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© 2012 American Chemical Society 5598 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
We also report here on the encapsulation of citrate-stabilized particles suspended in hexane. The resulting data were analyzed
magnetic nanoparticles and nanoparticles with adsorbed with the aid of Topas4 and EVA software to determine the
doxorubicin inside the polypyrrole microvessels. The role of average crystallite size of the ferrites according to the
the capsules is to provide the proper environment for the drug literature.21
molecules and nanoparticles and to protect them from SQUID. The characterization of magnetic properties was
degradation when they travel through the tissues to the performed using a Superconducting Quantum Interference
targeted site. Moreover, the capsules allow much higher loading Device (SQUID) magnetometer (Quantum Design MPMS
of the drug molecules when compared to, e.g., covalent grafting 7T) over the magnetic field range from −7.0 to +7.0 T at
onto the nanoparticle surface.7 Additionally, polymer shells various temperatures. The sample was weighted and placed in
influence the antifouling characteristics of the nanoparticles and the paraffin capsule. The known diamagnetic contribution of
also may contribute to their effective hydrodynamic size, one of the paraffin was taken into account, and the magnetization data
the key factors in avoiding the response by the ReticuloEndo- were corrected during the analysis.
thelial System (RES).13,16 Introducing double functionality to IR Spectroscopy. Infrared spectra were collected with a
the capsules is a novel idea, which opens up new perspectives Nicolet iN10 MX IR microscope (Thermo Scientific),
for commercial applications. To the best of our knowledge, equipped with DTGS detector.
there have been only few examples of such assemblies in the Raman Spectroscopy. Raman spectra were collected with a
literature. Abbasi et al. prepared polyelectrolyte capsules loaded LabRAM HR Raman confocal microscope (Horiba Jobin
with γ-Fe2O3 nanoparticles and fluorescein isothiocyanate.17 Yvon), equipped with an LPF Iridia edge filter, a 600 groove/
Poly(methacrylic acid) microspheres with embedded magnetic mm holographic grating, and a 1024 × 256 pixel Peltier-cooled
nanoparticles were used to entrap daunorubicin followed by Synapse CCD detector. The sample was excited with a
controlled release to the external solution.18 Gorin et al. semiconductor laser (Spectra-Physics) operating at 532 nm.
reported on coencapsulation of gold and magnetic nano- Fluorescence Measurements. The fluorescence measure-
particles in polyelectrolyte capsules.19 These structures ments were carried out on a Fluorolog 3-2-IHR320 (Horiba-
possessed two functions: they were optically addressable and Jobin Yvon) spectrofluorometer. The aqueous solutions of
susceptible to the magnetic field. Lately, Liu at al. reported doxorubicin were excited at 468 nm with emission collected at
alginate-templated polyelectrolyte multilayer microcapsules 590 nm. The ethyl acetate solutions of doxorubicin were
containing maghemite nanoparticles for controlled release of excited at 468 nm, and emission was collected at 586 nm. A
doxorubicin under the stimulus of a high-frequency magnetic short path quartz cuvette was used (0.1 cm, Starna Cells 52-Q-
field.20 The application of the high-frequency magnetic field 1). To minimize the effect of lamp fluctuation over time, each
significantly accelerated the drug release, which might be time the fluorescence signal was divided by the simultaneously
related to the induced heating effect and/or mechanical damage detected lamp intensity signal.
to the capsules’ walls by oscillating magnetic nanoparticles. UV−vis Measurements. The absorption spectra within the
range of 600−400 nm were obtained on a Perkin-Elmer
2. MATERIALS AND METHODS Lambda-25 spectrometer with small-volume cuvettes.
2.1. Reagents. Iron(III) chloride hexahydrate (p.a., Sigma- Confocal Fluorescence Microscopy. Confocal fluorescence
Aldrich), iron(II) chloride tetrahydrate (99%, Sigma-Aldrich), images were acquired using an optical microscope (Leica model
oleic acid (70−80%, Fluka), ammonia solution (p.a. 25%, TCS SP2, Wetzlar, Germany) with an argon ion laser (488 nm)
Chempur), o-chlorotoluene (97%, Fluka), N,N-dimethylforma- as an excitation source, emission collected at 550−620 nm.
