[System] polypeptide self-assembly literature information extraction

[User]
 acid and L-
phenylalanine were purchased from Aldrich. HOBt,
dihydrogen phosphate, and disodium hydrogen phosphate were
purchased from Merck. DCC, NaOH, MeOH, silica gel (100−200
mesh), Et2O, petroleum ether, EtOAc, and DMF were purchased from
SRL (India). The water used in all experiments was of Millipore Milli-
Q grade. Dulbecco Modiﬁed Eagle Medium (DMEM), 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), kana-
mycin sulfate, trypsin−EDTA solution, sodium chloride, potassium
chloride, and fetal bovine serum were purchased from Sigma-Aldrich.
2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)
was purchased from Himedia. Penicillin−streptomycin was purchased
from Invitrogen. Sodium bicarbonate was purchased from Merck. All
compounds were used without further puriﬁcation. MCF-7 (human
breast cancer cell line) was purchased from NCCS, Pune (India), and
cultured in Dulbecco Modiﬁed Eagle medium (DMEM) containing
10% (v/v) heat-inactivated fetal bovine serum at 37 °C and 5% CO2
atmosphere in our lab. Details of the synthetic procedures of gelator
peptide and instrumentation details are given in the Supporting
Information.
Morphology. To image the internal structure of
hydrogel, morphological studies were conducted with ﬁeld
emission scanning electron microscopy (FE-SEM) and high-
resolution transmission electron microscopy (HR-TEM). The
FE-SEM and HR-TEM images (Figure 2) revealed a dense
Figure 2. (a) Field emission scanning electron microscope (FE-SEM)
and (b) high-resolution ﬁeld emission scanning electron microscope
(HR-FE-SEM) at 9.375 mM concentration. (c) Transmission electron
microscope (TEM) and (d) high-resolution transmission electron
microscope (HR-TEM) images at 2.34 mM concentration.
nanoﬁbrilar network for
the xerogel (prepared from the
hydrogel at pH 7.46). The TEM images show that the width
of the ﬁbers varies between 25 and 45 nm while in the SEM
images this range is 50−90 nm. The diﬀerence in diameter of
the nanoﬁbers obtained from the two diﬀerent microscopic
techniques is due to the diﬀerence in sample preparation used
these two techniques (FE-SEM and HR-TEM). Self-
assembled peptide ﬁbers are several micrometers in length. The
entangled ﬁbrilar network provides a cage-like environment
where small water molecules can entrap to give a self-
supporting hydrogel. The three-dimensional network also
oﬀers enough room for entrapment of relatively large drug
FT-IR Study. Fourier transform infrared spectroscopy (FT-
IR) of the xerogel reveals a hydrogen-bonded supramolecular
network within the hydrogel. Signiﬁcant peaks were obtained at
3371, 1688, 1639, and 1535 cm−1 in the FTIR spectrum
(Figure S6). The peak at 3371 cm−1 indicates a hydrogen-
bonded N−H stretching while the peaks at 1639 and 1535
cm−1 are signature peaks for CO stretching (amide I) and
N−H bending (amide II), respectively. A sharp peak at 1688
cm−1 clearly suggests the presence of an antiparallel β-sheet
arrangement of the gelator peptide molecules within the gel
phase material.13
X-ray Diﬀraction Studies. To gain insight about
molecular arrangement within the supramolecular network
structure, SAXS on gels and wide-angle powder XRD of the
xerogel were carried out. From the small-angle X-ray scattering
it appears that the
(SAXS) measurements for the wet gel,
length of a single molecule in the self-assembled structure is
about 31 Å (D) (Figure S7). Taking this into consideration, in
the wide-angle XRD pattern periodic peaks at 5.25 Å (2θ =
16.86°), 4.61 Å (2θ = 19.23°), 3.88 Å (2θ = 22.90°), and 3.34
Å (2θ = 26.65°) can be assigned as D/6, D/7, D/8, and D/9,
respectively (Table 1 and Figure 3). This result indicates a
molecules (Table 1 and Figure 3). In this study, d = 4.92 Å
is the distance between the peptide chains within a β-sheet,
while the d spacing of 8.44 Å can be characterized as the
distance between two stacked β-sheets.13 Another peak at d =
3.76 Å signiﬁes the π−π interaction between the phenyl rings of
the molecules in the three-dimensional network structure
(Figure 3).56 Combining these results obtained from X-ray
diﬀraction and FT-IR analyses, it can be concluded that the
gelator peptide molecules are self-assembled in the gel state to
form a β-sheet-like structure. A probable mechanistic model is
shown in Figure S8. This indicates the various noncovalent
interactions involved in the self-assembled gel state material.
Fluorescence Spectroscopic Study for π−π Interac-
tion. Fluorescence spectroscopy is a very useful tool to know
the existence of π−π interactions within the self-assembled
molecules.57,58 To carry out this study, eight solutions were
taken in phosphate buﬀer (pH 7.46), where the gelator
concentration was varied from 0.0005% (w/v) to 0.1% (w/v).
Fluorescence emission spectra of these solutions were recorded
with excitation at 261 nm (Figure S9). A new broad peak
begins to develop around 358 nm, when the concentration is
increased from 0.0025% (w/v) to 0.005% (w/v), and this peak
gradually becomes signiﬁcant as the concentration increases up
to 0.1% (w/v). This new peak at 358 nm arise from the π−π
interaction.59,60 This indicates the presence of a strong aromatic
interaction between the phenyl
side chain moieties of
phenylalanine residues in these interacting gelator molecules.
the Thixotropic Property. A glass vial
containing 5 mg of P1 was taken and dissolved in 1 mL of
phosphate buﬀer having pH 7.46, and it was left standing for a
few minutes. The gel thus formed was shaken vigorously by
hand to obtain a clear solution. This solution was then allowed
to rest, and a self-supportive gel was regained within 40 min
(Figure 4). This thixotropic property of the peptide hydrogel
can be utilized for injectable purpose with an application in
drug delivery system (Figure 5).
investigations were carried out to
gain knowledge about the mechanical strength of the hydrogel
as well as to check whether the thixotropic nature observed
with naked eye can be explained through analytical measure-
ments. At ﬁrst a frequency sweep experiment (Figure 6) was
done with a 0.8% (w/v) hydrogel, where the storage modulus
(G′) and loss modulus (G″) were measured as a function of
angular frequency keeping strain constant at 0.1%. In the low-
frequency region (6−100 rad/s) the G′ and G″ values are only
weakly dependent on frequency, indicating formation of a good
stable gel.
A continuous strain time dependent study of the hydrogel
was then performed by using 0.5% (w/v) hydrogel, and it was
subjected to a continuous strain increased from 0.1% to 50% to
rupture the gel matrix. Then its restoration phenomenon was
recorded with time under a constant strain of 0.1%. During
lamellar arrangement of
the molecules within the self-
assembled network structure.54,55 Moreover, the peaks at d
values of 4.92 Å (2θ = 18.00°) and 8.44 Å (2θ = 10.47°) are
indicative of a β-sheet-like arrangement of
these gelator
Figure 5. Illustration of the injectable nature of the hydrogel and its vitamin release phenomenon with vitamin B12.
Figure 6. Frequency sweep rheological analysis of the hydrogel [(0.8%
(w/v)] at a constant strain of 0.1%.
application of continuous strain the G′ line gradually goes
below the G″ line to conﬁrm the transformation of the gel to a
sol like material, and then with re-establishment of gel strength
after cessation of high applied strain the G′ line again moves
over G″. The process is repeated over three cycles to verify its
reproducibility (Figure S10).
The frequency sweep experiment and step strain experiment
at 0.5% w/v have also been given in Figure S11 and Figure S12,
respectively.
Though the thixotropic property of the hydrogel can be
nicely explained from the previous experiment, the injectable
nature of a gel-like material can be best conﬁrmed through a
time dependent step-strain rheological experiment.35,61−63 In
this process the 0.8% (w/v) gel was ﬁrst kept under low
constant strain of 0.1% for the ﬁrst 200 s, and then it was
suddenly subjected to a higher strain of 40%; at this point G″
values greater than the corresponding G′ values were observed.
