Water soluble fluorescent-magnetic perylenediimide-containing maghemite-nanoparticles for bimodal MRI/OI imaging

Article abstract of DOI:10.1016/j.jinorgbio.2012.09.003

The protein shell of apoferritin-encapsulated maghemite nanoparticles was functionalized with two different red-emitting perylenediimide fluorophores (PDI). One glycosacharide-PDI complex has been synthesized for the first time to be labeled to apoferriti

Full text of DOI:10.1016/j.jinorgbio.2012.09.003

Journal of Inorganic Biochemistry 117 (2012) 205–211
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Water soluble fluorescent-magnetic perylenediimide-containing
maghemite-nanoparticles for bimodal MRI/OI imaging
Natividad Gálvez a, ,1, Ewelina J. Kedracka a,1, Fernando Carmona a,1, F. Javier Céspedes-Guirao b,2
,
⁎
,2
Enrique Font-Sanchis b, ,2, Fernando Fernández-Lázaro b,
,
⁎
⁎
Ángela Sastre-Santos b, ,2, José M. Domínguez-Vera a,
Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
División de Química Orgánica, Instituto de Bioingeniería, Universidad Miguel Hernández, 03202 Elche, Spain
⁎
⁎
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 April 2012
Received in revised form 3 September 2012
Accepted 3 September 2012
Available online 12 September 2012
The protein shell of apoferritin-encapsulated maghemite nanoparticles was functionalized with two different
red-emitting perylenediimide fluorophores (PDI). One glycosacharide-PDI complex has been synthesized for the
first time to be labeled to apoferritin-encapsulated maghemite nanoparticles. Bifunctionality of maghemite@
perylenediimide was demonstrated by both magnetic-core and fluorescent-labeled shell properties. SQUID
measurements confirmed superparamagnetic behavior above 35 K. Fluorescence of perylenediimides is retained
once attached to the magnetic nanoparticle. Moreover, fluorescence microscopy showed that one of these
fluorescent-magnetic nanoparticles was specifically internalized in bifidobacteria without affecting cell viability.
These results revealed that the dual-modal imaging probes of maghemite@perylenediimide nanoparticles have
the potential to be used as optical/MR dual imaging agents.
Keywords:
Magnetic nanoparticles
Fluorescent nanoparticles
Perylenediimides
Bimodal imaging
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
modalities in a way that leverages the respective strengths of each imag-
ing modality. One of these examples is the MRI-OI (optical imaging)
Nanoparticles are being now widely exploited in many biomedical
applications, such as ex vivo biosensors, in vivo bioimaging agents for
diagnosis and as drugs for therapy [1–3]. The basic rationale is that the
metal or metallic nanoparticles display properties and functionalities
that are not available from bulk counterparts or individual molecules.
Among the metallic nanostructures, the preparation of magnetic-
fluorescent nanoparticles is of special significance, because the combina-
tion of both properties into a single nanoparticle would greatly increase
its application potential in biomedical and bioimaging fields. Thus for
example, superparamagnetic iron oxide nanoparticles (SPIO) are consid-
ered the most promising nanosized objects for biomedical applications,
including magnetic resonance imaging (MRI), image-guided drug deliv-
ery and hyperthermia [4–6]. In fact, several SPIOs are currently marketed
and others are under clinical investigation [7,8]. The incorporation of
fluorescence to SPIOs is of considerable value in the biomedical field
since the new magnetic-fluorescent nanoentity can simultaneously
serve as MRI and fluorescent probe. At this point, it must be noted that
multimodal bioimaging is considered the most promising approach for
early diagnosis. The strategy is to combine two or more distinct imaging
modality [9–11], which can generate results superior to both modalities
operating separately: high sensitivity of OI and excellent spatial resolu-
tion of MRI.
In this context, it is a challenge to develop methods for producing
water soluble nanostructures exhibiting at a time magnetic and fluores-
cent properties. A feasible route is the decoration of a SPIO nanoparticle
with fluorescent species, either quantum dots (QD) or organic dyes.
