Self-Assembly of Synthetic and Biological Polymeric Systems of ...

exhibit an important contrast imaging effect, which confirms
their potential use as MRI contrast agents. Future work will
involve the refinement of the synthetic process to achieve
perfect control of magnetic coverage, and thus hybrid fibril
widths, magnetic nanowire lengths, population distribution
and in vivo assays.
Experimental Section
Materials: Human serum albumin, lysozyme, FeCl2 and FeCl3 were obtained
from Sigma Chemical. Proteins were used after further purification
by liquid chromatography over a Superdex 75 column equilibrated
with phosphate (0.01 m). All other chemicals were of the highest purity
available. Experiments were carried out with doubly distilled, deionised
and degassed water.
Preparation of HSA and lysozyme fibril solutions: Protein fibril solutions
were prepared by well-established protocols. [16] Briefly, HSA fibril stock
solution was made by dissolving the protein to a final concentration typically
of 2 mg mL 1 in phosphate buffer (pH 7.4, ionic strength 10 mm)
and brought to pH 2.0 by addition of HCl. Lysozyme fibrils were obtained
by forming a stock solution (2 mg mL 1 ) in glycine buffer (pH 2.0,
ionic strength 10 mm). Both protein stock solutions were dialysed extensively
against pure buffer. Protein concentrations were determined spectrophotometrically,
with use of molar absorption coefficients of 35219
and 37 609 m 1 cm 1 at 280 nm for HSA and Lys, respectively. [40] Before incubation,
the solutions were filtered through a 0.2 mm filter into sterile
test tubes. Samples were incubated at 65 8C in a reactor under reflux for
10 and 3 days for HSA and Lys, respectively.
Characterisation of the fibrils: Protein suspensions were applied to
carbon-coated copper grids, blotted, washed, negatively stained with
phosphotungstic acid (2%, w/v), air dried and then examined with a Phillips
CM-12 transmission electron microscope operating at an accelerating
voltage of 120 kV. Samples were diluted 20–200-fold when needed prior
to deposition on the grids.
Magnetic nanowire synthesis: A stock solution (typically 2 g L 1 ) of
either lysozyme or HSA fibrils was diluted 600-fold to avoid the presence
of undesired aggregates during the synthesis of the magnetite nanowire.
Different [Fe]/ACHTUNGTRENUNG[protein] molar ratios (10:1, 25:1 50:1, 100:1, 200:1 and
400:1) and sequential additions (one to five) of the iron salts were tested
to enable the formation of magnetite nanoparticles on the biotemplate
surface and to ensure full coverage of the fibril surface. In a typical synthesis,
an aliquot (100 mL) of lysozyme fibril solution (2 gL 1 , 1.34 ”
10 4 m) was placed in a flask reactor and diluted with doubly distilled, deionised
water (60 mL). Fibril solution was left to incubate at 65 8C for 1 h
with magnetic stirring. Separately, two aqueous solutions containing
FeCl2 (25.4 mg, 0.1 m) and FeCl3, respectively, (64.9 mg, 0.1 m) were prepared
and mixed in a 1:2 molar ratio. After the incubation period at
658C, the temperature of the fibril solution was raised to 80 8C, and a
specific volume of the iron salt mixed solution suitable to get the desired
[Fe]/ACHTUNGTRENUNG[protein] molar ratio was then added under nitrogen. The solution
was left to incubate for 3 h before the addition of NH4OH (1.5 m, 2 mL);
the resulting solution was allowed to react for an additional 1 h and allowed
to cool to room temperature. Stabilisation of the nanowires was
additionally achieved by addition of sodium citrate solution (10 mm,
1 mL) to the resulting nanowires at 80 8C and allowing to incubate for
90 min. The resulting biohybrid solution was dialysed through a dialysis
membrane (SpectraPor, Netherlands, 100 kDa) to remove remaining iron
salts, NH4OH and sodium citrate. If required, fresh additions of the same
volume of Fe salt solutions were carried out under the same conditions
as previously specified to ensure the full magnetic coverage of fibrils and
to create a nanowire.
Characterisation of magnetic 1D assemblies and nanowires: TEM images
were obtained with a transmission electron microscope (Phillips CM-12)
operating at an accelerating voltage of 120 kV as previously described.
HR-TEM images and SAED patterns were obtained with a transmission
7372
electron microscope (Carl-Zeiss Libra 200 FE-EFTEM) operating at
200 kV. X-ray diffraction experiments were carried out with a rotating
anode X-ray generator (Siemens D5005). Twin Gçbel mirrors were used
to produce a well-collimated beam of CuKa radiation (l = 1.5418 A). Xray
diffraction patterns were recorded with an imaging plate detector
(AXS F.Nr. J2–394). FTIR spectra of HSA in aqueous solutions were determined
with a FTIR spectrometer (model IFS-66v from Bruker) with a
horizontal ZnS ATR accessory. The spectra were obtained at a resolution
of 2 cm 1 and generally 200 scans were accumulated to provide a reasonable
signal-to-noise ratio. Magnetic susceptibility measurements were carried
out with a SQUID magnetometer (Quantum Design MPMS5, San
Diego, CA). Iron concentrations of each sample for SQUID, relaxivity
and MRI were determined by inductively coupled plasma atomic emission
spectroscopy (ICP-AES, Varian).
Magnetic resonance measurements and MRI imaging: Transverse and
longitudinal relaxation times were also measured at 9.4 T (400 MHz) and
378C with a Bruker Biospin USR94/20 instrument (Ettlingen, Germany).
An inversion-recovery pulse sequence was used to measure the longitudinal
relaxation times with 16 inversion recovery times logarithmically
spaced. Both Carr–Purcell–Meiboom–Gill (CPMG) and spin-echo pulse
sequences were used to measure transverse relaxation times. MRI phantoms
were constructed in agarose solutions (Sigma–Aldrich, 4%, w/v),
heated and stirred at 80 8C until complete solution of the solid agarose.
Then, with the system still remaining fluid, six HPLC vials (Cromlab,
Spain, 250 mL) were filled with the agarose solution (100 mL) and allowed
to cool to room temperature. Protein solution (5, 10, 15, 25, 50 or
100 mL) was then deposited over the surfaces of the agarose gels, and distilled
water (49, 45, 40, 35, 25 and 0 mL, respectively) was added to the
solutions to make the final volumes even at 150 mL per vial. After a waiting
period of at least 30 min, most of the deposited fluid had been absorbed
by the agarose gels, after which semi-gelificated agarose solution
(50 mL, 10 %, w/v, heated to 80 8C for solution and cooled to about 40–
508C before addition) was added to each phantom to seal them. Imaging
of the phantoms was performed with the previously described Bruker
Biospin system with 440 mT m 1 gradients. A 3D-Gradient Echo image
was acquired (T2*-weighting) with the following parameters: field of
view: 28.8 ”19.2 ”2 mm, matrix size: 288 ” 192 ”40 points giving a spatial
resolution of 100 ”100 ”200 mm, echo time TE = 8 ms, repetition time
TR=100 ms and flip angle = 30.
Acknowledgements
P. Taboada et al.
The authors thank the Ministerio de Ciencia e Innovación (MICINN) for
research project MAT 2010–17336, the Xunta de Galicia for research
project INCITE09206020PR and for European Regional Development
Funds (research project 2010/50), and the Fundación Ramón Areces for
additional financial support. We acknowledge the contributions of B. Argibay,
P. Ramos-Cabrer and J. Castillo from the Clinical Neurosciences
Research Laboratory of the Clinical Hospital of Santiago de Compostela
for the MR images acquired with the 9.4 T MR system.
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