[System] polypeptide self-assembly literature information extraction

[User]
prior to entrapment
into the hydrogel) at levels that are higher than their solubility limit
at neutral pH.
In order to test this hypothesis, we chose the self-assembling
peptide DLeuePheePhe and the antibiotic ciproﬂoxacin (CIP) as
a model hydrophobic drug that is sparingly soluble at physiological
pH. To the best of our knowledge, this is the ﬁrst report of self-
assembly of a drug and a tripeptide into a hydrogel with distinc-
tive nanostructure that allows for controlled release of the drug.
These materials were used to challenge cultures of Staphylococcus
aureus, Escherichia coli, and a clinical isolate of Klebsiella pneumo-
niae [34]. Sustained drug release from the hydrogels was reﬂected
by antibiotic concentrations of the surrounding solutions and by
PheeWang resin, O-Benzotriazole-N,N,N,N0-tetramethyl-uronium-hexaﬂuoro-
phosphate (HBTU), and Fmoc protected L-phenylalanine were purchased from GL
Biochem Ltd (China). Fmoc-protected D-leucine and D-valine were purchased from
Mimotopes (Australia). All solvents purchased were of analytical grade from Merck
(Australia). Piperidine, triﬂuoroacetic acid (TFA), diisopropyl ethyl amine (DIPEA)
were from Acros (Australia). Sodium dihydrogen phosphate, disodium hydrogen
phosphate, and sodium hydroxide were from BDH AnalaR (Australia). Ciproﬂoxacin
was purchased from Biochemika (SigmaeAldrich, Australia). L-phenylalanine (Phe),
D-leucine (DLeu), and Thioﬂavin T were from Sigma (Australia). High purity Milli-Q-
water (MQ water) with a resistivity greater than 18 MU cm was obtained from an in-
line Millipore RiOs/Origin system. Silicon wafers (M.M.R.C. Pty Ltd., Australia) were
cleaned by ultrasonication (1 h) in a surfactant solution of 2% ethanol with 2% RBS 35
(Pierce) followed by rinsing with copious amounts of MQ water and dried with
nitrogen, then cleaned for 1 h in a UV/ozone Procleaner(cid:3) (Bioforce Nanosciences,
US). The HPLC (Agilent Inﬁnity, Australia) was equipped with a quaternary pump
(G1311B), analytical C-18 column (Luna, 5 microns, 100 Å, 150 (cid:2) 4.60 mm, Phe-
nomenex, Australia), autosampler (G1329B), diode array detector (G4212B), ther-
mostat (G1330B) and thermostatted column compartment (G1316A). The gradient
consisted of acetonitrile (AcN)/water with 0.1% TFA with the following program:
t ¼ 0e3 min. 5% AcN; t ¼ 18 min. 95% AcN; t ¼ 18e20 min. 95% AcN; tR ¼ 8 min for
ciproﬂoxacin; tR ¼ 11 min for DLeuePheePhe.
The tripeptide DLeuePheePhe gelled immediately following a pH trigger [32].
Brieﬂy, the peptide (4.0 mg) was added to a solution of sodium phosphate 0.1 M at pH
11.8 (buffer A, 0.3 ml), and dissolution was achieved with sonication (5 min).
Gelation was triggered by subsequent addition of an equal volume of 0.1 M sodium
phosphate at pH 5.7 (buffer B, 0.3 ml), resulting in a ﬁnal pH of 7.4.
(cid:3)1 to obtain a ﬁnal gel with 0.1% w/v
or 0.2% w/v CIP, respectively) before adding the peptide. The resulting solution was
then used following the procedure described above (Section 2.3). Gelation was
allowed to occur for 2 h, after which any residues of supernatant solution that may
have been present (<2% total volume, containing <1% of total CIP) were gently
aspirated with a pipette.
The interaction between CIP and the gelling peptide DLeuePheePhe was
observed by ﬂuorescence spectroscopy. Fluorescence measurements were made
using a FlexStation 3 spectroﬂuorimeter (Molecular Devices). In order to avoid CIP
precipitation in the control samples (without the peptide), all measurements were
(cid:3)1 (i.e. 0.05% w/v)
carried out on samples with a ﬁnal CIP concentration of 0.5 mg ml
and increasing amounts of the gelling peptide (0, 3.9 mM, 7.9 mM, 11.8 mM, or 15.7 mM
corresponding to 0%, 25%, 50%, 75%, or 100% of the peptide amount used in our
release studies), following the procedure described in section 2.3. Brieﬂy, 100 ml of
each precursor solutions were added onto each well of a black 96-well plate.
Emission spectra were recorded after 2 h from 380 nm to 500 nm with an excitation
wavelength of 340 nm. At this timepoint, only samples with 50%, 75%, or 100%
peptide formed hydrogels. Higher amounts of peptide could not be tested as they
would lead to peptide precipitation. Experiments were repeated twice in triplicates.
Spectra were corrected by subtraction of the corresponding buffer signal (i.e. 0.1 M
sodium phosphate buffer, 100 ml at pH 11.8, and 100 ml at pH 5.7).