Although QDs show many unique optical properties, especially narrow
near infrared emissions, toxicology and biodegradation of QD still
remains a serious drawback for in vivo applications. An easy way to
overcome these limiting factors consists in the use of a given magnetic
nanoparticle carpeted with highly fluorescent low energy-emitting
dyes.
Previously, we have developed water soluble apoferritin-
encapsulated maghemite nanoparticles as novel contrast agents
for MRI [12], and we realized that their protein capsid can be used
as a platform for the incorporation of adequate dyes to produce
water soluble fluorescent-magnetic nanoparticles with potential
as optical/MR dual imaging agents.
Perylenediimide (PDI) chromophores have proven to be a remark-
able class of dyes characterized by exceptional thermal and photo-
chemical stabilities, as well as fluorescence quantum yields close to
unity in organic solvents and water [13–15]. Taking advantage of
these outstanding properties they have been exploited mainly in or-
ganic electronics [16,17] and, more recently, in biological applications
⁎
Corresponding authors. Fax: +34 958248526.
E-mail addresses: asastre@umh.es (Á. Sastre-Santos), josema@ugr.es
(J.M. Domínguez-Vera), fdofdez@umh.es (F. Fernández-Lázaro).
1
Fax: +34 958248526.
Fax: +34 966658351.
2
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.09.003

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N. Gálvez et al. / Journal of Inorganic Biochemistry 117 (2012) 205–211
HO
OH
H
O
H
O
HO
N
O
N
O
N
O
N
H
N
O
O
N
N
H
O
O
N
HO
O
O
O
O
O
H
SO3C
N
N
3
O
O
O
O
O
O
O
O
CH
3SO3
N
N
H
SO3C
3
CH
3SO3
N
N
O
O
N
O
O
N
N
N
N
H
O
N
O
H
O
O
H
O
HO
HO
H
O
H
O
PDI-2
H
O
O
PDI-1
H
O
Chart 1. Structure of PDIs used in this study.
[18,19]. Among other bioapplications, PDIs have been used for
biolabeling cardiac cells [20,21] and His-tagged proteins [22]. More-
over, we have used them as selective biolabels of the estrogen recep-
tor [23].
Therefore, the magnetic properties of the maghemite core and the fluo-
rescent properties of the grafted perylenediimides make the bifunction-
al maghemite@PDI-1 and maghemite@PDI-2 nanostructures good
choices to produce optical/MR dual imaging contrast agents.
Herein, we report the covalent coupling of water soluble PDI deriv-
atives, namely PDI-1 and PDI-2 (Chart 1), to the apoferritin capsid sur-
rounding the maghemite nanoparticle through the lysine residues
naturally found in the protein. The synthesis of the water-soluble
PDI-galactose conjugate PDI-2 will be also reported for the first time.
2. Material and methods
Solvents and reagents were obtained from commercial sources and
used as received. Column chromatography: SiO2 (40–63 μm). TLC plates
Scheme 1. Synthesis of compound PDI-2.

N. Gálvez et al. / Journal of Inorganic Biochemistry 117 (2012) 205–211
207
SDS Gel Electrophoresis of maghemite, maghemite@PDI and a
freshly mixture of maghemite and PDI samples (2 mg/mL) were
dissociated by heating at 100 °C for 10 min in 10% (w/v) glycerol, 5%
(v/v) ß-mercaptoethanol, 10% (w/v) SDS, and 0.5 M Tris–HCI, pH 6.8.
Samples were electrophoresed using a SDS-PAGE protocol with a sepa-
rating slab gel containing 12% polyacrylamide and run at constant
current (200 V). The gels were stained with Coomassie blue. Wells
containing maghemite and a mixture of maghemite and PDI were run
as controls.
Lyophilized bifidobacteria breve were obtained from BIOSEARCH
SA and cultivated on MRS-cys for 18 h.
