracted in ethyl
acetate,  to  yield  Boc–Phe–DPhe-COOH.  The
Boc group was deprotected using anhydrous TFA.
The  peptide  was  purified  on  a  preparative
reverse-phase C18 column (Deltapak, C18, 15 µm
internal diameter [i.d.] 300 × 19 mm) using an
acetonitrile–water linear gradient 5–95% ace-
tonitrile  (0.1%  TFA)/water(0.1%  TFA)  at  a
flow rate of 4ml/min over 55 min. The puri-
fied  peptide  was  reinjected  into  an  analytical
reverse-phase C18 column (Phenomenex, C18, 5µ,
i.d. 250 × 4.6 mm) using an acetonitrile–water
linear gradient 5–95% acetonitrile (0.1% TFA)/
water (0.1% TFA) at a flow rate of 1 ml/min over
25 min and was found to be 98% pure with a
retention time of 15 min. The purified peptide
was analyzed by MS (Applied Biosystems QStar
[Q-TOF]). Observed mass: 310.32 Da, expected
mass: 310 Da.
Assembly of dipeptides
Stock solutions of dipeptides were prepared by
dissolving 0.1–1 mg of them in 50 µl of HFIP.
In some cases mild sonication or heating was
necessary to dissolve peptides. Assembly was
initiated by addition of double-distilled water
(1 ml) to the stock solution. Samples were aged
24–48 h before experiments.
  Transmission electron microscopy
For transmission electron microscopic studies,
peptide samples were prepared at a concentra-
tion of 0.1–10 mg/ml in double-distilled water
and  incubated  for  30–60  min  to  make  sure
that  the  process  of  assembly  was  complete.
Samples  were  adsorbed  on  a  300-mesh  cop-
per  grid  with    a  carbon-coated  formvar  sup-
port and stained with 1% uranyl acetate and
viewed under 120 kV mode of a transmission
electron microscopy (Tecnai 12 BioTWIN, FEI
Netherlands). Photomicrographs were digitally
recorded using a Megaview II (SIS, Germany)
digital camera. Image ana lysis was carried out
using Analysis II (Megaview, SIS, Germany)
and ImageJ [101] software packages.
  Dynamic light-scattering studies
Light-scattering  studies  were  performed  in
Photocor complex using a multiple Tau digi-
tal correlator. All studies were done at room
temperature. Dynamic light scattering (DLS)
experiments  were  carried  out  at  an  angle  of
90°  using  a  32  nm  laser.  For  DLS  measure-
ment  samples  were  prepared  in  a  dust-free
environment.
  Drug entrapment
Drug entrapment in nanostructures that did
not show visible assembly
Sterile stocks of dipeptide nanostructures of con-
centration 1 mg/ml were prepared in double-
distilled water, filtered using Nalgene filters with
0.2 µm pore size and kept at 4°C. Drug stocks
were prepared at a concentration of 20 mg/ml
and diluted to respective concentrations in water
(niacin [2.5 mg/ml], amodiaquin [5 mg/ml],
vitamin B12 [5 mg/ml], mitoxantrone [2.5 mg/
ml] and ampicillin [10 mg/ml]) and kept at 4°C.
Relevant bioactive molecules were mixed with
preformed nanostructures (100 µg of drugs/ml
of nanostructures) and incubated for 48 h with
constant shaking. Samples were then ultracen-
trifuged at 120,000 rpm for 5 h at 10°C in a
Beckman Coulter OptiMax MLA-130 rotor. A
small volume was removed from the superna-
tant and the concentration of free test molecules
was estimated spectrophotometrically. The per-
centage  entrapment  was  calculated  using  the
f ollowing formula:
Drug entrapment in nanostructures that
showed visible assembly
The relevant bioactive molecules were added to
preformed  nanostructures  at  a  concentration
of  100  µg  of  drugs/1  ml  nanostructures  and
mixed  well  by  vortexing.  Samples  were  then
incubated for 48 h with constant shaking and
then centrifuged at 13,000 rpm for 30 min at
25°C in a table top centrifuge. A small volume
was removed from the supernatant and free-drug
concentration was estimated spectrophotometri-
cally. The percentage entrapment was calculated
as described above.