After that, the strain was suddenly decreased and ﬁxed to a
constant low strain of 0.1% (same as ﬁrst interval), and from
this point (400 s) the restoration of gel strength was observed
(Figure 7). It is evident from Figure 7 that the hydrogel takes
about 360 s after the withdrawal of the large strain to almost
for G′ value to retain its
recover its original strength,
low strain of 0.1%. All
original value, under a constant
Figure 7. Step-strain time dependent rheological analysis of
hydrogel [(0.8% (w/v)] at a ﬁxed angular frequency of 1 rad/s.
rheological experiments were performed at a constant temper-
ature of 25 °C. The recovery of the original gel strength is
crucial for injectable applications. In real situations, the three
intervals of this experiment are analogous to (i) a viscoelastic
material within a syringe, (ii) the material within the needle of
the syringe, and (iii) after its injection into a target system.
To check whether the gel-sol transformation occurs just on
mechanical shaking by hand, the frequency sweep experiment
has been performed both in gel state and in the sol state after
mechanical shaking of the 0.8% w/v gel at a ﬁxed strain of 15%.
In the case of gel, G′ > G″ in our studied frequency region
(0.5−6 rad/s). However, after mechanical shaking of the gel, it
is transformed into a low viscous liquid, and G′ < G″ in the low
and our studied frequency region (0.5−6 rad/s). This study
clearly indicates the breaking of
the gel upon mechanical
shaking and the transformation of the gel phase material to a
low viscous liquid/solution (Figure S13).
Sustained Drug Release from the Injectable Gel. The
peptide P1 forms a hydrogel at physiological pH (7.46) and
temperature (37 °C). The nanoﬁbrillar network structure
within the hydrogel has many void spaces as it is evident from
FE-SEM and HR-TEM images, and it provides a cage-like
environment that can act as a template for the entrapment and
release of small drug molecules. The supramolecular three-
dimensional ﬁbrous network structures within the hydrogel,
conﬁrmed from FE-SEM and HR-TEM images, provides a
cage-like environment, and this can work as a template for the
entrapment and release of small drug molecules. Vitamin B12
and vancomycin are the two biomolecules which were used in
the present study to observe their release kinetics from the
hydrogel. Vitamin B12 (cyanocobalamin) plays a very important
role in normal functioning of nerves and brain and for the
formation of blood.14 On the other hand, vancomycin is an
antibiotic used in the treatment of infections caused by Gram-
positive bacteria like penicillin-resistant Staphylococcus aureus.63
It is interesting to observe the release of these two biologically
important molecules from the peptide-based hydrogel at
physiological pH and temperature (37 °C and pH 7.46). In
this regard, two 1 mL gels having the same concentration of
0.8% (w/v) were prepared and loaded with vitamin B12
(hydrogel 1) and vancomycin (hydrogel 2) separately. These
gels (1 mL each) were encapsulated with 1.14 mg of vitamin
B12 and 0.24 mg of vancomycin separately. The loaded
hydrogels were covered with phosphate buﬀer over the gels
and kept at 37 °C in an incubator. The gradual enhancement of
color intensity of the buﬀer layer and subsequent decrease in
color intensity of the gel layer were observed for the vitamin B12
containing hydrogel (Figure 5). However, this observation was
missing from the other hydrogel as the vancomycin solution is
colorless. For the kinetic study, small aliquots of the buﬀer
solution were taken from time to time, and their UV/vis spectra
were recorded to note the absorbance values at 550 nm for
vitamin B12 and 280 nm for vancomycin. The absorbance data
of aliquots from hydrogel 1 (the hydrogel containing only
vitamin B12) and hydrogel 2 (the hydrogel containing only
vancomycin) were taken at the same time up to 47 h. The
entire process was repeated ﬁve times for each sample, and a
percentage release proﬁle of drugs with error bars is obtained
Figure 8. Percentage release plot of some important biomolecules
from the hydrogel at physiological pH (7.46) and temperature (37
°C), where concentration of the drugs loaded in the hydrogel was 1.14
mg/mL for cyanocobalamin (vitamin B12) and 0.24 mg/mL for
vancomycin.
where Mt, M∞, and λ are the total amount of drug released
during the measurement, the total amount of drug that was
kept within the gel matrix, and hydrogel thickness, respectively.
The time of measurement (in seconds) and the diﬀusion
constant of the drug molecule are represented by t and D,
respectively. The drug loaded hydrogels in glass vials remained
stable in the presence of buﬀer for about 47 h for both hydrogel
1 and hydrogel 2 under physiological pH and temperature. It
has been observed that about 90% and 80% drug have been
released from the hydrogels 1 and 2, respectively, before these
gels start to break and start to diﬀuse into the respective buﬀer
layers. The calculated diﬀusion constants for hydrogels 1 and 2
are 9.47 × 10−11 and 7.48 × 10−11 m2 s−1, respectively. This
slow and sustained release of crucial biomolecules from
hydrogels existing in physiological conditions holds future
promise for wide applications in drug delivery systems.
The practical applicability of this type of hydrogel formed in
physiologically friendly environment lies in the point whether
they are injectable or not. Injectable hydrogels can be delivered
to a target in vivo via a simple syringe without taking the pain of
surgical treatment. A simple demonstration of the injectable
nature of the vitamin B12 loaded hydrogel formed from P1 is
shown in Figure 5.
Inhibition of cancer cells (breast
cancer cell line, MCF7) proliferation was evaluated by the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction.64 MCF-7 cells were seeded at a density of
10 000 cells per well in a 96-well plate for 18−24 h before
treatment with the hydrogelator. Then cells were treated with
various concentrations of the compound (stock concentration
was 10 mM in autoclaved water) in DMEM medium containing
10% fetal bovine serum for 24h in 96-well plate (Figure 9).
Following the termination of the experiment, cells were washed
and promptly assayed for viability using MTT. Results were
expressed as percent viability = [A550 (treated cells) −
background/A550 (untreated cells) − background] × 100.
Study of the Change in the Cellular Morphology after
the Compound Treatment. Change in the cellular
morphology of MCF-7 cells after the compound treatment
was evaluated by following method. MCF-7 cells were seeded at
a density of 50 000 cells per well in a 6-well plate for 18−24 h
before the compound treatment. Then the cells were treated
with 0.025, 0.05, and 5 mM concentration of compound (stock
concentration was 10 mM in autoclaved water) in DMEM
medium containing 10% fetal bovine serum for 24 h in diﬀerent
6-well plate. Following the termination of the experiment, the
morphologies of
the cells were observed in the inverted
microscope (Model Nikon Eclipse Ti-U) in DIC mode.
Dose-dependent MTT assay with varying concentration of
the gelato

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[System] polypeptide self-assembly literature information extraction

[User]
We demonstrate an effective strategy to prepare highly stable nanoemulsions
using ferrocene-modified tripeptides. Compared with traditional nanoemulsions,
bioorganometallic peptide nanoemulsions are appealing for a number of reasons,
including long-term and outstanding thermal stability, redox activity and
biocompatibility. The formed nanoemulsions could remain stable for more than
four months at room temperature, which is the highest stability reported so far
for peptide and protein emulsifiers. The phase behaviour and size distribution
of the emulsions could be precisely tailored by altering the temperature,
solvent ratio and redox state of the ferrocene moiety. In this process, we
observed a unique enthalpy-driven phase transition from nanoemulsions to
hydrogels, which could be attributed to the competition between the interfacial
free energy and the association energy among the self-assembling peptides.
Moreover, we could impart catalytic activity to the nanoemulsions through
rationally altering the sequence of the tripeptides. The structurally tunable,
functional bioorganometallic nanoemulsions offer new opportunities in many areas
including drug delivery, and the food and cosmetic industries.