Confocal Laser Microscopy Assay. Probiotic bacteria were seeded
onto sterile polylysine coverslip plates. After 24 h, bacteria were incu-
bated with maghemite@PDI nanoparticles for 30 min at room tem-
perature after being washed twice with phosphate-buffered saline
(PBS). The side of coverslip with fixed bacteria was topped by a
glass slide with 10 μL drop of 50% glycerol/PBS (v/v). Then the glass
slide was inverted and placed above a 63× objective on the confocal
microscope.
Fig. 1. UV–vis spectra of maghemite@PDI-1 and maghemite@PDI-2.
coated with SiO2 60 F254 were visualized by UV light. NMR spectra
were recorded at 25 °C using a Bruker AC300 spectrometer. The sol-
vents for spectroscopic studies were of spectroscopic grade and used
as received. High resolution mass spectra were obtained from a Bruker
Reflex II matrix-assisted laser desorption/ionization time of flight
(MALDI-TOF) using dithranol as matrix. Thermospectronic UV300 and
Helios Gamma spectrophotometers were used for UV–visible measure-
ments. Fluorescence emission and excitation spectra were recorded on
a Jasco FP-6500 spectrofluorometer. Spectra were recorded at room
temperature. For the emission spectra, samples were excited at
350 nm with slit widths of 5 and 10 nm for both excitation and emis-
sion monochromators.
Samples for transmission electronic microscopy (TEM) were pre-
pared by placing a drop onto a carbon-coated Cu grid. TEM images of
samples negatively stained with uranyl acetate (to visualize the protein
shell) confirmed that the particles were actually encapsulated by the
apoferritin capsid. Electron micrographs were taken with a Phillips
CM-20 HR analytical electron microscope operating at 200 keV.
Magnetic susceptibility measurements were performed on lyoph-
ilized samples using a magnetometer (Quantum Design MPMS-XL-5)
equipped with a SQUID sensor. Variable-temperature measurements
were carried out in the temperature range 2–300 K.
2.1. Experimental
2.1.1. Synthesis of N,N′-bis-(2,6-diisopropylphenyl)-1,6,7,12-tetra-(4-
prop-2-ynyloxy-phenoxy)perylene-3,4:9,10-tetracarboxydiimide (3)
305 mg (2.2 mmol) of K2CO3 and 283 mg (1.91 mmol) of 4-prop-
2-ynyloxyphenol were added to a solution of 180 mg (0.21 mmol) of
N,N′-bis-(2,6-diisopropylphenyl)-1,6,7,12-tetrachloroperylene-3,4:9,
10-tetracarboxydiimide in 10 mL of N-methyl-2-pyrrolidone, and the
mixture was heated 8 h under argon atmosphere at 90 °C. Then, the
mixture was cooled to room temperature, poured into 2 N HCl and
the resulting precipitate was filtered and washed with water. The
obtained solid was purified by column chromatography on silica gel
(8/2, CH2Cl2/petroleum ether) to afford 151 mg (56%) of compound
3.
1H NMR (300 MHz, CDCl3): δ(ppm)=8.20 (s, 4H), 7.43 (t, 2H, J=
7.7 Hz), 7.29 (d, 4H, J=7.7 Hz,), 6.96 (d, 8H, J=9.4 Hz), 6.91 (d, 8H,
J=9.4 Hz), 4.65 (d, 8H, J=2.4 Hz), 2.69 (m, 4H), 2.54 (t, 4H, J=
2.4 Hz), 1.12 (d, 24H, J=6.8 Hz). HR-MS (MALDI-TOF, dithranol):
m/z [M]+ calcd. for C84H66N2O12 1294.4610; found 1294.4687. UV–
vis (DMF): λmax nm (log ε) 286 (4.75), 450 (4.25), 546 (4.52), 589
(4.74).
Fig. 2. SDS gel electrophoresis showing the tracks corresponding to maghemite, a mixture maghemite+PDI-2 and maghemite@PDI-2. Prussian blue stain was used for iron. A similar
pattern was observed for maghemite@PDI-1.