mide (HPLC 99,9%, Sigma-Aldrich), citric acid monohydrate 2.3. Synthesis. Oleic-Acid-Coated Nanoparticles. The
(p.a., Chempur), iron(III) citrate monohydrate (p.a., Fluka), first step of our synthesis was to obtain a stock sample of oleic-
disodium phosphate (p.a., POCH, Poland), ethyl acetate acid-coated iron oxide magnetic nanoparticles. These nano-
(HPLC 99,9% CHROMASOLV Plus, Sigma-Aldrich), doxor- particles were synthesized using the bottom-up coprecipitation
ubicin hydrochloride (Selleck Chemicals), pyrrole (Sigma- approach. Amounts of 5.18 g of FeCl2·4H2O and 14.1 g of
Aldrich), o-phenanthroline, and DMF were used as received. FeCl3·6H2O (molar ratio 1:2) were dissolved in 150 mL of
For preparation of polypyrrole microvessels, it is critical to use water to prepare solution “A”. Solution “B” was obtained by
high purity bromoform (Aldrich, >99%). All aqueous solutions mixing 20 mL of concentrated ammonia solution (25% NH3 aq)
were prepared with the Milli-Q-Plus water. with 4 mL of oleic acid, resulting in colloid formation of oleic
2.2. Instrumentation. TEM. The studies were performed acid in basic solution. The colloid “B” was added to solution
in the Laboratory of Structural and Physicochemical Research, “A” and stirred for 1.5 h. Thereafter, the magnetic nanoparticles
University of Warsaw, Warsaw, Poland, equipped with Energy were precipitated by a magnet and washed several times (∼10)
Filter Transmission Electron Microscope (EF-TEM) Zeiss with acetone. No presence of Fe2+ ions in the supernatant was
Libra 120 Plus, operating at 120 kV. A drop of dilute colloid of confirmed by addition of o-phenanthroline. The precipitate
magnetic nanoparticles was placed on Formvar/carbon grids collected by a magnet was dried to remove acetone. Part of the
(S162-3, Agar Scientific), and solvent was allowed to evaporate. magnetic nanoparticles was sonicated in hexane leading to
DSC. The differential scanning calorimetry measurement was ferrofluid formation. The ferrofluid was subsequently filtered
performed on a DSC-50 Shimadzu instrument. An argon flow through a 200 nm membrane (Filtropur S 0.2, SARSTEDT)
rate was set to 30 mL/min. The temperature was scanned from and analyzed.
20 to 300 °C with a rate of 10 °C/min. Citric-Acid-Coated Nanoparticles. The synthesis of citric-
PXRD. The diffraction pattern was collected on a Bruker D8 acid-coated nanoparticles was a modified version of the ligand
Discover diffractometer under Cu Kα radiation (λ = 1.540598 exchange approach presented in the literature.5 An amount of
Å), covering a range of 2θ from 20° to 70° at scan rate of 1°/ 414 mg of oleic-acid-coated nanoparticles from the previous
min in 0.012° steps. The measurement was performed in the step was dispersed in a 1:1 by volume mixture of DMF/o-
capillary filled with the oleic-acid-stabilized colloidal nano- chlorotoluene with the help of a sonicator. The 3.13 g of citric
5599 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
Figure 1. Powder X-ray diffraction pattern of iron oxide nanoparticles. Bars represent peak shifts and relative intensities for maghemite (JCPDS card
no. 39-1346).