  Cytotoxicity assays
For cytotoxicity experiments, HeLa cells were
grown  in  RPMI-1640  supplemented  with
10% fetal calf serum. Cells were maintained
on  tissue  culture  treated  polystyrene  plates
(TCTP)  at  37°C  in  a  humified  atmosphere
containing 5% CO2. Sub-confluent cells were
trypsinized, harvested and counted by a try-
pan  blue  dye  exclusion  test  using  a  hemocy-
tometer.  The  resulting  cell  suspensions  were
diluted using RPMI-1640 (supplemented with
10% fetal calf serum) and seeded in 96-well
plates at a cell density of 2 × 104 cells per well.
Cells were plated in triplicates. After 12 h of
growth, approximately 20 µl of nanostructures
(prepared at a concentration of 1 mg/ml water)
were added to each well and incubated for a
period of 24 h. Cells incubated with 20 µl of
phosphate-buffered saline were taken as a con-
trol. After this, cell viability was determined
using a traditional MTT assay. The percent-
age viability was calculated using the equation
given below:
Percentage viability of cells = ([absorbance
of MTT formazan produced by cells grown in
presence of nanostructures/absorbance of MTT
formazan produced by cells grown on TCTP
control plate]) × 100
  Cellular uptake of
dipeptide nanostructures
HeLa cells were grown and maintained in TCTP
plates as described above. Sub-confluent cells
were trypsinized, harvested and counted. The
resulting  cell  suspensions  were  diluted  using
RPMI-1640  (supplemented  with  10%  fetal
calf serum) and seeded in 6-well plates at a cell
density of 1 × 105 cells per well. 2 µl of Alexa-
488  (Invitrogen,  from  stock  of  0.1  mg/20  µl
dimethylsulfoxide), was added to sterile stocks
of nanostructures prepared at a concentration of
0.1–1 mg/ml and incubated for 2 h at room tem-
perature. Free dyes were removed by centrifuging
nanostructures in 3 kDa cut-off plates. 200 µl of
these labeled nanostructures were added to HeLa
cells grown in 6-well plates and incubated for
18–24 h. After this, cells were washed 2–3-times
with phenol red-free media and observed under
fluorescence microscope.
  In vivo biodistribution of
dipeptide nanostructures
Radiolabeling of nanostructures
99mTc is the most widely used radioisotope in
nuclear  medicine,  commonly  available  and
much less expensive when compared with 111In
or 123I. Nanostructures were labeled with 99mTc
by direct labeling method using stannous tar-
tarate  and  sodium  pertechnetate  (sodium
pertechnetate separated from Molybdenum-99
by  solvent  extraction  method  was  procured
from Regional center for Radiopharmaceutical
division  [Northern  Region],  Board  of
Radiation  and  Isotope  Technology,  Delhi,
India. All other chemicals and solvents were
of analytical reagent grade). 0.1 ml of dipep-
tide nanostructures (from 1 mg/ml stock) were
mixed with 0.1 ml of stannous tartarate solu-
tion (2.5 mg/ml) and the pH was adjusted to
pH7  using  sodium  bicarbonate  solution.  To
this  mixture,  1  ml  of  99mTc  (2  mCi/ml)  was
added and incubated for 30 min at room tem-
perature. Quality control was performed as per
the method reported earlier  [25]. The labeling
efficiency of 99mTc with peptide nanostructures
was estimated by ascending thin-layer chroma-
tography (TLC)  using silica gel-coated fiber
sheets (Gelman Sciences Inc, MI, USA). The
instant  TLC  was  performed  using  acetone
or  0.9%  saline  as  the  mobile  phase.  A  total
of  2–3  µl  of  the  radio labeled  complex  was
applied  at  a  point  1  cm  from  one  end  of  an
instant-TLC–silica  gel  strip.  The  strip  was
developed in acetone or 0.9% saline and the
solvent front was allowed to reach 8 cm from
the origin. The strip was cut into two halves
and  the  radioactivity  in  each  segment  was
determined in a well-type g-ray counter (g-ray
spectrophotometer, Type GRS23C, Electronics
Corporation of India Ltd, Mumbai, India). The
free  pertechnetate  that  moved  with  the  sol-
vent (Rf = 0.9) was determined. The reduced/
hydrolyzed tech netium along with the labeled
complex remained at the point of application.
The amount of reduced/hydrolyzed 99mTc was
determined using saline as the mobile phase.