Ferrocene-L-Phe-L-Phe-L-His (Fc-FFH, 98% in purity), ferrocene-L-
Phe-L-Phe-L-Asp (Fc-FFD, 98% in purity), ferrocene-L-Phe-L-Phe-L-
Phe (Fc-FFF, 98% in purity), and ferrocene-L-Phe-L-Phe-L-Ser (Fc-
FFS, 98% in purity) were synthesized by GL Biochem Ltd
(China). All other chemicals were of analytical grade, which
were obtained from commercial sources. Phosphate buﬀer
(50 mM, pH 7.20) was prepared by mixing a certain volume of
50 mM NaH2PO4 solution (prepared by dissolving 600 mg
NaH2PO4 in 100 mL water) and 50 mM Na2HPO4 solution (pre-
pared by dissolving 1.79 g Na2HPO4·12 H2O in 100 mL water).
Preparation of nanoemulsions. In a typical experiment, 1 mg
Fc-FFH (Fc-FFD, Fc-FFF, and Fc-FFS) was first dissolved in
50 mM pH 7.20 phosphate buﬀer at 60 °C, then diﬀerent
volumes of ethyl acetate were added into Fc-FFH (Fc-FFD, Fc-
FFF, and Fc-FFS) buﬀer solution (the volume ratio of phos-
phate buﬀer to ethyl acetate was altered from 1 : 9, 3 : 7, 5 : 5,
7 : 3, to 9 : 1, and the total volume was always 1 mL) at room
temperature. The mixtures were homogenized for 30 s, leading
stable nanoemulsions at diﬀerent
to the formation of
aqueous/organic volume ratios.
Preparation of hydrogels. Fc-FFH nanoemulsions were firstly
prepared as described above. The nanoemulsions were incu-
bated at temperatures below 25 °C without any disturbance,
leading to the formation of self-supporting hydrogels.
Determination of Fc-FFH monomers’ concentration during
the phase transition. In the experiment, we used D2O to
prepare pH 7.20 phosphate buﬀer. Then the Fc-FFH nanoe-
mulsions and hydrogels were prepared as described above.
The two kinds of samples were tested by nuclear magnetic res-
onance (NMR) analysis. The characteristic peaks of D2O and
Fc could be integrated, and the ratio corresponded to the
monomers’ concentration in two phases.
Electrochemistry oxidation experiment. Electrochemical oxi-
dation of Fc-FFH nanoemulsions prepared in 7 : 3 aqueous/
organic volume ratio was conducted at room temperature
using an LK2005A electrochemical workstation (LANLIKE,
China). The amperometric i–t curve was measured under a
nitrogen atmosphere, and a three-electrode cell was used to
maintain a constant potential of 500 mV between the working
electrode and reference electrode. A platinum plate electrode,
a platinum wire electrode, and an Hg/Hg2Cl2 electrode were
used respectively as the working, counter and reference electro-
des, and 50 mM pH 7.20 phosphate buﬀer was used as the
supporting electrolyte. The progress of oxidation was followed
by monitoring the current.
Catalytic experiment. Fc-FFH nanoemulsions as catalysts
were prepared at a 7 : 3 aqueous/organic volume ratio as
described above. 10 mM p-NPB was mixed with 850 μL nanoe-
mulsions, 122.5 μL 50 mM phosphate buﬀer, and 17.5 μL
acetonitrile. The mixture solution was homogenized for 30 s
and then added into a quartz cuvette. The production of p-NP
was determined based on the absorbance at 400 nm. In
addition, at the end of each catalytic reaction, the nanoemul-
sions were recycled simply by injecting another equivalent of
PNPB to the next batch.
Scanning electron microscopy. 10 μL of prepared nanoemul-
sions and a slice of hydrogel were deposited neatly on a micro-
scope glass coverslip and dried in air. All samples were
sputter-coated with platinum using an E1045 Pt-coater
(Hitachi High-technologies CO., Japan), and then imaged with
an S-4800 field emission scanning electron microscope (SEM,
Hitachi High-technologies CO.,
Japan) at an acceleration
voltage of 3 keV.
Transmission electron microscopy. The morphology of the
tripeptide assemblies was further assessed using a JEOL 100CX-II
transmission electron microscope (TEM, JEOL Ltd, Japan) oper-
ated at 80 keV. To prepare samples, a 10 μL aliquot of nanoemul-
sions was placed onto a 200 mesh carbon-coated copper grid,
and air dried. And for hydrogels, the sample was dispersed in
ddH2O, and 10 μL aliquot of aqueous dispersion was placed onto
a 200 mesh carbon-coated copper grid, and air dried.
Dynamic light scattering. The nanoemulsions prepared as
described above were diluted in ddH2O and left to stand
without any disturbance at 20 °C, 25 °C, 30 °C, 40 °C, 50 °C,
60 °C, and 70 °C. The size distribution of the samples was
measured using a Zetasizer Nano AS (Malvern Instruments,
British) at the given temperature.
Fourier transform infrared spectroscopy. The as-prepared
nanoemulsions and hydrogels were frozen at −45 °C and sub-
sequently dried under vacuum. The spectra of samples were
recorded on a Nicolet-560 FTIR spectrometer (Nicolet Co.,
USA) with a KBr pellet method across
the range of
−1. A total of 16 scans were accumulated with a
400–4000 cm
resolution of 4 cm
Diﬀerential scanning calorimetry. Thermal analysis of Fc-
FFH nanoemulsions prepared in the 7 : 3 aqueous/organic
volume ratio was performed by diﬀerential scanning calorime-
try (DSC 1/500, Mettler Toledo, Switzerland), under the protec-
tion of a nitrogen atmosphere at constant temperatures of 5,
10 and 15 °C. The sample cell was filled with 20 μL Fc-FFH
nanoemulsions. The sample should be prepared immediately
prior to its introduction into the instrument. The area of the
exothermic peak was integrated to yield the enthalpy of the
phase transition from nanoemulsions to hydrogels.

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{"peptide_sequence": "FFF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "32.38", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}, {"peptide_sequence": "FFS", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "90/10 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "29.27", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}, {"peptide_sequence": "FFS", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "70/30 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "29.27", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}, {"peptide_sequence": "FFS", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "29.27", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}, {"peptide_sequence": "FFS", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "30/70 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "29.27", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}, {"peptide_sequence": "FFS", "self-assembly_phase": "vesicle", "N-terminal_modification": "Ferrocene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 PB/Ethyl acetate", "peptide_concentration(mg/ml)": "29.27", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "0.5"}]


[System] polypeptide self-assembly literature information extraction

[User]
 of
supramolecular interactions, chirality and orientation, which depends on the
peptide sequence and solvent environment. This is the first successful
demonstration of using oscillating pressure waves to generate anisotropic
organo- and hydrogels consisting of oriented tripeptides structures.

A number of approaches have been used to align both
inorganic and organic materials. These include Langmuir–
Blodgett techniques, lithography, chemical vapor deposition,
magnetic and electric fields.3 Several successful examples have
been reported to generate anisotropic gels but few methods
allow for in situ alignment in solution or the gel-phase. Stupp’s
group demonstrated a thermal pathway to process self-assembling
peptide amphiphiles into highly aligned macrostructures.4a Yan
and co-workers used heating to trigger covalent crosslinking of
cyclo-dipeptides leading to highly ordered peptide crystals.4b,c Xu
and co-workers reported an enzymatic self-assembly approach
a Advanced Science Research Center (ASRC) and Hunter College, City University of
New York, New York, NY 10031, USA. E-mail: Rein.Ulijn@asrc.cuny.edu
b WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde,
Thomas Graham Building, 295 Cathedral Street, G1 1XL Glasgow, UK.