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N. Gálvez et al. / Journal of Inorganic Biochemistry 117 (2012) 205–211
Fig. 3. TEM image of maghemite@PDI-1 unstained (left) and stained with uranyl acetate (right). Similar images were obtained for maghemite@PDI-2.
2.1.2. Synthesis of N,N′-bis-(2,6-diisopropylphenyl)-1,6,7,12-tetra-{4-
[1-(2,2,7,7-tetramethyltetrahydro-bis [1,3] dioxolo[4,5-b;4′,5′-d]pyran-
5-ylmethyl)-1H-[1–3] triazol-4-ylmethoxy]phenoxy}perylene-3,4:9,10-
tetracarboxydiimide (4)
8.10 (s, 4H), 7.43 (t, 2H, J=7.7 Hz), 7.31 (d, 4H, J=7.7 Hz,), 7.06
(d, 8H, J=9.4 Hz), 7.00 (d, 8H, J=9.4 Hz), 5.51 (d, 4H, J=4.8 Hz),
5.18 (s, 8H), 4.61–4.62 (m, 8H), 4.16–4.32 (m, 16H), 2.67 (m, 4H),
1.11 (d, 24H, J=6.8 Hz). HR-MS (MALDI-TOF, dithranol): m/z
[M+Na]+ calcd. for C108H110N14O32Na 2138.7334; found 2138.7407.
UV–vis (DMF): λmax nm (log ε) 286 (4.72), 455 (4.22), 547 (4.46),
588 (4.67).
A mixture of 63.5 mg (0.049 mmol) of compound 3, 71.25 mg
(0. 25 mmol) of 6-azido-6-deoxy-1,2:3,4-di-o-isopropylidene-α-D-
galactopyranose, 4.46 mg (0.022 mmol) of sodium ascorbate, 2.80 mg
(0.011 mmol) of CuSO4·5H2O, 12 mL of chloroform, 1 mL of ethanol
and 1 mL of water was refluxed for 24 h, cooled to room temperature
and the solvents were evaporated. The residue obtained was purified
by column chromatography on silica gel (8/2, CH2Cl2/acetone) to afford
119 mg (63%) of compound 4. 1H NMR (300 MHz, CDCl3): δ(ppm)=
8.17 (s, 4H), 7.81 (s, 4H), 7.41 (t, 2H, J=7.7 Hz), 7.27 (d, 4H, J=
7.7 Hz,) 6.94 (s, 16H), 5.51 (d, 4H, J=4.8 Hz), 5.18 (s, 8H), 4.61–4.62
(m, 8H), 4.16–4.32 (m, 16H), 2.70 (m, 4H), 1.47 (s, 16H), 1.35 (s, 16H),
1.28 (s, 16H), 1.26 (s, 16H), 1.12 (d, 24H, J=6.8 Hz). High Resolution
Mass Spectrometry (MALDI-TOF, dithranol): m/z [M]+ calcd. for
2.1.4. Preparation of perylene-maghemite nanoparticles
Magnetite was synthesized according to Massart's method, with
slight modifications [24] by coprecipitation of Fe(II) and Fe(III) salts.
After oxidation with HClO4, a colloid of 6 nm-sized maghemite was
obtained.
Horse spleen apoferritin was purchased from Sigma and purified
by chromatography.
The encapsulation of magnetic iron oxide nanoparticles in apoferritin
was carried out following a previous procedure [12,25] based on the
dissociation–association of the protein capsid by monitoring the pH.
Thus, the pH of a apoferritin solution (5 mL, 5 mg/mL) was slowly
lowered to pH 2 with 0.1 M HCl and then, 50 μL of a solution of
maghemite nanoparticles (0.1 M Fe) was added and the mixture stirred
for 30 min. The pH of the resulting solution was then adjusted to 7 with
0.1 M NaOH.
The resulting clear reddish dark solution was purified to remove un-
folded subunits or large nanoparticles aggregates by centrifugation
(10,000 rpm, 1 h) then chromatographied on Sephadex G-25 column
to isolate the protein-containing fractions. Once purified, the
apoferritin-encapsulated maghemite nanoparticles were incubated in
water with the perylene dye in a large excess (118 PDI-1/ApoFt and
164 PDI-2/ApoFt) over 24 h at 4 °C. The resulting solution was exhaus-
tively dyalized against milli-Q water, at 4 °C, to remove the unbound
fluorophore. Finally, the sample was chromatographied by G-25
Sephadex chromatography and fluorescent fractions were collected
and mixed.