The  reduced/hydrolyzed  99mTc  remained  at
the  point  of  application  while  both  the  free
pertechnetate and the labeled complex moved
away with the solvent front. The net amount of
99mTc–peptide nanostructures was calculated by
subtracting the activity moved with the solvent
front using acetone and that when using saline.
Figure 2. Self-assembly of the dipeptides into different architectures.
HFIP: 1,1,1,3,3,3-hexa-fluoro-isopropanol.
Figure 3. Transmission electron microscopic images of self-assembled
nanostructures formed by the dipeptides at a concentration of 1 mg/ml in
1,1,1,3,3,3-hexa-fluoro-isopropanol/water.
RDF: Arg–DPhe; EDF: Glu–DPhe; FDF: Phe–DPhe; LDF: Lue–DPhe.
acetonitrile/water as the mobile phase. Purified
peptides were checked for purity by analytical
reverse-phase HPLC and were found to be 98%
pure with varying retention times depending
on their physical property  (table  1). Purified
peptides were further analyzed by MS (table 1).
  Self-assembly of ∆Phe-containing
dipeptides
Inspired by the self-assembly of Phe–DPhe into
stable nanotubular structures, we investigated
the self-assembling behavior of other DPhe con-
taining dipeptides, having positive (Arg), nega-
tive (Glu), hydrophobic (Lue) or aromatic (Phe)
amino  acids  at  their  N-terminus  (FiguRe  1).
Self-assembly of dipeptide nanostructures was
initiated by dispersion of 1 mg of peptides in a
small volume (50 µl) of HFIP and subsequent
addition of 1 ml of water at near-neutral pH
(FiguRe 2). Self-assembled structures formed by
dipeptides Phe–DPhe, Lue–DPhe, were visible
to the naked eye, whereas those formed by Arg–
DPhe and Glu–DPhe were not visible (table 2).
A critical ana lysis revealed that dipeptides
having hydrophobic groups at their N-terminus
formed visible assemblies whereas those with
hydrophilic  N-termini  formed  assembled
s tructures that were not visible to the naked eye.
  Electron microscopic studies
The morphology of peptide nanostructures was
ascertained using transmission electron micro-
scopy with uranyl acetate staining  (FiguRe 3).
Amphiphilic dipeptides such as Glu–DPhe and
Arg–DPhe assembled into vesicular structures
(table 2 & FiguRe 3). Arg–DPhe, which did not
show  any  visible  assembly,  formed  pleomor-
phic vesicular structures with an average dia-
meter of 40 nm at a concentration of 1 mg/ml
(FiguRe 3). However, the dipeptide Glu–DPhe
needed  a  higher  concentration  (5  mg/ml)  to
form vesicular nanostructures. Vesicles formed
by  the  dipeptide  were  pleomorphic  with  a
mean diameter of 110 nm [26]. At a concentra-
tion of 1 mg/ml, Glu–DPhe formed irregular
structures with no proper shape (FiguRe 3). As
reported earlier, Phe–DPhe, which has an addi-
tional aromatic group, formed ordered nano-
tubes [23] with a mean diameter of 15–20 nm
and length in microns where as the dipeptide
Leu–DPh

[Assistant] [{"peptide_sequence": "RF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "4.8", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2160.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "4.8", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2160.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "4.8", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2160.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
We demonstrate the use of dipeptide amphiphiles that, by hand shaking of a
biphasic solvent system for a few seconds, form emulsions that remain stable for
months through the formation of nanofibrous networks at the organic/aqueous
interface. Unlike absorption of traditional surfactants, the interfacial
networks form by self-assembly through π-stacking interactions and hydrogen
bonding. Altering the dipeptide sequence has a dramatic effect on the properties
of the emulsions formed, illustrating the possibility of tuning emulsion
properties by chemical design. The systems provide superior long-term stability
toward temperature and salts compared to with sodium dodecyl sulfate (SDS) and
can be enzymatically disassembled causing on-demand demulsification under mild
conditions. The interfacial networks facilitate highly tunable and stable
encapsulation and compartmentalization with potential applications in cosmetics,
therapeutics, and food industry.