acid monohydrate was added, and the solution was stirred for Preparation of Polypyrrole Microvessels Loaded with
24 h. The magnetic nanoparticles were recovered by a magnet Magnetic Nanoparticles. To 5 mL of bromoform, 25 μL of
and washed several times with acetone and dried. Dried aqueous suspension of nanoferrites (ca. 4 mg/mL) with
nanoparticles were suspended in 35 mL of water and sonicated adsorbed doxorubicin was added and sonicated for 30 s at
for 25 min in a water bath and an additional 3 min at 400 W 400 W with an ultrasonic processor. Once the emulsion was
with a USP400 sonicator. To separate citric acid nanoparticles formed, 200 μL of pyrrole was added. The emulsion was placed
from larger aggregates, the solution was centrifuged 5 min at in a cylindrical quartz container, rotated at 20 rpm, and
5000 rpm (ca. 2500 g). The dark with deep ruby hue solution irradiated with a UV light for three minutes (mercury lamp
over the precipitate was collected and filtered through a 200 nm Polamp-5, Poland, 80 W, placed 10 cm from the reaction
membrane. This solution was stable in time and could not be vessel). After completion of the reaction, the resulting
precipitated with a solid magnet. The concentration of the microvessels filled with nanoparticles were separated from the
obtained colloid was 4 mg/mL. supernatant by centrifugation and washed with bromoform.
Adsorption of Doxorubicin onto Magnetic Nanoparticles. The centrifugation and washing steps were repeated three
The series of doxorubicin water solutions with the following times.
concentrations, 2.5, 6.25, 12.5, 18.75, 25, 27.7, 33.3, 50, 62.5,
75, 87.5, and 100 μM, were prepared. For the adsorption 3. RESULTS AND DISCUSSION
studies, 0.5 mL of each solution was transferred to the separate Figure 1 shows the PXRD pattern of the stock sample of
vial and mixed with 50 μL of colloid of citric-acid-stabilized iron magnetic nanoparticles (NPs). The diffraction pattern consists
oxide nanoparticles (concentration: 4 mg/mL). Subsequently, 4 of a series of peaks revealing a cubic spinel structure that fits
mL of ethyl acetate was added to each vial, forming a ternary relatively well to the γ-Fe2O3 phase (maghemite JCPDS card
system (water/nanoparticles/ethyl acetate). The vials were no. 39-1346) diffraction pattern.
sealed and shaken vigorously. After 2 h of equilibration, the The full width at half-maximum (fwhm) value of the most
organic solution was placed in the cuvette for fluorescence pronounced reflection (311) shown on the PXRD pattern was
measurements. To validate the above approach of relating the used to evaluate the average diameter of nanoferrites, according
amount of DOX that partitions to the organic phase with the to the Scherrer equation.22 This procedure yielded ca. 11 nm
amount of DOX in the aqueous solution, the calibration curve particle diameter, a value in good agreement with results
was prepared in the same manner, but instead of iron oxide obtained in the literature.2,23
colloid, 50 μL of water was added. However, the spinel crystal structure of γ-Fe2O3 (maghe-
Desorption of Doxorubicin. Freshly prepared samples of mite) and Fe3O4 is very similar, differing only by ca. 1% in the
nanoparticles with adsorbed doxorubicin (100 μL of colloid of cubic lattice constant; therefore, PXRD is relatively insensitive
citric-acid-stabilized iron oxide nanoparticles mixed with 1 mL to the difference between maghemite and magnetite. Thus, we
of 345 μM doxorubicin, equilibrated for 2 h) were centrifuged; used the Raman spectroscopy as an alternative way of
supernatant was removed; and the remaining pellets were evaluation of the stoichiometry of magnetic nanoparticles.24−26
washed briefly in a small amount of water and then Shown in Figure 2 is the Raman spectrum of the nanoferrites.
resuspended into 3 mL of 0.1 M NaClaq (pH 6.3) at room Characteristic lattice vibrations of mainly maghemite (384, 487,
temperature. At controlled times, 1 mL of sample suspension and 680−720 cm−1) are visible.27
was precipitated in the field of a solid magnet, and the However, great care is required when using the Raman
supernatant absorbance was measured within the range of 600− spectroscopy for the case of nanoferrites. It has been reported
400 nm. After that, it was returned to the stock 3 mL in the literature that the structure of nanoferrites is very
suspension. The suspension was gently rocked during the sensitive to phase transformations due to the local heating of
whole desorption time course. the sample by laser irradiation during the spectra acquisition.
5600 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
Figure 3. TEM image and histogram of citrate-stabilized nanoparticles. Scale bar corresponds to 50 nm.