C132H142N14O32 2435.9948; found 2436.0305. UV–visible (UV–vis)
Dimethylformamide (DMF): λmax nm (log ε) 285 (4.74), 455 (4.24),
548 (4.48), 588 (4.69).
2.1.3. Synthesis of N,N′-bis-(2,6-diisopropylphenyl)-1,6,7,12-tetra-{4-
[1-(3,4,5,6-tetrahydroxytetrahydropyran-2-ylmethyl)-1H-[1–3]triazol-
4-ylmethoxy]phenoxy}perylene-3,4:9,10-tetracarboxydiimide (PDI-2)
40.0 mg (0.016 mmol) of compound 4 was dissolved in 4.5 mL of
trifluoroacetic acid and 0.5 mL of water, and the solution was stirred
under argon atmosphere at room temperature for 24 h. Solvents were
evaporated, and the residue obtained was purified by column chro-
matography on silica gel (8/2, CH2Cl2/methanol) to afford 30 mg
(87%) of PDI-2. 1H NMR (300 MHz, CH3OD): δ(ppm)=8.12 (s, 4H),
10
3. Results
For this study, we have chosen two different water soluble PDIs.
The unsymmetrically substituted PDI-1 presents a carboxylic group
at one of the imide positions that could allow the link with the nano-
particle, and four methylpyridinium groups at the bay positions, to
avoid face-to-face aggregation and, as consequence, fluorescence
quenching. This molecule also could permeate the nanoparticle shell
and stabilized either into the central or hydrophobic cavity between
the 4-helix bundles as it has been recently shown [26]. On the other
hand, PDI-2 (specially designed and synthesized for the current
study) presents four galactopyranose residues at the bay positions
to take advantage of the interaction of this kind of moieties with the
proteic cover of the magnetic nanoparticle.
5
0
0
100
200
300
T (K)
Fig. 4. ZFC and FC magnetization of maghemite@PDI-1. Similar curves were obtained
for maghemite@PDI-2 (Supplementary Information).

N. Gálvez et al. / Journal of Inorganic Biochemistry 117 (2012) 205–211
209
using absorbance values of the peaks at 547 and 592 nm for PDI-1 and
PDI-2, respectively, and 280 nm for protein capsid (once corrected by
subtraction of the absorbance of PDI-1 and PDI-2 at this wavelength).
Values of 5 (for maghemite@PDI-1) and 7 (for maghemite@PDI-2) PDI
residues per capsid were obtained. The difference in the number of la-
beled dyes could be attributed either to the different coupling reaction
with the capsid lysines of PDI-1 and PDI-2 or to a more favorable access
(due to shape, cross section, charge) to the protein cavity of PDI-2 when
compared to PDI-1. As previously reported, ferritin subunits contain
several lysine residues at the external surfaces, with lys97 and lys104
being the most exposed and reactive [29–31]. In this sense, the presence
of 5 and 7 molecules of PDIs per nanoparticle (about 10–15% of labeling)
is significatively low when compared with other dye-labeled ferritin
samples: about 15 Cy5.5 [32], 22 Red Orange 16, 40 Remazol Brillian
blue or 44 NBD molecules [29–31]. There is considerable evidence in
the literature that small molecules permeate the ferritin shell and
stuck inside the capsid, either at the large central cavity or at the hydro-
phobic cavity existing between the 4-helix bundles [26]. To evidence if
PDIs are conjugated at the lysines or stabilized inside the cavity, dena-
turing gel electrophoresis of maghemite nanoparticles and maghemite
containing PDIs was performed. As it can be observed in Fig. 2, the
PDI-2 of maghemite@PDI-2 does not run with ferritin subunits but
runs similar to free PDI-2, suggesting that PDIs are associated to the
maghemite nanoparticle without reacting with exterior lysines.