Materials. Fmoc-Tyr (97%), H-Leu-OtBu 3 HCl (g98.0%), Ala-Ot-
Bu 3 HCl (g98.0%), O-tert-Butyl-L-Ser-tert-Butyl ester hydrochloride
(g98.0%), N,N-Diisopropylethylamine (DIPEA) (>99.5%), Trifluoro-
acetic acid (99%), Sodium hydroxide, 1-Pyreneacetic acid, Fluor-
escein isothiocyanate isomer I (g90%), Thioflavin T, Sodium
phosphate monobasic monohydrate (ACS reagent, 98.0%(cid:1)
102.0%), Sodium phosphate dibasic heptahydrate (ACS reagent,
98.0%(cid:1)102.0%), Hexadecane (99%) and Mineral oil were pur-
chased from Sigma-Aldrich and used as received. Fmoc-Phe-Phe-
Phe-OH was purchased from BACHEM. 2-(1H-Benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was
purchased from Novabiochem. Phosphate buffer solution (pH 8)
was prepared by dissolving 94 mg of NaH2PO4 3 H2O and 2.5 g of
Na2HPO4 3 7H2O in 100 mL of water.
Preparation of Emulsions. 10 mM Fmoc-YL solution was pre-
pared by dissolving 5.32 mg of Fmoc-YL in 1 mL of phosphate
buffer solution. Different volumes of chloroform were added
to Fmoc-YL buffer solution at 80 (cid:1)C (the volume ratio of buffer
solution to chloroform is altered from 1:9, 3:7, 5:5, 7:3, to
9:1, total volume was always 1 mL), after hand-shaking for
5 s emulsions form in vials. For SEM, IR, stability, average particle
size, critical emulsion concentration and demulsification mea-
surements, the volume ratio of buffer solution to chloroform
was fixed at 7:3. The concentration of all aromatic peptide
amphiphiles studied (Fmoc-YA, Fmoc-YS, Pyr-YL, Fmoc-FF and
Fmoc-FFF) was 10 mM, except for in the determination of the
critical emulsion concentration (0.1(cid:1)10 mM) and demulsifica-
tion measurements (2 mM).
Characterization. The structures of Fmoc-YL microcapsules
were determined by Hitachi S800 field emission scanning elec-
tron microscope (SEM) at an accelerating voltage of 10 keV. The
transfer of Fmoc-YL, YA and YS were analyzed by UV(cid:1)vis
spectroscopy (JAS.C.O V-660 spectrophotometer). The formation
of Fmoc-YL gels and labeling of ThT were carried out by
fluorescence spectroscopy (JAS.C.O FP-6500 spectrofluorometer).
High resolution mass spectra (HRMS) were recorded on a Thermo
Electron Exactive. 400.1 (1H) NMR spectra were recorded on
Bruker Avance 400 spectrometer at room temperature using
perdeuterated solvents as internal standards.
The ﬁbrous structures of Fmoc-YL, YA, YS were determined
by atomic force microscopy (AFM). The images were obtained
by scanning the mica surface in air under ambient conditions using
a Veeco diINNOVA Scanning Probe Microscope (VEECO/BRUKER,
Santa Barbara, CA, USA) operated in tapping mode. 20 μL of solu-
tions were placed on a trimmed and freshly cleaved mica sheet
(G250(cid:1)2 Mica sheets 100 (cid:3) 100 (cid:3) 0.00600; Agar Scientiﬁc Ltd., Essex,
UK) attached to an AFM support stub and left to air-dry overnight in
a dust-free environment. The AFM scans were taken at 512 (cid:3) 512
pixels resolution. Typical scanning parameters were as follows: tapp-
ing frequency 308 kHz, integral and proportional gains 0.3 and 0.5,
respectively, set point 0.5(cid:1)0.8 V and scanning speed 1.0 Hz.
The β-sheet-like arrangement was determined by infrared
absorption spectra, which were recorded on a Bruker Vertex 70
spectrometer, averaging 25 scans per sample at a resolution
of 1 cm(cid:1)1. Samples were sandwiched between two 2 mm CaF2
windows separated with a 50 μm polytetraﬂuoroethylene
(PTFE) spacer.
Conﬂict of
ﬁnancial interest.
Supporting Information Available: Synthesis of peptides,
ﬂuorescence spectra of peptide before and after gelation,
UV(cid:1)vis spectra of peptides in water and oil phases, transfer
between two phases, AFM images of peptide ﬁbers, and
photographs of critical emulsion concentration measurements
were showed. This material is available free of charge via the
Internet at http://pubs.acs.org/.