The secondary structure of the peptides was analysed using a 0.1 cm quartz cell
on a Jasco J815 Spectropolarimeter, with 1 s integrations, 1 accumulation and a step
size of 1 nm with a band width of 1 nm over a range of wavelengths from 200 to
270 nm. Peptide samples freshly prepared in buffer A were diluted with buffer B
directly in the CD cell, and spectra were recorded after at 15e30 min. Measurements
FT-IR spectra were collected on a Nicolet 6700 FT-IR spectrometer in ATR mode.
Freshly prepared samples were left to settle for 2 h in a glass vial. A portion of the gel
was then transferred on a clean piece of silicon wafer (1 cm (cid:2) 1 cm), gently spread
by pressing a coverslip on top, and dried under vacuum for 24 h. Dried samples on
the silicon wafers were placed directly to the ATR crystal, facing down. Scans were
between 1800 and 600 cm
Dynamic sweep rheological analysis was conducted on an Ares rheometer (TA
Instruments, USA) with 25 mm aluminium parallel plate geometry. A Peltier tem-
perature controller was connected to the rheometer to maintain a temperature of
25 (cid:4)C. Hydrogels were analysed after 24 h. A gap of 300 mm was set. Strain sweeps
(cid:3)1. Frequency sweeps were recorded
were recorded using a frequency of 10 rad s
using a controlled strain of 1%. Average measurements (n ¼ 3) have been plotted
with Excel.
Hydrogels were prepared as described above and after 2 h approximately 20 ml
were immediately placed on wells of a “m-Slide Angiogenesis” uncoated (Ibidi,
Germany, through DKSH Australia). 20 ml of a solution of Thioﬂavin T (200 mM in
50 mM GlycineeNaOH pH 7.5, 0.2 mm-ﬁltered) were placed on top. After 15 min, the
slides were imaged on a Leica SP5 microscope (63(cid:2) water immersion objective, NA
1.2, excitation 458 nm, emission 468e600 nm). Samples treated and stained with an
identical protocol but without the peptide, were used as control and did not reveal
any of the ﬂuorescent structures described in the text (data not shown). The video
was obtained with Imaris software from a z-stack of a peptide gel with 0.2% w/v CIP
after 48 h of self-assembly.
A laboratory-built humidity-controlled vitriﬁcation system was used to prepare
the hydrogels for imaging in a thin layer of vitriﬁed ice using cryo-TEM. Humidity
was kept close to 80% for all experiments, and ambient temperature was 22 (cid:4)C. 200-
Mesh copper grids coated with perforated carbon ﬁlm (Lacey carbon ﬁlm: ProSci-
Tech, Qld, Australia) were used for all experiments. Grids were glow discharged in
nitrogen for 5 s immediately before use. Hydrogels were prepared as described
above and analysed after 2 h. Hydrogels were agitated gently with the pipette tip in
order to liquefy them slightly if possible. Approximately 4 ml aliquots of sample were
pipetted onto each grid prior to plunging. In the case of samples which could not be
liqueﬁed adequately, the gel was smeared gently onto the grid. After 30 s adsorption
time the grid was blotted manually using Whatman 541 ﬁlter paper, for approx-
imately 6 s. Blotting time was optimised for each sample. The grid was then plunged
into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid ni-
trogen until required. The samples were examined using a Gatan 626 cryoholder
(Gatan, Pleasanton, CA, USA) and Tecnai 12 Transmission Electron Microscope (FEI,
Eindhoven, The Netherlands) at an operating voltage of 120 KV. At all times low dose
(cid:3)2 for all
procedures were followed, using an electron dose of 8e10 electrons Å
imaging. Images were recorded using an FEI Eagle 4kx4k CCD camera (FEI, Eind-
hoven, The Netherlands).
Carbon-coated 300-mesh copper grids were glow-discharged in nitrogen to
render the carbon ﬁlm hydrophilic. Hydrogels were prepared as described above
and analysed after 2 h. A 4 ml aliquot of sample was pipetted onto each grid. After
30 s adsorption time the excess was drawn off using Whatman 541 ﬁlter paper,
followed by staining with 2% aqueous potassium phosphotungstate at pH 7.2, for
10 s. Grids were air-dried until needed. The samples were examined using a Tecnai
12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an
operating voltage of 120 KV. Images were recorded using a Megaview III CCD camera
and AnalySIS camera control software (Olympus). Each grid was systematically
examined and imaged to reﬂect a representative view of the sample. This was
important as samples of this type can be very non-homogeneous on the grid; they
had the tendency not to disperse evenly and the amyloid ﬁbrils occured in distinct
320 kHz. Typical scan settings involved the use of an applied piezo deﬂection voltage
of 0.6e0.7 V at a scan rate of 0.7 Hz. All images were processed (1st order ﬂattening
algorithm) using Igor Pro software. Freshly prepared hydrogel samples were prepared
as described above and left to settle for 2 h. A portion of each sample was then spread
onto a clean square of silicon wafer (1 cm (cid:2) 1 cm) by gently pressing a glass coverslip
on top. Samples were then dried in a vacuum oven at room temperature for 24 h.