E-mail: shuo.bai@strath.ac.uk
c Centre for Ultrasonic Engineering, Department of Electronic & Electrical
Engineering, University of Strathclyde, 204 George Street, Glasgow G1 1XW, UK
d Institute of Molecular, Cell and Systems Biology College of Medical,
Veterinary and Life Sciences, University of Glasgow, G128QQ, UK
which, by nature of the aromatic side chains of peptides used,
resulted in alignment of fibers through aromatic–aromatic
interactions for generating anisotropic hydrogels.4d Further-
more, the shear-flow and magnetic field were also used to align
macroscopic domains within peptide hydrogels.4e–f
Ultrasound, i.e. high frequency (>20 kHz) pressure waves, is
commonly used in supramolecular chemistry to overcome
energy barriers and disrupt intermolecular interactions, which
has been used routinely to control self-assembly and gelation
processes,5 including most recently the demonstration of dynamic
and transient reconfiguration of peptide nanostructures.5g We
reasoned that the directionality of sound waves – longitudinal
pressure waves which lead to oscillating compression of the
solvent volume in the same direction as the wave itself – may
provide opportunities to introduce anisotropy in gel-phase,
especially if the ultrasound is applied directionally. Thus,
in this work we demonstrate the use of ultrasound to trigger
the self-assembly and alignment of tripeptide nanostructures
resulting in thermodynamically stable anisotropic organo- and
hydrogels (Scheme 1).
The D–L–L stereochemistry in tripeptides was previously shown
to give rise to self-supporting hydrogels by Hartley’s group6
without the need for modification with aromatic1a or aliphatic1b
groups normally required for short peptide gelation. Here, we use
tripeptides DFFD (D-phenylalanine–L-phenylalanine–L-aspartic
acid) and DFFI (D-phenylalanine–L-phenylalanine–L-isoleucine),
which could form gel-phase materials in methanol and aqueous
solution triggered by ultrasonic effect, respectively. Tripeptides
were synthesized by using the standard Fmoc (fluorenylmethyloxy-
carbonyl)–tBu strategy (for details on synthesis, see Experimental
section). 10 mg of DFFD was fully dissolved in 1 ml methanol
(25 mM) by heating up to 50 1C. After cooling down to room
temperature, a precipitate was observed (Fig. 1a). The solubility
of DFFD in methanol determined by UV-Vis spectroscopy is
20 mM. Transmission electron microscopy (TEM) images show
that the precipitate consists of random, short and curved nano-
fibres (Fig. 1a). For the ultrasonic experiments, we used a purpose-
built setup, consisting of a glass beaker with a piezo-transducer
(a) Chemical structures of tri-peptide derivatives of DFFI and
DFFD. (b) Cartoon of self-assembly of DFFD and DFFI altering from disordered
short fibers to highly oriented fibrous clusters after ultrasound exposure in
methanol (DFFD) and buﬀer solution (pH 8, DFFI).
of micro-structures in the gel-phase, verifying that structures
observed by electron microscopy were not drying induced
artefacts during sample preparation (Fig. 1e).
To gain insights into the chiral organization of the peptide
structures, the presence of chirally organized phenyl moieties
and orientation of self-assembled structures within the tripeptide
before and after ultrasound, circular (CD) and linear dichroism
(LD) spectroscopy were used. As Fig. 2a shows, a CD and LD
silent spectrum can be obtained for DFFD before ultrasound.
Remarkably, sound waves induced supramolecular chirality by
significantly enhancing the peaks at 240 and 270 nm in the
organogel, which may be attributed to the supramolecular
organization of chiral centers within p–p stacking of phenyl-
alanines.6a,7 LD spectra show that a minimum at 205 nm and
a maximum at 255 nm are observed which indicates both
perpendicular and parallel orientation of phenyl moieties to
the axis exist in self-assembled structures (Fig. 2b).8
Infrared spectroscopy in the amide I region of the spectrum
(1600–1700 cm(cid:2)1) was used to determine the hydrogen bonding
networks within the fibrous structures. Spectra of the samples
with and without ultrasound treatment are displayed in Fig. 2c.
The suspended precipitate of DFFD exhibits a weak, broad peak
around 1680 cm(cid:2)1, suggesting the presence of unstructured or
solvated amide groups. Notably, a strong redshift of the amide
absorption around 1650 cm(cid:2)1 was observed upon ultrasound
exposure, together with a second peak around 1694 cm(cid:2)1. These
Fig. 1 TEM images of DFFD in methanol (a) before and (b) after ultrasound
exposure. Inset photographs are DFFD forming precipitation or gel without
or with ultrasound in methanol. (c, d) SEM and (e) two-photon microscopy
images of the ultrasonicated DFFD peptide gels. The scale bars are (a) 100 nm,
(b) 100 nm, (c) 10 mm, (d) 5 mm and (e) 5 mm. The concentration is 25 mM.
(80 kHz) attached to the bottom, which could provide one-
directional ultrasonic pressure with constant amplitude, rather
than general sonication bath with varied frequencies and direc-
tions (for details on ultrasonic setup, see Experimental section).
Upon ultrasound exposure during cooling of the peptide solution
for 60 seconds, a gel-phase material may be observed (Fig. 1b).
Scanning and transmission electron microscopy (SEM and TEM)
showed formation of aligned peptide structures consisting of
tube-like nanostructures of approximately 15 nm in diameter
that, in turn, organize into bundles of microtubes (approximately
1 mm in diameter) of tens of mm in length (Fig. 1c and d). Two-
photon microscopy of structures labelled with thioflavin T (ThT)
(dissolved with DFFD in methanol)1f further supported formation
Fig. 2 (a) CD, (b) LD and (c) FT-IR spectra of DFFD in methanol before and
after ultrasound irradiation. (d) Rheology of the elastic modulus (G0) and
viscous modulus (G00) of DFFD organogel after ultrasound irradiation. The
concentration is 25 mM.
observations suggest a more ordered H-bonding pattern.1a,9 Fig. 2d
shows the linear viscoelastic responses of the DFFD organogels. The
storage modulus (G0) exceeds the loss modulus (G00) by factor of 12
demonstrating that the tripeptides formed viscoelastic gels. Overall,
these results revealed that the size transition of peptide self-
assembled structures from nano- to micron scale was acousti-
cally induced, where supramolecular chirality and orientation
of peptide microtubes and hydrogen bonding interactions were
dramatically enhanced (Scheme 1).
In water, gelation was not observed for DFFD, most likely
due to the hydrophilicity of charged aspartic acid. The peptide
sequence was therefore adapted to increase the hydrophobicity.
By changing aspartic acid (DFFD) to more hydrophobic isoleucine
(DFFI), the ultrasonic alignment can be used to fabricate aniso-
tropic hydrogels, indicating that the anisotropic arrangement
of supramolecular interactions is highly related to the peptide
sequences and solvent environment. 12.5 mg of DFFI was
dissolved in 1 ml phosphate buﬀer solution (pH 8) (30 mM).
The solubility was enhanced by heating up to 80 1C. After
cooling down to room temperature, a precipitate consisting of
entangled short nanofibers was observed (Fig. 3a). The solubility
of DFFI in buﬀer solution determined by UV-Vis spectroscopy is
15.5 mM. When in situ ultrasound treatment was introduced to
the DFFI buﬀer solution for 30 seconds during the cooling
process, a hydrogel was obtained. TEM images show that the
self-assembled peptide structure changes to bundles of highly
oriented microfibers (Fig. 3b). SEM images indicate that the
macroscopic alignment of peptide fibers up to 1 mm of thickness
and 100 mm of length are achieved (Fig. 3c). Two-photon micro-
scopy image showed the formation of micro-structure of DFFI in
the gel-phase (Fig. 3d).
To acquire more insights at the supramolecular level in the
aqueous media, the samples were characterized by spectro-
scopic methods. Significant enhancement of CD and LD signal
at 215 nm revealed the presence of chiral centers within b-sheet
type arrangement and perpendicular orientation of phenyl
moieties to the axis in self-assembled structures after ultrasonic
exposure (Fig. 4a and b). IR absorption spectra were taken by
using deuterated phosphate buﬀer at pD 8 and show a broad
band from 1635 to 1650 cm(cid:2)1 (Fig. 4c), indicating a less ordered
H-bonding pattern between the peptide backbones.9 Fig. 4d
shows the linear viscoelastic responses of the DFFI hydrogels.
The storage modulus (G0) exceeds the loss modulus (G00) by factor
of 22 demonstrating that the tripeptides formed viscoelastic gels.