5. Crossley, S.; Faria, J.; Shen, M.; Resasco, D. E. Solid Nano-
particles That Catalyze Biofuel Upgrade Reactions at the
Water/Oil Interface. Science 2010, 327, 68–72.
6. Muller, R. H.; Mader, K.; Gohla, S. Solid Lipid Nanoparticles
(SLN) for Controlled Drug Delivery;A Review of the State
of the Art. Eur. J. Pharm. Biopharm. 2000, 50, 161–177.
7. Chen, L. Y.; Yu, S. Z.; Wang, H.; Xu, J.; Liu, C. C.; Chong, W. H.;
Chen, H. Y. General Methodology of Using Oil-in-Water
and Water-in-Oil Emulsions for Coiling Nanoﬁlaments.
J. Am. Chem. Soc. 2013, 135, 835–843.
10. Platzman, I.; Janiesch, J. W.; Spatz, J. P. Synthesis of Nano-
structured and Biofunctionalized Water-in-Oil Droplets as
Tools for Homing T Cells. J. Am. Chem. Soc. 2013, 135,
3339–3342.
14. Helgeson, M. E.; Moran, S. E.; An, H. Z.; Doyle, P. S.
Mesoporous Organohydrogels
from Thermogelling
Photocrosslinkable Nanoemulsions. Nat. Mater. 2012, 11,
15. Zhang, W. B.; Zhu, Y. Z.; Liu, X.; Wang, D.; Li, J. Y.; Jiang, L.;
Jin, J. Salt-Induced Fabrication of Superhydrophilic and
Underwater Superoleophobic PAA-g-PVDF Membranes
for Eﬀective Separation of Oil-in-Water Emulsions. Angew.
Chem., Int. Ed. 2014, 53, 856–860.
22. Morikawa, M.; Yoshihara, M.; Endo, T.; Kimizuka, N. Alpha-
Helical Polypeptide Microcapsules Formed by Emulsion-
Templated Self-Assembly. Chem.;Eur. J. 2005, 11, 1574–
25. Hermanson, K. D.; Huemmerich, D.; Scheibel, T.; Bausch,
A. R. Engineered Microcapsules Fabricated from Recon-
stituted Spider Silk. Adv. Mater. 2007, 19, 1810–1815.
26. Wu, C.; Bai, S.; Ansorge-Schumacher, M. B.; Wang, D. Y.
Nanoparticle Cages for Enzyme Catalysis in Organic Media.
Adv. Mater. 2011, 23, 5694–5699.
27. Cui, M. M.; Emrick, T.; Russell, T. P. Stabilizing Liquid Drops
in Nonequilibrium Shapes by the Interfacial Jamming of
Nanoparticles. Science 2013, 342, 460–463.
28. Miesch, C.; Kosif, I.; Lee, E.; Kim, J. K.; Russell, T. P.; Hayward,
R. C.; Emrick, T. Nanoparticle-Stabilized Double Emulsions
and Compressed Droplets. Angew. Chem., Int. Ed. 2012, 51,
29. Kosif, I.; Cui, M. M.; Russell, T. P.; Emrick, T. Triggered In Situ
Disruption and Inversion of Nanoparticle-Stabilized Dro-
plets. Angew. Chem., Int. Ed. 2013, 52, 6620–6623.
Jiang, J. H.; Zhu, Y.; Cui, Z. G.; Binks, B. P. Switchable
Pickering Emulsions Stabilized by Silica Nanoparticles
Hydrophobized In Situ with a Switchable Surfactant. An-
gew. Chem., Int. Ed. 2013, 52, 12373–12376.
31. Yang, H. Q.; Zhou, T.; Zhang, W. J. A Strategy for Separating
and Recycling Solid Catalysts Based on the pH-Triggered
Pickering-Emulsion Inversion. Angew. Chem., Int. Ed. 2013,
52, 7455–7459.
36. Wiese, S.; Spiess, A. C.; Richtering, W. Microgel-Stabilized
Smart Emulsions for Biocatalysis. Angew. Chem., Int. Ed.
2013, 52, 576–579.
41. Raeburn, J.; Alston, B.; Kroeger, J.; McDonald, T. O.; Howse,
J. R.; Cameron, P. J.; Adams, D. J. Electrochemically-
Triggered Spatially and Temporally Resolved Multi-
Component Gels. Mater. Horiz. 2014, 1, 241–246.