Figure 5. (A) Magnetization as a function of magnetic field at temperatures ranging from 2 to 300 K. The hysteresis loop is shown in the inset. (B)
Magnetization at 300 K (points) and theoretical approach (solid line) proposed by Millan et al.28 for parameters: diameter 12 nm, Ms = 35 emu/g, χ
= 1.7e−4 emu/(g·Oe); see text for details.
order of 56 emu/g. This value compares well with maghemite However, the obtained estimate must be considered with care
nanoparticle characteristics found in the literature (55−70 as it results from drastic assumptions: size distribution of
emu/g, depending on size, crystallinity, and surface modifica- nanoparticles was neglected, and the spherical shape of the
tion28−31). However, this relatively low value can result either nanoparticles was used for calculations. Moreover, magnet-
from poor crystallinity of our system or from the under- ization of a nanoparticle is most probably smaller than that of
estimation of the thickness of the nonmagnetic (organic) layer bulk material due to the surface effects. Nevertheless, the
of citrates covering the crystalline core and its paramagnetic obtained values of the diameter d and parameters Ms agree with
contribution to the magnetization.29 We opt for the latter the general observation of their relative dependence at room
explanation. Over 50 K the sample becomes superparamag- temperature, presented by Millan et al.28
netic; the hysteresis disappears; and magnetization decreases at It should be stressed, however, that although eq 1 provides a
higher temperatures. reasonable fit to the experimental data the obtained parameters
Such behavior can be interpreted as resulting from Ms and χ are purely phenomenological. Moreover, in our case
magnetizing the system of ferromagnetic nanoparticles, where they appeared to be temperature dependent, while one set of
individual spins of Fe ions are FM coupled and form very large parameters should be applicable for different temperatures. On
magnetic moments of nanoparticles. The moments of different the other hand, it was possible to describe the data in the entire
nanoparticles are uncoupled, so the entire sample behaves as a temperature range assuming a certain distribution of size of
superparamagnet. Fast saturation at low magnetic field (B < 1 nanoparticles. The detailed analysis, taking into account
T) is characteristic for magnetization of very large magnetic different models, will be presented separately.
moments. Such a system should saturate at low temperatures Figure 6 presents the zero-field cooling (ZFC) and the field
and field of a few tesla, which is not the case (Figure 5). cooling (FC) curves for the sample, plotted from 2 to 350 K.
Instead, a slow, nearly linear increase for higher fields is The magnetization of the FC protocol increases monotonously
observed (B > 1 T), which may be attributed to the presence of with decreasing temperature, while the magnetization on the
different magnetic phases such as, e.g., the antiferromagnetic ZFC protocol initially increases with temperature decrease to
one, which magnetizes linearly with the magnetic field below reach a maximum and then decreases with further temperature
the Néel temperature. decrease. The difference between FC and ZFC magnetization
According to Millan et al.,28 the measured magnetization M results from anisotropy of single nanoparticles and is typical for
can be decomposed into the superparamagnetic part (described superparamagnetic systems. The temperature at which ZFC
by a Langevin function) and a linear component starts to deviate from FC (ca. 65 K in our case) is called the
blocking temperature, TB, and can be used to estimate the
⎛ μB ⎞ nanoferrite anisotropy. According to Néel theory, the blocking
M = MsL⎜ ⎟ + χB temperature is related to the particle volume V and the
⎝ kBT ⎠ (1) magnetic anisotropy constant Ka that includes contributions
from magnetocrystalline (bulk), shape, strain, and surface
where L is the Langevin function; μ is the average magnetic anisotropies.32 For a single domain nanoferrite, this relation
moment of the ferromagnetic core; kB is the Boltzmann reads
constant; and Ms and χ being adjustable parameters represent
the saturation magnetization and linear contribution due to the 25kBTB
presence of antiferromagnetic layer, respectively. Applying eq 1 Ka =
V (2)
to our data, we obtained a volume of a nanoparticle, which for a
spherical nanoparticle corresponds to the diameter of 12.5 nm, where kB is the Boltzmann constant. For a nanoparticle of ca.
in reasonable agreement with PXRD and TEM results. 10 nm (vide supra), the value of Ka can be estimated as 4.3 ×
5602 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
Figure 7. FTIR spectra of citric-acid-stabilized nanoferrites (spectrum a), the same nanoferrites after adsorption of doxorubicin (spectrum b), and
free doxorubicin (spectrum c).