The results indicate that using one-directional ultrasound, which
provides diﬀerential influences of self-assembly of peptides in
both organic and aqueous media by dramatically increasing the
hydrophobic stacking and hydrophilic hydrogen bonding inter-
actions, gives rise to the enhancement of chirality, orientation
and gelation process.
Following the same experimental procedure as mentioned
above, we used a standard sonication bath (the frequency is around
40 kHz with varied acoustic pressure and amplitude) as a control
experiment. Fig. S2 (ESI†) shows that the sonication bath can also
trigger the gelation of DFFD in methanol and DFFI in buﬀer
solution. However, disordered fibrous structures are observed by
SEM (Fig. S2, ESI†), which proves that oscillating pressure waves
give rise to the highly oriented peptide self-assembly.
In conclusion, we demonstrate the use of ultrasonic waves as
an in situ approach to induce the self-assembly and alignment of
tripeptides forming highly ordered micro-fibrous structures in
both organic and aqueous media. Two diﬀerent peptides were
studied, DFFD and DFFI with the former forming an anisotropic
organogel in methanol, while the latter forms an anisotropic
hydrogel, showing that the anisotropic arrangement of supra-
molecular interactions is highly related to the peptide sequences
and solvent environment. These results indicate that the influence
Fig. 3 TEM images of DFFI in phosphate buﬀer (pH 8) (a) before and (b) after
ultrasound irradiation. Inset photographs are DFFI forming solution or gel without
or with ultrasound in buﬀer. (c) SEM image and (d) two-photon microscopy
images of the ultrasonicated DFFI peptide gels. The scale bars are (a) 100 nm,
(b) 1 mm, (c) 10 mm and (d) 2 mm. The concentration is 30 mM.
Fig. 4 (a) CD (circular dichroism), (b) LD (linear dichroism) and (c) FT-IR spectra
of DFFI in phosphate buﬀer (pH 8) before and after ultrasound irradiation.
(d) Rheology of the elastic modulus (G0) and viscous modulus (G00) of DFFI
hydrogel after ultrasound irradiation. The concentration is 30 mM.
We thank Dr Margaret Mullin for SEM and TEM measure-
ment, Prof. Gail McConnell for help with two-photon micro-
scopy measurement and Craig Irving for the DOSY experiments.
The research leading to these results has received funding from
the European Research Council under the European Union’s
Seventh Framework Programme (FP7/2007–2013)/EMERgE/ERC
grant agreement no. [258775]. CGP would like to thank Linn
Products Ltd for funding. Y. M. A. acknowledges financial
support by FP7 Marie Curie Actions of the European Commission,
via the initial training network ReAd (no. 289723).
2 (a) A. W. Feinberg, A. Feigel, S. S. Shevkoplyas, S. Sheehy, G. M.
Whitesides and K. K. Parker, Science, 2007, 317, 1366–1370;
(b) A. Merzlyak, S. Indrakanti and S. W. Lee, Nano Lett., 2009, 9,
846–852; (c) C. J. Bettinger, R. Langer and J. T. Borenstein, Angew. Chem.,
Int. Ed., 2009, 48, 5406–5415; (d) R. Nelson, M. R. Sawaya, M. Balbirnie,
A. Ø. Madsen, C. Riekel, R. Grothe and D. Eisenberg, Nature, 2005, 435,
773–778; (e) C. M. Dobson, Nature, 2003, 426, 884–890.
4 (a) S. M. Zhang, M. A. Greenfield, A. Mata, L. C. Palmer, R. Bitton,
J. R. Mantei, C. Aparicio, M. O. de la Cruz and S. I. Stupp, Nat. Mater.,
2010, 9, 594–601; (b) X. H. Yan, J. B. Li and H. Mo¨hwald, Adv. Mater.,
2011, 23, 2796–2801; (c) X. Yan, Y. Su, J. Li, J. Fru¨h and H. Mo¨hwald,
Angew. Chem., Int. Ed., 2011, 50, 11186–11191; (d) J. Zhou, X. Du,
Y. Gao, J. Shi and B. Xu, J. Am. Chem. Soc., 2014, 136, 2970–2973;
(e) B. D. Wall, S. R. Diegelmann, S. Zhang, T. J. Dawidczyk,
W. L. Wilson, H. E. Katz, H. Q. Mao and J. D. Tovar, Adv. Mater.,
2011, 23, 5009–5014; ( f ) M. Wallace, A. Z. Cardoso, W. J. Frith,
J. A. Iggo and D. J. Adams, Chem. – Eur. J., 2014, 20, 16484–16487.
5 (a) Y. B. Wang, C. L. Zhan, H. B. Fu, X. Li, X. H. Sheng, Y. S. Zhao,
D. B. Xiao, Y. Ma, J. S. Ma and J. N. Yao, Langmuir, 2008, 24, 7635–7638;
(b) A. M. Hung and S. I. Stupp, Langmuir, 2009, 25, 7084–7089;
(c) N. Komiya, T. Muraoka, M. Iida, M. Miyanaga, K. Takahashi and
T. Naota, J. Am. Chem. Soc., 2011, 133, 16054–16061; (d) L. Sambri,
F. Cucinotta, G. De Paoli, S. Stagni and L. De Cola, New J. Chem., 2010,
34, 2093–2096; (e) A. Kotal, T. K. Paira, S. Banerjee and T. K. Mandal,
Langmuir, 2010, 26, 6576–6582; ( f ) G. Cravotto, E. C. Gaudino and
P. Cintas, Chem. Soc. Rev., 2013, 42, 7521–7534; (g) C. G. Pappas,
T. Mutasa, P. W. J. M. Frederix, S. Fleming, S. Bai, S. Debnath, S. M.
Kelly, A. Gachagan and R.

[Assistant] [{"peptide_sequence": "fFD", "self-assembly_phase": "organogel", "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": "fDI", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "12.5", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
BACKGROUND: Curcumin, the principal curcuminoid of the popular Indian spice
turmeric, has a wide spectrum of pharmaceutical properties such as antitumor,
antioxidant, antiamyloid, and anti-inflammatory activity. However, poor aqueous
solubility and low bioavailability of curcumin is a major challenge in its
development as a useful drug. To enhance the aqueous solubility and
bioavailability of curcumin, attempts have been made to encapsulate it in
liposomes, polymeric nanoparticles (NPs), lipid-based NPs, biodegradable
microspheres, cyclodextrin, and hydrogels.
METHODS: In this work, we attempted to entrap curcumin in novel self-assembled
dipeptide NPs containing a nonprotein amino acid, α, β-dehydrophenylalanine, and
investigated the biological activity of dipeptide-curcumin NPs in cancer models
both in vitro and in vivo.
RESULTS: Of the several dehydrodipeptides tested,
methionine-dehydrophenylalanine was the most suitable one for loading and
release of curcumin. Loading of curcumin in the dipeptide NPs increased its
solubility, improved cellular availability, enhanced its toxicity towards
different cancerous cell lines, and enhanced curcumin's efficacy towards
inhibiting tumor growth in Balb/c mice bearing a B6F10 melanoma tumor.
CONCLUSION: These novel, highly biocompatible, and easy to construct dipeptide
NPs with a capacity to load and release curcumin in a sustained manner
significantly improved curcumin's cellular uptake without altering its
anticancer or other therapeutic properties. Curcumin-dipeptide NPs also showed
improved in vitro and in vivo chemotherapeutic efficacy compared to curcumin
alone. Such dipeptide-NPs may also improve the delivery of other potent
hydrophobic drug molecules that show poor cellular uptake, bioavailability, and
efficacy.

Materials and methods
Materials
Curcumin (diferuloyl methane), tetrahydrofuran, N-methyl
morpholine,  isobutyl  chloroformate,  trifluoroacetic  acid,
methanol,  1,1,1,3,3,3-hexafluoro-2-propanol  (HFIP),
phenol,  and  DL-threo-phenylserine  were  from  Sigma-
Aldrich  (Munich,  Germany).  N-[(tert-butoxy)carbonyl]-
L- methionine,  N-[(tert-butoxy)carbonyl]-L-leucine,
N-[(tert-butoxy)carbonyl]-L-and isoleucine, L-phenylalanine
were  from  Novabiochem  (Merck,  Darmstadt,  Germany).