TEM images of all fluorescent-magnetic nanoparticles mainly
showed spherical nanoparticles (Fig. 2). The protein shell was visual-
ized by negative staining with uranyl acetate, confirming that the
nanoparticles were actually encapsulated within the apoferritin cap-
sid (Fig. 3 right). The average particle size was 6.0 1.2 nm.
Magnetization values per Fe gram of maghemite@PDI-1 and
maghemite@PDI-2 were found to be close, within experimental uncer-
tainty, to those of unsubstituted maghemite [12]. M(T) and M(H)
sligthly decreased due to the presence of the non-magnetic perylene
dyes.
Fig. 5. Fluorescence emission spectra of maghemite@PDIs.
PDI-1 was prepared as described elsewhere [23]. On the other
hand, the synthesis of PDI-2 starts with the substitution on the N,
N′-bis-(2,6-diisopropylphenyl)-1,6,7,12-tetrachloroperylene-3,4:9, 10-
tetracarboxydiimide [27] with 4-prop-2-ynyloxyphenol to obtain the
tetraalkynyl substituted 3. Click reaction of the latter with 6-azido-
6-deoxy-1,2:3,4-di-o-isopropylidene-α-D-galactopyranose [28] yields
compound 4, which after acetal deprotection with TFA affords PDI-2 in
moderate yield (Scheme 1). All new compounds were characterized by
UV–vis and NMR spectroscopies, together with high resolution
MALDI-TOF mass spectrometry.
We have previously reported water soluble 6 nm-sized maghemite
nanoparticles with excellent MRI properties [12]. These nanoparticles
are coated by the apoferritin capsid, which contains lysine residues capa-
ble of further functionalization [29–31]. Thus, we have added to the
maghemite nanoparticles two PDI dyes: PDI-1 with fluorescence
emission at 592 nm and PDI-2, with emission at 625 nm. These
maghemite@PDI complexes have high fluorescence and can be
used in biological applications for cell and protein staining.
The coupling reaction between maghemite and PDI was carried
out by incubation of the maghemite nanoparticles with an excess of
the corresponding PDI, exhaustive dialysis and chromatograpy to re-
move the unreacted dye.
The temperature dependences of the zero-field-cooled (ZFC) and
field-cooled (FC) magnetization of maghemite@PDI-1 are shown in
Fig. 4. ZFC and FC curves coincide at high temperature and begin to
separate as the temperature decreases, showing a maximum (in the
case of the ZFC curve) at TB =35 K. A consequence of the blocking
of the magnetic moments below TB is the existence of a marked hys-
teresis loop of magnetization with a coercitive field of 332 Oe at 2 K.
Similar behavior was exhibited by maghemite@PDI-2. The shape of
the hysteresis loop of magnetization is similar to that of maghemite
nanoparticles [12].
UV–vis spectra of maghemite@PDIs consist of the superposition of
the spectra of each component of the conjugates, which indicates that
there is no interaction between both moieties in the ground state
after covalent coupling of the PDI unit to the apoferritin capsid surfaces
(Fig. 1). The amount of dye per maghemite capsid was determined by
TEM and magnetic studies indicate that, as expected, functionalization
of the nanoparticle has a negligible effect on the magnetic properties of
Fig. 6. Confocal laser scanning microscopy of bifidobacteria incubated with maghemite@PDI-2: (right) bright-field images; (left) fluorescent images (red).

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N. Gálvez et al. / Journal of Inorganic Biochemistry 117 (2012) 205–211
Fig. 7. Z-axis scanning image of internalized maghemite@PDI-2 recorded by confocal microscope. Micrographs were taken while the focal plane was moved in incremental steps
from the bottom of the cell up to the coverslip.
the core. This is therefore a simple and powerful route for chemically
modifying the water soluble maghemite nanoparticle without losing its
superparamagnetic properties.