Johnson, E. K.; Adams, D. J.; Cameron, P. J. Directed
Self-Assembly of Dipeptides to Form Ultrathin Hydrogel
Membranes. J. Am. Chem. Soc. 2010, 132, 5130–5136.
43. Braun, H. G.; Cardoso, A. Z. Self-Assembly of Fmoc-Diphe-
nylalanine inside Liquid Marbles. Colloids Surf., B 2012, 97,
44. Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A. K.;
Ulijn, R. V. Enzyme Assisted Self-Assembly under Thermo-
dynamic Control. Nat. Nanotechnol. 2009, 4, 19–24.
45. Bai, S.; Debnath, S.; Gibson, K.; Schlicht, B.; Bayne, L.;
Zagnoni, M.; Ulijn, R. V. Biocatalytic Self-Assembly of
Nanostructured Peptide Microparticles Using Droplet Mi-
croﬂuidics. Small 2014, 10, 285–293.
46. Mann, S. The Origins of Life: Old Problems, New Chemis-
tries. Angew. Chem., Int. Ed. 2013, 52, 10746–10750.
47. Zou, Q.; Zhang, L.; Yan, X.; Wang, A.; Ma, G.; Li, J.; Möhwald,
H.; Mann, S. Multifunctional Porous Microspheres Based
on Peptide-Porphyrin Hierarchical Co-Assembly. Angew.
Chem., Int. Ed. 2014, 53, 2366–2370.
49. Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Rigid,
Self-Assembled Hydrogel Composed of a Modiﬁed Aro-
matic Dipeptide. Adv. Mater. 2006, 18, 1365–1370.
50. Hirst, A. R.; Roy, S.; Arora, M.; Das, A. K.; Hodson, N.; Murray,
P.; Marshall, S.; Javid, N.; Sefcik, J.; Boekhoven, J.; van Esch,
J. H.; Santabarbara, S.; Hunt, N. T.; Ulijn, R. V. Biocatalytic
Induction of Supramolecular Order. Nat. Chem. 2010, 2,
1089–1094.
51. Fleming, S.; Debnath, S.; Frederix, P. W. J. M.; Hunt, N. T.;
Ulijn, R. V. Insights into the Co-Assembly of Hydrogelators
and Surfactants Based on Aromatic Peptide Amphiphiles.
Biomacromolecules 2014, 10.1021/bm240170z.
52. Zhang, Y.; Yang, Z. M.; Yuan, F.; Gu, H. W.; Gao, P.; Xu, B.
Molecular Recognition Remolds the Self-Assembly of Hy-
drogelators and Increases the Elasticity of the Hydrogel by
10(6)-fold. J. Am. Chem. Soc. 2004, 126, 15028–15029.
53. Yang, J. J.; Van Wart, H. E. Kinetics of Hydrolysis of Dansyl
Peptide Substrates by Thermolysin: Analysis of Fluores-
cence Changes and Determination of Steady-State Kinetic
Parameters. Biochemistry 1994, 33, 6508–6515.
54. Roy, S.; Javid, N.; Frederix, P. W. J. M.; Lamprou, D. A.;
Urquhart, A. J.; Hunt, N. T.; Halling, P. J.; Ulijn, R. V. Dramatic
Speciﬁc-Ion Eﬀect in Supramolecular Hydrogels. Chem.;
Eur. J. 2012, 18, 11723–11731.
55. Roy, S.; Javid, N.; Sefcik, J.; Halling, P. J.; Ulijn, R. V. Salt-
Induced Control of Supramolecular Order in Biocatalytic
Hydrogelation. Langmuir 2012, 28, 16664–16670.
57. Fleming, S.; Frederix, P. W. J. M.; Ramos-Sasselli, I.; Hunt, N.;
Ulijn, R. V.; Tuttle, T. Assessing the Utility of Infrared
Spectroscopy as a Structural Diagnostic Tool for β-sheets
in Self-Assembling Aromatic Peptide Amphiphiles. Lang-
muir 2013, 29, 9510–9515.
59. Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.;
Turner, M. L.; Saiani, A.; Ulijn, R. V. Fmoc-Diphenylalanine
Self Assembles to a Hydrogel via a Novel Architecture
Based on pi-pi Interlocked beta-Sheets. Adv. Mater. 2008,
20, 37–41.