Figure 8. Adsorption isotherm of doxorubicin on the aqueous suspension of nanoferrites. Average of 10 measurements R = 0.9956. Inset: Calibration
curve of DOX fluorescence intensity measured in ethyl acetate vs drug concentration in the aqueous phase; no nanoferrrites present; R = 0.9983.
accurate, quantitative determination of doxorubicin interactions aqueous doxorubicin solutions in contact with the ethyl acetate
with iron oxide nanoparticles. Since the citrate-stabilized phase in a volume ratio identical to the adsorption experiments
hydrophilic nanoferrites do not partition into the organic with nanoferrites. Here, the differences in the absorption
phase, this approach eliminated problems associated with light intensities before and after ethyl acetate addition were very
scattering on nanoparticles and therefore provides higher small, nevertheless allowing for the evaluation of the partition
sensitivity than typical measurements directly in an aqueous coefficient of doxorubicin into the organic phase at the level of
suspension of nanoparticles. 1%. Therefore, we assumed that the equilibrium partitioning of
As compared to its affinity to water, doxorubicin also has a doxorubicin into the organic phase, though easily detected by
relatively low affinity toward organic (immiscible with water) spectrofluorometry as an increase of fluorescence intensity of
solvents such as ethyl acetate. Therefore, this approach the organic phase, does not affect substantially the concen-
eliminated fluorescence nonlinearity at higher aqueous tration of the drug in the aqueous phase. A similar effect has
concentrations.39 Nevertheless, the amount of doxorubicin been reported in the literature.39 We are aware that this is a
transferred to the organic phase could be easily detected by rather simplistic assumption; nevertheless, we think that the
spectrofluorometry with linear dependence of fluorescence fluorescence of ethyl acetate solution of doxorubicin at
intensity detected for the organic phase for the whole range of equilibrium with aqueous solution could be directly related to
doxorubicin concentration studied in the aqueous solution. The the concentration of the drug in this aqueous phase.
rationale behind this is that at equilibrium the chemical Furthermore, the fluorescence intensity of doxorubicin in
potentials of DOX in both phases must be equal (we assumed ethyl acetate being in equilibrium with the aqueous phase is
that 2 h is sufficient to equilibrate both phases in contact with sufficiently high to allow the drug detection with high
respect to DOX partitioning). This results in the direct relation sensitivity. Therefore, we used this ternary system to study
of concentrations of this solute in ethyl acetate and in aqueous the drug interactions with nanoparticles and were able to
phases. Under constant thermodynamic conditions, the ratio of determine its adsorption isotherm onto the surface of citric-
these concentrations, the partition coefficient, should be acid-stabilized nanoparticles. This isotherm is shown in Figure
constant because it is defined by the difference between the 8.
standard chemical potentials of doxorubicin in both contacting Apparently, the adsorption isotherm follows the Henry’s
solvents. To validate this relation, we performed the calibration approach within the large concentration range of our
curve under the same experimental conditions as for the case of experiments. The Henry’s law, which is valid for most
the adsorption experiments, except for the presence of isotherms at low adsorbate concentration regimes (Henry’s
nanoferrites in the aqueous phase. The linearity of the obtained region), relates the concentration of the adsorbing species in
calibration curve with constant slope (see inset in Figure 8) the solution with the amount of the same species adsorbed
confirms our approach and allows us to unambiguously onto the surface, by a simple linear equation
determine the amount of DOX adsorbed onto the surface of cads = Kads·csol (3)
nanoparticles, by measuring its fluorescence in the ethyl acetate
phase. where cads and csol are the concentrations of species adsorbed
The question that arises is to what extent the partition of onto the surface and “free” in the solution, respectively, and
doxorubicin into the organic phase lowers its concentration in Kads is the adsorption equilibrium constant. Since we do not
an aqueous suspension of nanoferrites. To answer this question, observe a plateau on the adsorption isotherm, we think that in
we studied the UV−vis absorption spectra (not shown) of the concentration range studied (up to 100 μM) we still remain
5604 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
Figure 9. Binding of doxorubicin to citrate-stabilized magnetic nanoparticles: drug loading vs initial doxorubicin/nanoferrites weight ratio. Inset:
doxorubicin loading efficiencies in water and 0.1 M NaClaq. See text for details.