Self-assembled peptide gels with 0.1% w/v, 0.2% w/v or without CIP were prepared
directly in a glass vial (diameter ¼ 1.8 cm, height ¼ 6.5 cm, 10 ml total volume), fol-
lowing the procedure described above in Section 2.3 (ﬁnal gel volume of 0.6 ml). After
2 h, 10 ml of PBS buffer pH 7.4 were very gently added from the side of the vial,
carefully avoiding disruption (and subsequent ﬂoating) of the gel disc. Vials were
incubated at 37 (cid:4)C, 50 rpm, 70% humidity, and at selected time points 0.5 ml samples
were taken and replaced with 0.5 ml of fresh buffer. Samples were analysed by HPLC
and absorbance was monitored over time. HPLC signal integration was used to
quantify the peptide (Abs at 215 nm) and CIP (Abs at 273 nm). Fresh solutions of
peptide and CIP were used as standards. Each experiment was repeated at least twice
in triplicates, and mean and standard deviation values were calculated in Excel.
Concentration of ciproﬂoxacin released from the gel system over a time course
was also determined by a modiﬁcation of broth micro-dilution assay from CLSI (M2-
A8) [33]. Bacterial suspensions of S. aureus ATCC 29213, E. coli ATCC 25922,
K. pneumoniae B5055 [34] were prepared in Mueller-Hinton broth (MHB) to reach
a density of 1 (cid:2) 106 CFU/ml. Ciproﬂoxacin solutions were sampled from the gel-
releasing vials at 4 h, 8 h, 24 h, 48 h, day 4, and day 6 and prepared as serial double
dilutions (100 ml) in 96-well microplates with MHB. 100 ml of the bacterial suspen-
sions were plated into microwells containing ciproﬂoxacin and the plates were
incubated at 35 (cid:4)C overnight. Minimum Inhibitory Concentration (MIC) of cipro-
ﬂoxacin against of S. aureus ATCC 29213, E. coli ATCC 25922, K. pneumoniae B5055 [34]
were also examined using broth micro-dilution assay and commercial ciproﬂoxacin
powder. This was to provide the baseline to determine the concentration of cipro-
ﬂoxacin in the gel solution. Concentration of ciproﬂoxacin ingredients released by the
gel system at each time points were calculated, by using the formula:
(cid:3)dilution factor. MIC was obtained as mentioned before. The
Concentration ¼ MIC/2
dilution factor was obtained by counting the number of wells without visible growth.
Micro-gel well diffusion assay was performed to examine the potential anti-
bacterial activities of ciproﬂoxacin-gel system, by following the method described
by Magaldi et al. [35] Inocula of either S. aureus ATCC 29213, E. coli ATCC 25922, or
K. pneumoniae B5055 [34] were prepared by growing cells to a log phase (5e6 h).
The suspensions were adjusted with 0.85% NaCl to ﬁnal optical density (OD) read-
ings of 0.1 and 10 with a spectrophotometer at 600 nm. These two OD readings
represented bacterial cultures used for conventional antibiotic susceptibility tests
and bacterial populations of higher resistance to antibiotics (i.e. bioﬁlms) respec-
tively [36,37]. 20 ml of MuellereHinton agar were melted, cooled to 55 (cid:4)C and then
inoculated with 1 ml of the bacterial suspension. The inoculated agar was poured
into the Petri dish plate, and allowed to cool down on a levelled surface. Once the
medium had solidiﬁed, 2 wells, each of 6 mm in diameter, were cut out of the agar
with a biopsy puncture. 70 ml of the gel with 0.2% w/v or without ciproﬂoxacin were
placed into wells in the plate. The plates were incubated in a humid chamber for 20 h
at 35 (cid:4)C. The plates with inhibition zone of bacterial growth were imaged.
Haemolysis assay was performed as described by Ciornei et al. [38] Blood was
centrifuged at 800(cid:2) g for 10 min, and then the supernatant (plasma) and ‘buffy coat’
(white blood cells) were removed. Erythrocytes were rinsed three times and then
resuspended in phosphate-buffered saline (pH 7.4). Next, 400 ml of solutions
obtained from the in vitro release study for hydrogels with 0.2% w/v ciproﬂoxacin at
different time points (4 h, 8 h, 24 h, 48 h, day 4, and day 6 respectively) were used to
replace the PBS in the erythrocyte suspension and were followed by incubation of
2 h at 37 (cid:4)C with gentle end-over-end rotation. Triton X-100 at 2% (SigmaeAldrich)
served as a positive control, and DMSO at 2% served as a negative control. After
incubation, the samples were centrifuged at 800(cid:2) g for 10 min. The release of
haemoglobin was monitored by measuring the absorbance of the supernatant at
540 nm and was expressed as a percentage of the value for Triton X-100-induced
haemolysis.
Peptide self-assembled hydrogels without drug were prepared as described
above (Section 2.3) by using 10 ml of each precursor solution which were ﬁltered
(0.2 mm) and added to each microwell of a m-slide Angiogenesis uncoated (Ibidi)

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