Sodium   acetate,  ethyl  acetate,  and  acetonitrile  were  from
 Spectrochem Pvt Ltd (Mumbai, India). Anhydrous sodium
sulfate and citric acid were from Merck (Munich, Germany).
RPMI-1640,  Dulbecco’s  modified  Eagle’s  medium,  fetal
bovine  serum  (FBS),  penicillin,  and  streptomycin  were
from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylth-
iazolyl-2)-2,5-diphenyltetrazolium  bromide  (MTT)  was
from   Sigma-Aldrich. The  apoptosis  assay  kit  was  from
 Invitrogen. HeLa (human cervical cancer), MCF-7 (human
breast   cancer),  Huh-7  (human  hepatoma),  and  L-929
(human fibroblast) cells were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). HeLa and
L-929 cells were cultured in RPMI medium containing 10%
FBS and MCF-7 and Huh-7 cells were cultured in Dulbecco’s
modified  Eagle’s  medium  supplemented  with  10%  FBS.
B6F10 cells (mouse melanoma cells) cells were a generous
gift from Dr Kundu’s laboratory (NCCS, Pune, India) and
were maintained in RPMI medium containing 10% FBS. All
culture media were supplemented with 100 U/mL penicillin
and 100 µg/mL streptomycin. Cells were maintained on tissue
culture-treated polystyrene plates (Corning, Corning, NY) at
37°C in a humidified atmosphere containing 5% CO2.
Preparation of dipeptide NPs
Stock  solutions  were  prepared  by  dissolving  2  mg  of
peptide(s)  in  50  µL  of  methanol.  In  some  cases,  mild
sonication or heating was necessary to dissolve the peptides.
 Assembly  was  initiated  by  addition  of  1  mL  of  aqueous
methanol (50% v/v) to the stock. Samples were incubated
for 24 hours before experiments.
Characterization of NPs using
transmission electron microscopy (TEM)
TEM of NPs was carried out using uranyl acetate negative
staining  method.  Briefly,  after  evaporating  the  methanol,
drops  of  NPs  were  adsorbed  on  300  mesh  copper  grids
with  carbon-coated  formvar  support  (TAAB   Laboratories
 Equipment, Berks, UK) and stained with 1% uranyl acetate
(Sigma-Aldrich). Excess fluid was removed from the grid
surface with a filter paper (Whatman No 1;  Whatman, Kent,
 England)  and  the  grid  was  air-dried  at  room  temperature
before being loaded onto the microscope. Pictures of NPs were
acquired in a TEM (Tecnai 12 BioTWIN; FEI,  Eindhoven,
 Netherlands) operating at 120 kV.  Photomicrographs were
digitally  recorded  using  a  Megaview  III  (SIS,  Münster,
Germany) digital camera. Image analysis to measure particle
dimensions was carried out using the iTem software package
(FEI, Hillsboro, OR).
Loading of curcumin in dipeptide NPs
and determination of curcumin-loading
efficiency and drug-loading content
Curcumin  solution  in  methanol  was  added  to  preformed
NPs at a ratio of 1:1 w/w, and the mixtures were incubated
for  48  hours  at  room  temperature  with  constant  shaking.
Methanol was evaporated under a vacuum and free curcumin
was separated from that entrapped in NPs by centrifugation
at  800  ×  g  for  15  minutes  in  a  MultiScreen  96-well  filter
plate  (Millipore,  Billerica,  MA).  Curcumin  content  in  the
filtrate was quantified using UV-vis spectroscopy at 426 nm.
The percentage entrapped curcumin in dipeptide NPs was
determined using the following equation:
To determine the exact quantity of curcumin in dipep-
tide NPs, curcumin was loaded in NPs as described above
(1:1  w/w  of  curcumin  in  1  mL  of  dipeptide  NPs).  Free
curcumin  was  separated  from  that  entrapped  in  NPs  by
centrifugation at 800 × g for 15 minutes in a MultiScreen
96-well filter plate (Millipore). Samples were washed three
times  with  PBS  and  NPs  were  then  dissolved  by  adding
200 µL of neat methanol. Curcumin content was determined
by comparing the sample with curcumin standard curve in
methanol using the following equation:
Analysis of photophysical properties
of curcumin and curcumin loaded
in dipeptide NPs
To determine whether entrapment in dipeptide NPs had an
effect on curcumin’s photophysical properties, spectroscopic
analysis was condcuted.20 The absorbance and fluorescence
spectra of both native curcumin and (Cm)NPs at a concentra-
tion of 1 mg/mL were measured in a 50:50 (v/v) methanol:
water solution. Absorption spectra were recorded from 350
to 600 nm (Lambda 35; Perkin Elmer, Waltham, MA) and
fluorescence  emission  spectra  were  recorded  from  450  to
700 nm with an excitation wavelength of 420 nm (LS 55;
Perkin Elmer).
In vitro curcumin release
Curcumin release studies were carried out using the dialysis
bag method.21 A known quantity (1.0 mg/mL) of (Cm)NPs
was placed in a dialysis membrane tube with a molecular
weight  cut-off  of  3000  Da  (SnakeSkinTM  Pleated  Dialysis
Tubing; Thermo Scientific, Rockford, IL). Because of the low
solubility of curcumin in aqueous media, the release study
was carried out by immersing the tube in a beaker containing
50 mL of 50:50 (v/v) methanol: water solution to facilitate
sink-like conditions and stirred constantly at room tempera-
ture for 36 hours. At various time points, 1 mL of solution
was withdrawn from the release medium and replaced with
fresh PBS. Samples were lyophilized and dissolved in 100 µL
of  methanol  to  determine  curcumin  content  using  UV-vis
spectroscopy (Lambda 35; Perkin Elmer).
Fluorescence and flow cytometric
analysis for cellular uptake of NPs
Taking  advantage  of  the  intrinsic  green  fluorescence  of
curcumin,  we  studied  its  cellular  uptake  by  fluorescence
microscopy and fluorescence-activated cell sorting (FACS)
analysis. HeLa cells were grown in 6-well culture plates at
37°C  in  a  5%  CO2  incubator  up  to  80%  confluency.  Cells
were  then  treated  with  30 µM  native  curcumin,  (Cm)NPs,
and void NPs for 24 hours and analyzed for curcumin uptake.
For  microscopic  analysis,  cells  were  fixed  using  fixative
( Sigma-Aldrich), washed three times with PBS, and analyzed
under a fluorescence microscope (Eclipse TE2000-U; Nikon,
Tokyo, Japan) to measure intracellular curcumin  fluorescence.
For FACS analysis, cells were trypsinized, washed with PBS
three times, and resuspended in 500 µL sheath fluid (Becton
Dickinson, Franklin Lakes, NJ) and examined using FACS-
Caliber flow cytometer (Becton Dickinson).
Cytotoxicity of (Cm)NPs towards
different cancerous cell lines
HeLa, MCF-7, or HUH-7 cells were plated (1 × 104 cells/
well)  in  triplicate  in  96-well  sterile  microtiter  plates
(Corning) and allowed to grow for 12 hours for proper cell
adherence. Cells were then treated with different concen-
trations (10–30 µM of free curcumin, (Cm)NPs, and void
dipeptide NPs for 24 hours. Cells incubated with 20 µL of
PBS were used as a control. After incubation, the media
was discarded and replaced with 180 µL of fresh growth
medium. Subsequently, 20 µL of MTT reagent, (5 mg/mL)
was added to each well. The plate was then incubated at
37°C under 5% CO2 for 4 hours. The medium was removed,
and 100 µL of dimethylsulfoxide was added to solubilize
the formazan crystals. Optical density was then taken at
572 nm, subtracting for plate absorbance at 690 nm. The
percentage  of  viable  cells  was  calculated  based  on  the
 following equation:
taken at 572 nm,  subtracting for plate absorbance at 690 nm.