in the Henry’s region. To explain this result, we evaluated the the nanocarriers. Less precise is the definition of the drug
specific area of citrate-stabilized colloidal suspension and the loading, sometimes referred to as drug loading content. Here,
area available for adsorption of doxorubicin under our we will define it as weight percentage of drug with respect to
experimental conditions. Setting 4.87 g/cm3 as the maghemite the total weight of nanoparticles with attached drug (% w/w)
density,29 and the average particle diameter of 9 nm (assumed
to be uniform and spherical), we obtained ca. 550 m2/g of the Wads
drug loading content (%) = × 100%
specific area of our maghemite, comparable with that of silica WNP
gel (560−455 m2/g).40 Since for the adsorption experiments
we used suspension containing 4 mg/mL (vide supra), this where Wads is the mass of doxorubicin adsorbed onto the
yielded ca. 0.5 m2 of the surface of citrate-stabilized maghemite nanoparticles and WNP is the mass of magnetic nanoparticles
available for DOX adsorption. Even though such simplistic with attached doxorubicin.
calculations should be considered with the utmost care, they Recalculating the isotherm data from Figure 8, we can now
strongly support the observed linearity of the adsorption show the doxorubicin loading content vs the doxorubicin/
isotherm within the concentration range of our experiments. magnetic nanoparticle mass ratio used for the adsorption
One has also to consider the presence of a finite thickness of (please keep in mind that the mass of nanoparticles is kept
citrate shell providing an additional space for the adsorption constant; only the amount of drug varies). The results are
process. shown in Figure 9.
Thus, from the slope of this isotherm, the equilibrium However, for the drug feed higher than 0.5 (w/w)
constant of interactions between doxorubicin and nanoparticles
doxorubicin/nanoparticles mass ratio, we observed an extensive
can be calculated, yielding the value Kads = 1.7 × 104. From the
aggregation with subsequent fast precipitation. To our opinion,
relation ΔG0 = −RT ln Kads, the Gibbs free energy of
such a behavior drastically hinders the use of larger
adsorption was determined as ΔG0 = −24.1 kJ/mol. The above
results indicate rather high affinity of doxorubicin to the surface concentrations of doxorubicin in the feeding solution for
of nanoparticles. If we consider the ratio of DOX concentration further applications for the prospective drug delivery system,
on nanoferrites and doxorubicin concentration in an aqueous even though one can reach the load of 60% (w/w). This is not
solution, we obtain ca. 2 × 103 higher surface concentration of surprising, given the literature data, where there is a strong
doxorubicin (mol) per gram of nanoparticles than its volume tendency of the drug to form aggregates with citrates (cf. ref
concentration (mol/g). We think that the observed high surface 45), and deterioration of the stability of colloid due to the
concentration of doxorubicin is mainly due to a relatively thick screening of surface charge is reported. Therefore, the
(ca. 25% w/w) citric acid shell around the crystalline core of concentration range up to 4 × 10−7 mol/g of doxorubicin in
nanoferrites. This thermodynamic approach allows one to water (0.53 w/w doxorubicin/nanoparticle mass ratio) seems
evaluate the equilibrium constant of the adsorption process. to be quite sufficient from the point of view of system stability
From the practical point of view, the drug−nanocarrier data are and load, allowing us to obtain a relatively stable nanoferrite
typically presented in terms of drug loading (loading content) colloidal system with drug load up to 30%. Such a system was
and/or loading efficiency, the latter being defined as the used for doxorubicin release studies below.
percentage of drug on the nanoparticles with respect to the Having said that, we can relate our results to other similar
overall amount of drug involved in the process. In other words, studies on doxorubicin loading content on magnetic nano-
it shows how efficient the process of drug immobilization is on particles. This is presented in the Table 1 below.