60. Li, X.; Kuang, Y.; Lin, H. C.; Gao, Y.; Shi, J.; Xu, B. Supra-
molecular Nanoﬁbers and Hydrogels of Nucleopeptides.
Angew. Chem., Int. Ed. 2011, 50, 9365–9369.
61. Reches, M.; Gazit, E. Casting Metal Nanowires within
Discrete Self-Assembled Peptide Nanotubes. Science 2003,
300, 625–627.
62. Moitra, P.; Kumar, K.; Kondaiah, P.; Bhattacharya, S. Eﬃcacious
Anticancer Drug Delivery Mediated by a pH-Sensitive
Self-Assembly of a Conserved Tripeptide Derived from
Tyrosine Kinase NGF Receptor. Angew. Chem., Int. Ed. 2014,
53, 1113–1117.

[Assistant] [{"peptide_sequence": "YL", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "5.53", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "YL", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Chloroform", "peptide_concentration(mg/ml)": "5.53", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "YA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "5.11", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "YA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Chloroform", "peptide_concentration(mg/ml)": "5.11", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "YS", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "5.27", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Self-assembly of aromatic peptide amphiphiles is known to be driven by a
combination of π-π stacking of the aromatic moieties and hydrogen bonding
between the peptide backbones, with possible stabilisation from the amino acid
side chains. Phenylalanine-based Fmoc-dipeptides have previously been reported
for their characteristic apparent pKa transitions, which were shown to coincide
with significant structural and morphological changes that were peptide sequence
dependent. Here, phenylalanine was replaced by leucine and the effect on the
self-assembling behaviour of Fmoc-dipeptides was measured using potentiometry,
fluorescence and infrared spectroscopy, transmission electron microscopy, X-ray
scattering and shear rheometry. This study provides additional cues towards the
elucidation of the sequence-structure relationship in self-assembling aromatic
peptide amphiphiles.

Materials: All Fmoc-dipeptides were purchased from C S
Bio Company (Menlo Park, USA) and used without fur-
ther puriﬁcation. The purity of the compounds was ver-
iﬁed by HPLC (> 98%) and mass spectrometry. HPLC
grade water and deuterated water (99.9 atom% D) were
purchased from Merck and Sigma-Aldrich, respectively.
Sample preparation: Depending on the desired concen-
tration, the required amount of Fmoc-dipeptide was sus-
pended into 2 mL of HPLC grade water. Sodium hydroxide
(0.5 M) was gradually added to the aqueous suspensions
of Fmoc-dipeptide until pH 10.5 was reached (∼ 55 μL
for 10 mmol L−1 sample). The samples were vortexed and
sonicated for one minute to fully dissolve the peptide am-
phiphiles. Depending on the concentration and on the
target pH, a required volume of dilute hydrochloric acid
(0.085 M) was then added dropwise while the solution was
vortexed and sonicated until the target pH was obtained.
Next, the samples were heated to 75–80 ◦C until fully dis-
solved (1.0, 2.5 and 1.5 minutes for Fmoc-LL, Fmoc-LG
and Fmoc-GL, respectively) and homogenised. The sam-
ples were subsequently cooled and maintained at 4 ◦C for
∼ 12 hours (overnight) to promote or maintain gelation.
To allow comparison between the studied samples, all were
investigated after this same aging time. Reported pH val-
ues were those measured after storage. They were found
to be identical to the pH values measured before heating
within ±0.3 units.
Potentiometry: pH measurements were performed using
a Hanna Instruments pH210 pH-meter equipped with
a Hamilton Spintrode pH-probe (reference system: Ag/
AgCl, electrolyte: 3 M KCl, diaphragm: ceramic, sensi-
tivity: 58 mV/pH unit at 25 ◦C). The pH-meter was cali-
brated before each experiment to check the response of the
electrode with two buﬀer solutions purchased from Fisher
Scientiﬁc: phthalate pH 4.01 and phosphate pH 7.01 buﬀer
solutions.