The percentage of viable cells was calculated based on the
following equation:
Apoptosis assay
HeLa cells were seeded (2.5–3.0 × 105 cells/plate) on 60-mm
plates and incubated until 80% confluency was reached. Cells
were then treated with 30 µM of free curcumin and the same
amount of (Cm)NPs based on the cytotoxicity results, which
was used to determine the maximum cell killing efficacy at a
concentration of 30 µM. As a control, cells were incubated
with complete growth medium. After 24 hours, cells were
harvested  by  quick  trypsinization  to  minimize  potentially
high annexin V background levels in adherent cells, washed
twice with cold PBS, and stained with annexin V-fluorescein
isothiocyanate  (FITC)  and  propidium  iodide  (PI)  in  bind-
ing  buffer  as  directed  by  the Vybrant  apoptosis  assay  kit
(V13241; Invitrogen). Stained cells were placed on ice and
protected  from  light  until  reading  using  flow  cytometry.
Processed single-cell suspensions were analyzed on a FAC-
SCaliber flow cytometer with a laser excitation wavelength
of 488 nm. The green signal from fluorescein isothiocyanate/
annexin V was measured at 525 nm and the red signal from PI
was measured at 620 nm. Cells that stained negative for both
annexin V and PI were viable. Cells that were annexin V+/
PI- were in early apoptosis, whereas cells that were necrotic
or in late apoptosis were annexin V+/PI+.
Cytotoxicity of NPs towards L-929
(human fibroblast) cells
L-929 cells were plated (1 × 104 cells/well) in triplicate in
96-well  sterile  microtiter  plates  (Corning)  and  allowed  to
grow  for  12  hours  for  proper  cell  adherence.  Cells  were
then  treated  with  different  concentrations  (20–50  µM)  of
void dipeptide NPs for 24 hours. Cells incubated with 20 µL
of PBS were used as a control. After incubation, the media
was  discarded  and  replaced  with  180  µL  of  fresh  growth
medium. Subsequently, 20 µL of MTT reagent (5 mg/mL)
was  added  to  each  well. The  plate  was  then  incubated  at
37°C  under  5%  CO2  for  4  hours. The  medium  was  then
removed  and  100  µL  of  dimethylsulfoxide  was  added  to
solubilize the formazan crystals. Optical density was then
In vivo antitumor efficacy
of curcumin-loaded NPs
Male mice (Balb/c), 4–6 weeks old, weighing 18–22 g were
housed in a temperature- and light-controlled room at the ani-
mal maintenance facility of International Centre of Genetic
Engineering  and  Biotechnology  at  our  institution. Animal
experiments were approved by the Institutional  Animal Care
and Use Committee and were in compliance with all regula-
tory guidelines. Cultured tumor cells B6F10 (mouse mela-
noma cells) were detached from flasks using trypsin/EDTA
followed by neutralization with RPMI containing 10% FBS
and pelleted at 300 × g for 5 minutes. The cells were then
washed three times with cold PBS and resuspended in PBS.
To initiate tumors, a total of 5.0 × 106 cells (0.1 mL) were
injected subcutaneously into the abdomen of 4–6-week-old
mice. Palpable solid tumors developed within 10–12 days
post-tumor  cell  inoculation.  Once  tumor  masses  became
established (as soon as tumor volume reached ∼125 mm3),
the animals were randomized to receive either physiologi-
cal saline, void NPs (at a dose of 20 mg/kg body weight),
curcumin-NPs in aqueous solution (at a dose of 25 mg/kg
body weight), curcumin alone [at a dose of 25 mg/kg body
weight in methanol: water solution (50:50; v/v)] in 100 µL for
28 days via intravenous injection (at 3-day intervals). Tumor
sizes were measured using calipers and tumor volume was
calculated using the equation:
In vivo toxicity studies of dipeptide NPs
Animal were intravenously administered with physiological
saline and void NPs in 100 µL (equivalent to the therapeutic
dose) for 28 days via intravenous injection (at 2-day intervals)
and monitored continuously for any behavioral changes and
body mass loss. After these animals were sacrificed, organs
were  excised,  sectioned,  and  studied  for  morphological
changes using hematoxylin and eosin staining.

[Assistant] [{"peptide_sequence": "MF", "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": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "LF", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "IF", "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": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
DOI: 10.1371/journal.pcbi.1003718
PMCID: PMC4091692
PMID: 25010703 [Indexed for MEDLINE]

The de novo protein/peptide design framework applicable to
multimeric systems consists of two stages [49,50,52,53,59,70–72].
The framework has been developed to handle flexible backbone
templates, since experimental structures are not often available for
multimeric systems. As such, a flexible backbone template must be
created through simulation.
associating tripeptides, MD simulations were performed for this
the plausible
purpose, which produced many snapshots of
multimeric complex. These snapshots were then used to produce
a flexible backbone template. The flexible backbone template was
subsequently used as the input for the design framework. The first
stage of the framework is Sequence Selection, which is based on a
global optimization method that minimizes the potential energy of
a designed sequence in the flexible template structure. The
potential energy used can either be based on an 8-bin Ca-Ca force
field or an 8-bin centroid-centroid force field [93,94]. A novel
aspect of this method is the mathematical connection of residues in
the design framework, so that identical chains in the template
structure remain identical throughout the design procedure. The
optimized sequences are then subjected to a Fold Specificity
calculation and screening. Fold Specificity assesses how energet-
ically favorable it is for the designed sequence to adopt the target
multimeric structure in comparison to the native sequence. In
cases where the native sequence is known to associate, Fold
Specificity aims to produce designed sequences that are more
energetically favorable in the target multimeric structure than the
native sequence. In cases where the native sequence does not
assemble, sequences with higher Fold Specificity are considered to
have a higher chance of adopting the novel multimeric structure.
Finally, a subset of high confidence sequences is subjected to an
PyRosetta [95] was used to generate the initial tripeptide models
for the template sequence Ac-IVD through a Monte Carlo (MC)
conformational
function ‘‘make_pose_from_se-
quence’’ was used in conjunction with the ‘‘fa_standard’’ Rosetta
force field [96]. A SmallMover object was constructed with the
backbone being allowed to move, with 5 MC perturbations per
cycle. The model was subjected to 60,000 MC cycles, with the
Metropolis criterion determining whether a move was accepted or
rejected. This procedure was used to generate 200 low energy
decoys for the template. The models were then clustered in
Rosetta. The four lowest energy models from the densest cluster
were centered at the origin.
In CHARMM, the four tripeptides were translated 8 A˚ in both
the y- and z- directions so as to form a square box with the
distance from the center of the box to the center of each peptide
being 11.31 A˚ . Each tripeptide was rotated randomly. ‘‘Hbuild’’
was used to construct the hydrogen atoms. Periodic boundary
conditions, which determine the length and (consequently) volume
of the box, were applied in CHARMM so that the concentration
the system was 20 mg/mL. The nonbonded cutoffs
CHARMM were set using the following options and values:
ctonnb 20, ctofnb 20, cutnb 24, and cutim 24. Implicit solvent was
invoked using the generalized Born with simple switching model
[97]. A half smoothing length of 0.3 A˚ , a non-polar surface tension
molA2, and a grid spacing of 1.5 A˚ were used.
The system was subjected to 2,000 steps of steepest descent,
followed by 2,000 steps of adopted basis-set Newton-Raphson, and
finally an additional 2,000 steps of steepest descent minimization.