5605 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
The Journal of Physical Chemistry C Article
Figure 11. Fluorescence confocal images of polypyrrole microvessel filled with (a) doxorubicin and (b) doxorubicin and nanoferrites with adsorbed
doxorubicin. Inset: normalized fluorescence intensity cross-section vs distance along the line marked on the image [μm]. Scale bar (bottom right)
corresponds to 2 μm. Inset prepared with ImageJ software (http://rsb.info.nih.gov/ij/).
is possible to use such structures to encapsulate both the A close inspection of the microscopic images reveals that the
nanoferrites and doxorubicin for the purpose of targeted drug presence of nanoferrites affects the “sharpness” of the right-
delivery. The adsorption of doxorubicin onto the surface of hand image, suggesting that for this case the distribution of the
nanoferrites can provide much higher payload of this drug as drug is more “diffused” into the microvessel interior. Here, the
compared to the same compound dissolved in an aqueous doxorubicin molecules are adsorbed onto the nanoparticles,
interior of the microvessels. In the encapsulation procedures, thus the effective concentration of the drug in the capsule’s core
the single emulsion or double emulsion methods are frequently is increased. On the other hand, the surrounding solution does
used.43 In the present work, we used an adaptation of the single not exhibit fluorescence, so leakage of the drug from the
emulsion method, where nanoferrites with adsorbed (and free) microvessel is not significant over the time scale of the
drug were suspended in the aqueous droplets of the appropriate experiment (consecutive confocal microscopy images were
water-in-bromoform emulsion, then coated with polypyrrole, as collected at varying Z and are provided in Figures SI and SII,
described above. On the basis of our previous work,55 we Supporting Information).
expected the nanoferrites to be encapsulated within the voids of For better visualization, the TEM image of several contacting
the hollow polymeric shell; however, we could not exclude the microvessels with void space between them is also shown in
possibility of their partial incorporation within the shell Figure 12. Black “specks” are the nanoferrites that are partially
itself.20,56
The encapsulated species were detected using confocal as
well as TEM microscopies. Figure 11 presents the confocal
fluorescence microscopy image of polypyrrole microcapsules
filled with doxorubicin (a) and filled with nanoferrites with
adsorbed doxorubicin (b). Close inspection of these two images
shows that for both cases doxorubicin is encapsulated within
the microvessels, being confined mainly within the shell of the
capsules. This is likely due to the interaction of doxorubicin
with polypyrrole. A similar partitioning of the fluorescent
probe, Rhodamine 6G, within the polypyrrole capsule’s shells
was reported by us recently.52 Even though the nature of this
interaction is not known, it seems the partitioning effect may be
governed mainly by stacking interaction between pyrrole rings
in the polymer and the ring system of doxorubicin. On the
other hand, doxorubicin was reported to be accumulated within
the capsule’s walls consisting of negatively charged polymers.57
As we expect polypyrrole to be neutral or positively charged
Figure 12. TEM image of contacting microvessels with void space
(due to the presence of polarons or bipolarons in the polymer
between them. Inset: smaller magnification of the same spot. Scale bar
backbone), we should rather exclude the possibility of attractive corresponds to 100 nm.
Coulombic interactions in our case.
5607 dx.doi.org/10.1021/jp2095278 | J. Phys. Chem. C 2012, 116, 5598−5609
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distributed also in the void volume. A majority of the (WKP_1/1.4.3./1/2004/72/72/165/2005/U). Confocal fluo-
nanoferrites, however, fill the microvessels and/or are rescence images were collected in the Laboratory of Confocal
incorporated/adsorbed on the polypyrrole shell. Microscopy, Nencki Institute of Experimental Biology, Warsaw,
■ CONCLUSIONS
We have synthesized magnetic nanoferrites with very good
Poland. K.N. and J.R. acknowledge support of the Foundation
of Polish Science MPD Program cofinanced by the EU
European Regional Development Fund.
magnetization behavior and relatively low contribution from
shape and surface magnetic anisotropies. Supported by PXRD
data and TEM images, these results point to rather good
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ASSOCIATED CONTENT Chubykalo-Fesenko, O.; Veintemillas-Verdaguer, S.; Parak, W. J.;
*
S Supporting Information Morales, M. P.; Gonzalez, J. M.; Hernando, A.; de la Presa, P. J. Phys.
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