“Titration” experiments: The 5, 10 and 20 mmol L−1 sam-
ples at pH 10.5 were prepared as described above. For
1 mmol L−1 samples, 5 mmol L−1 samples at pH 10.5 were
used as stock solutions and diluted to the desired con-
centration in a ﬁnal volume of 2 mL. The pH of the ﬁ-
nal solutions was then adjusted to pH 10.5. The “titra-
tion” experiments were performed by stepwise addition of
small volumes of diluted HCl (0.085 M; 2–60 μL depend-
ing on pH and system), up to a total added volume of
750 μL. After each addition the samples were heated to
75–80 ◦C for one minute, vortexed, sonicated, and subse-
quently cooled back to room temperature using a water
bath. pH values were recorded before and after heating of
the samples. The samples were deemed fully mixed when
no signiﬁcant diﬀerence (±0.3 pH unit) was observed be-
tween two pH measurements. Due to the strong and con-
stant agitation applied, samples were liquid at all times
during the “titration” experiments. As a control, water
was also titrated using the same methodology described
above; that is, NaOH was added to the water in order to
bring the pH of the solution to 10.5 and then HCl was
added stepwise. Experiments, including the characterisa-
tion experiments described below, were repeated a least 3
times to ensure reproducibility.
overnight. Emission and excitation slit widths were set at
3 and 10 nm, respectively. Emission spectra (excitation at
265 nm) were acquired at 25 ◦C in the 300–600 nm range
with a scan speed of 300 nm min−1 using FL WinLab soft-
ware. Changes in the pH conditions and therefore in the
self-assembled structures formed are likely to modify the
refractive index of the samples and the quenching eﬀect
resulting from self-assembly. These eﬀects would have a di-
rect eﬀect on the intensity of the transmitted light hence
on the emission peaks’ intensity. For this reason, ﬂuores-
cence spectra were all normalised as a function of their
respective maximum emission peak. The un-normalised
spectra are presented in the supplementary material.
Fourier-transform infrared spectroscopy (FTIR): Multiple
bounce attenuated total reﬂectance (ATR) FTIR experi-
ments were undertaken using samples prepared in deuter-
ated water. Spectra were recorded on a Thermo Nicolet
5700 spectrometer equipped with a trough plate compris-
ing of a zinc selenide crystal, which permitted 12 reﬂec-
tions with a 45◦ angle of incidence. The samples were
spread directly on the surface of the trough plate. Spec-
tra were acquired in the 4000–400 cm−1 range with a res-
olution of 4 cm−1 over 128 scans. The deuterated water
spectrum was used as background and subtracted from all
spectra (software used: Omnic version 7.2, Thermo Elec-
tron Corporation).
Mechanical properties: Mechanical properties were as-
sessed using a stress-controlled rheometer (Bohlin C-
VOR) equipped with a Peltier device (Bohlin Instru-
ments) to control temperature using a parallel-plate ge-
ometry (40 mm diameter). To ensure the measurements
were made in the linear regime, amplitude sweeps were
performed and showed no variation in G(cid:3) and G(cid:3)(cid:3) up to
a strain of 1%. The dynamic moduli of the hydrogel were
measured as a function of frequency in the range 0.01–
100 rad s−1 with a strain of 1%. To keep the sample hy-
drated, a solvent trap was used and the atmosphere within
the sample chamber was saturated with water. Unless oth-
erwise stated the experiments were performed at 25 ◦C
and repeated at least three times (each time on new sam-
ples) to ensure reproducibility. The standard deviation of
multiple experiments is represented by error bars in the
dynamic frequency sweeps.
Transmission electron microscopy (TEM) and image anal-
ysis: A carbon-coated copper grid (400 mesh, Agar Scien-
tiﬁc) was glow discharged for 30 seconds then placed on
10 μL of sample for 15 seconds. After blotting on What-
man 50 ﬁlter paper, the loaded grid was washed in double
distilled water for 30 seconds and blotted. The sample
was then stained with 10 μL of 2% (w/v) uranyl acetate
(centrifuged for 5 minutes beforehand) for one minute and
blotted for 10 seconds. Data were collected at high vac-
uum on a JEOL 1220 transmission electron microscope
connected to a high resolution (up to 3 ˚A/pixel resolution)
Gatan Orius CCD camera.

[Assistant] [{"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "4.3", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "4.8", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "5.3", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "5.8", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "6.3", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "6.8", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "8.0", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "8.5", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "9.0", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "9.5", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "10.0", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LL", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.443", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "7.6", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "4.3", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "4.56", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "4.82", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "5.08", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "5.34", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "1.882", "PH": "5.6", "temperature(celsius)": "4.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Ethanol", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Dichloromethane", "peptide_concentration(mg/ml)": "4.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}]