The system was heated to 300 K over 10 ps (stepsize: 0.5 fs) and
with harmonic constraints on all heavy atoms with force constant
molA2. The system (N,V,T ensemble) was equilibrated for
molA2 on all heavy
atoms. The system was subjected to 10 ns of molecular dynamics
(MD) at 300 K, with SHAKE constraints applied to all bonds
involving a hydrogen atom with a tolerance of 1–10. All
simulations were performed using Langevin dynamics with the
leapfrog integration scheme. The last 5 ns of
the simulation
trajectory were processed into pdb files, which were used as the
flexible template for design.
the template
sequence Ac-IVD, the motif [hydrophobic]-[hydrophobic]-[E/D]
was applied in the computational method. The hydrophobic
residues were allowed to mutate to Leu, Ile, Val, Ala as utilized in
the self-assembling hexapeptide Ac-LIVAGD [11], as well as Met,
Phe, Tyr, and Trp. Aromatic moieties have been observed to be
important for association due to p-stacking, so aromatic residues
were included to expand the scope of tripeptides available for
comparison. This resulted in a total of 128 candidate tripeptide
sequences, which is a small enough pool that no further biological
or mutational constraints were required. Previously developed 8-
bin Ca-Ca and centroid-centroid force fields
[93,94] were
available for use as the potential energy function in the Sequence
Selection stage. In this study, the 8-bin centroid-centroid force
field was employed, since unlike the Ca-Ca force fields,
centroid-centroid force field implicitly includes side-chain direc-
tionality in the potential energy calculation.
Since the template for tripeptide association was determined
through MD simulations,
the optimization-based Sequence
Selection method was used with a flexible template rather than
a rigid backbone template. Two previous methods were developed
for flexible template protein design: a Weighted-average method
and a Distance Bin method [70–72]. The Weighted-average
method takes into account each flexible template, such that the
potential energy of the system is the average of all the determined
templates in the flexible ensemble. The Distance Bin method
allows for the optimization method to design not only for the
sequence, but also for the optimal interaction distances for each
residue-residue interaction. The Distance Bin method represents
the most rigorous way of designing with a flexible backbone. For
this reason, the Distance Bin Sequence Selection framework for
multimeric system design was the chosen framework that was used
in this study.
The model minimizes the summation of pairwise interaction
energies Ejl
ik, which is the interaction between residue types j and l
in residue positions i and k whose distance apart falls in distance
bin d. The binary variable yj
i equals 1 if residue type j is in residue
position i, and 0 otherwise. The binary variable wjl
ik equals 1 if, and
only if, yj
k are both equal to 1, and is 0 otherwise. This
represents an exact linearization of the problem. The final binary
variable bikd is allowed to equal 1, if and only if, the distance
between positions i and k fall into distance bin d in at least one of
the flexible models in the template. In this way, the model is
allowed to select one, and only one, distance bin in which two
residues can fall, from the set of distance bins observed in the
flexible template. A new element of this model is the addition of a
mathematical parameter denoted here as SConnexik to connect
design constraints between multiple chains. This parameter is
defined as 1 if two design positions (i and k) are identical positions
in a design system. For example, in the design of a dimer, two
To further validate and analyze the 128 possible tripeptides, a
method capable of calculating the Fold Specificity [70] for sequences
in multimeric systems was developed. This method uses a constrained
annealing simulation in CYANA [98,99] to produce a set of initial
models. A local AMBER energy minimization using TINKER [100]
is then performed on each model to produce a set of 500 final models,
along with corresponding AMBER ff94 energy values [101]. Using
these AMBER energy values the Fold Specificity value is calculated as
where ‘‘New’’ is the set of models produced for the new sequence,
‘‘native’’ is the set of models produced for the reference sequence Ac-
IVD, and Ei is the AMBER energy value calculated for model i.
Physically, Fold Specificity assesses how energetically favorable it is
for the designed sequences to adopt the target multimeric structure in
comparison to the native sequence. The aim is to assess the specificity
of the designed sequences for the target structure using a more
detailed, atomistic potential energy than in the Sequence Selection
stage. Since the metric compares the energy values of the designed
sequence directly to those of the native sequence, a ‘‘favorable’’
sequence is one that has a Fold Specificity value greater than 1.
PyRosetta was used to construct initial coordinates of the subset
of the 128 tripeptide sequences that ranked highly in the Fold
Specificity metric. The MC and MD protocols, which were
described previously in the Template Generation section, were
used to generate a trajectory for each candidate sequence. The
ensemble of models generated through this dynamics run could
then be used in the calculation of an Approximate Association
Affinity of four tripeptides associating together in the simulations.
Since the tripeptides have high flexibility, they do not have a single
stable state. Thus, the simulations did not attempt to reproduce
the three-dimensional structure of an associate precursor [11], but
to provide an estimation of the affinity of a particular sequence to
itself through physics-based intermolecular interactions.
This metric was used in conjunction with the Jacobi logarithm
[102] to avoid numerical overflow in the calculation. K*
association was
calculated for each candidate sequence and rank-ordered from the
highest (most favorable spontaneous association) to the lowest
(Table S1). The simulation of each design was then visually
inspected to assess whether the tripeptides associated during the
simulation and thus fit to the model. Sequences that did not
associate were not considered regardless of
metric. The final set of designed sequences picked for experimen-
tal assessment was
selected via a combination of Potential
Energy, Fold Specificity, and Approximate Association Affinity.
The criteria for this selection are provided in more detail in the
Materials and preparations. The tripeptides (Ac-LVE, Ac-
YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were purchased from
American Peptide Company (Sunnyvale, CA, USA), while Ac-
YMD, Ac-DMY, Ac-IVE, and Ac-EVI were manually synthesized
via solid phase peptide synthesis [103] and purified to .95% via
HPLC. Amino acid content (AA %) analysis was performed and
the actual peptide content of each sample was determined by
calculating the net weight (gross weight6AA %). The peptides
were dissolved by vortexing in deionized water. The tripeptide
samples were prepared from 5 to 40 mg/mL, in steps of 5 mg/
to assess and compare the association properties of
tripeptides. To allow for proper self-assembly, all samples were left
untouched for 24 h before further analyses.
electron microscopy
studies. The peptide samples were flash-frozen at 280uC and
subsequently freeze-dried. The dried samples were adhered onto
copper conductive tape on a sample holder and sputtered with
Rheological measurements. The peptide solutions (20 mM,
250 mL) were pipetted into a polystyrene ring mold as described
previously [104]. The solutions were allowed to gel at ambient
temperature (22uC) over 24 hours before rheological measurements
were carried out. Ten hydrogel samples of each peptide candidate
were prepared and measured. The viscoelastic properties of the
hydrogels were measured at 25uC on an ARES-G2 rheometer (TA
Instruments, Piscataway, NJ) equipped with an 8.0 mm-diameter
titanium serrated plate. Upon loading the hydrogel on the rheometer
platform and adjusting the height of the measurement plate, the
system was allowed to equilibrate for 300 s. The oscillatory frequency
X-ray crystallography studies. Ac-YLD in a glass vial was
dissolved in water to 5 mg/mL, and was allowed to crystallize
spontaneously at ambient temperature for 24 h. The crystals were
then transferred into 25% (v/v) glycerol for 5 min before being
flash frozen in liquid nitrogen. X-ray diffraction data was collected
at 2173uC on a Bruker X8 PROTEUM system consisting of a
MICROSTAR micro-focus X-ray generator, a PLATINUM135
CCD detector, and a 4-circle KAPPA goniometer. Data reduction
was carried out using SAINT, SADABS, and XPREP, which are
part of the Bruker PROTEUM2 program suite (Bruker AXS inc.)
[105]. Ab initio structural solution was achieved using SHELXD
[106], and the model obtained was further refined using SHELXL
[107] (through the ShelXle graphical user interface [108]). Details
of crystallization, data collection and refinement are listed in
Table 5. The final structure was deposited at the Cambridge
Crystallographic Data Centre with the deposition numbers CCDC
974865 (Data S1 and Data S2).
Figure S1 Pictures of peptides (shuffled sequences) in
water. Row 1: (From left to right) Ac-YMD and Ac-DMY in

[Assistant] [{"peptide_sequence": "LVE", "self-assembly_phase": "fiber", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "35.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "2880.0"}, {"peptide_sequence": "LVE", "self-assembly_phase": "fiber", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "40.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "2880.0"}, {"peptide_sequence": "YYD", "self-assembly_phase": "fiber", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": 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"temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "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": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "12.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "19.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "26.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "33.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "LLE", "self-assembly_phase": "particle", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "40.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "20160.0"}, {"peptide_sequence": "VIE", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Acetylation", "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": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "2880.0"}, {"peptide_sequence": "VIE", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "10.0"}]