Fluorescence spectra of maghemite@PDI-1 and maghemite@
PDI-2 are shown in Fig. 5. Emission bands at 590 and 621 nm were
observed upon excitation at 350 nm. Therefore, as expected, fluores-
cence properties of PDI-1 and PDI-2 are not significantly modified
after coupling to the apoferritin capsid (PDI-1 λem. =592 nm; PDI-2
the mechanism of internalization still remains unclear, we tentatively
propose that the presence of carbohydrate moieties in PDI-2 and the
ionic character of PDI-1, explain the different internalization activity
of maghemite@PDI-1 and maghemite@PDI-2. Further investigations
are needed for clarifying this point and are currently in progress.
It must be noted that after staining by maghemite@PDI-2, the bac-
teria appeared healthy without any sign of structural degradation
and, moreover, fluorescence intensity remained for days, thus indi-
cating that the maghemite@PDI-2 nanoparticles are not metabolized
by the bacteria. In addition, the application of the Live/Dead Bacterial
Viability Kits SYTO9 (green) and Propidium Iodide (red) (Invitrogen)
showed an increase in the live(green)/dead(red) ratio with respect to
the control (see Material and methods), pointing out to the bacterial
continuous proliferation in the presence of maghemite@PDI-2
nanoparticles, which underlines their lack of toxicity.
λ
em. =625 nm).
The biological applicability of maghemite@PDI-1 and maghemite@
PDI-2 was tested with a bifidobacteria breve culture. Bifidobacterium
is a genus of Gram-positive anaerobic bacteria that colonizes the
gastrointestinal tract and therefore represents an ideal carrier for
fluorescent-magnetic material and to serve as bimodal oral contrast
agents [33], one of the most important challenges in Nanomedicine.
Bifidobacteria breve (400 μL of 2×109 CFU) was subjected to either
maghemite@PDI-1 or maghemite@PDI-2 (100 μL of 1 mg/mL solution)
for 10 min. As it can be observed in Fig. 6, these bacteria became fluo-
rescent in the presence of maghemite@PDI-2, showing the red emission
of PDI-2. This observation suggests an internalization of the PDI-2 dye
into the bacteria. This is not the case with maghemite@PDI-1.
Bacteria with maghemite@PDI-2 were imaged at different depths
along the Z axis. Micrographs were taken while the focal plane was
moved in incremental depths from the bottom of cells up to the cover-
slip. Typical section images (Fig. 7) demonstrated that part of
maghemite@PDI-2 was present inside bacteria.
Quantification of bacteria proliferation was performed by using
the Live/Dead Bacterial Viability Kits SYTO9 (green) and Propidium
Iodide (red) (Invitrogen), counting the number of live (green) and
dead (red) bacteria in a batch of three experiments by the software
Image-Pro Plus 6.0. The average live/dead ratio was used to quantify
the effect of maghemite@PDI nanoparticles into bacteria proliferation
by comparing with control experiments where no nanoparticles were
present. The presence of maghemite@PDI-2 nanoparticles resulted in
an increase of the live/dead ratio of 1.5 with respect to control exper-
iments whereas maghemite@PDI-1 nanoparticles did not affect bac-
teria proliferation.
5. Conclusions
We have prepared water soluble fluorescent-magnetic maghemite@
perylenediimide nanoparticles by a simple and flexible route. Magnetic
and fluorescent properties of both materials are retained in the final bi-
functional nanoparticle. MRI properties of the maghemite nanoparticles
and primary results in the internalization into bifidobacteria are encour-
aging in terms of the applicability of these maghemite@PDI nanoparticles
as MRI/fluorescent probes.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2012.09.003.
Acknowledgments
We are grateful to the MINECO and FEDER (projects CTQ2009-
09344, CTQ2010-20349 and Consolider-Ingenio 2010 project HOPE
CSD2007-00007), Junta de Andalucía (project FQM-02525) and
BIOSEARCH S.A. for financial support.
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