Journal of Controlled Release 130 (2008) 168174

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Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l

Cytokine adsorption/release on uniform magnetic nanoparticles for localized

drug delivery
Raquel Mejas a, Roco Costo b, Alejandro G. Roca b, Cristina F. Arias a, Sabino Veintemillas-Verdaguer b,
Teresita Gonzlez-Carreo b, Mara del Puerto Morales b,, Carlos J. Serna b,
Santos Maes a, Domingo F. Barber a,
a
Department of Immunology and Oncology, Centro Nacional de Biotecnologa/CSIC, Darwin, 3, Campus de Cantoblanco, E-28049 Madrid, Spain
b
Department of Particulate Materials, Instituto de Ciencia de Materiales de Madrid/CSIC, Sor Juana Ins de la Cruz 3, Campus de Cantoblanco, E-28049 Madrid, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Attachment of cytokines to magnetic nanoparticles has been developed as a system for controlled local drug
Received 10 April 2008 release in cancer therapy. We studied the adsorption/release of murine interferon gamma (IFN-) on
Accepted 31 May 2008 negatively charged magnetic nanoparticles prepared by three different methods, including coprecipitation,
Available online 7 June 2008
decomposition in organic media, and laser pyrolysis. To facilitate IFN- adsorption, magnetic nanoparticles
were surface modied by distinct molecules to achieve high negative charge at pH 7, maintaining small
Keywords:
Nanoparticle
aggregate size and stability in biological media. We analyzed carboxylate-based coatings and studied the
Drug delivery colloidal properties of the resulting dispersions. Finally, we incubated the magnetic dispersions with IFN-
Iron oxide and determined optimal conditions for protein adsorption onto the particles, as well as the release capacity at
Cytokines different pH and as a function of time. Particles prepared by decomposition in organic media and further
Immunomodulation modied with dimercaptosuccinic acid showed the most efcient adsorption/release capacity. IFN-
Surface modications adsorbed on these nanoparticles would allow concentration of this protein or other biomolecules at specic
sites for treatment of cancer or other diseases.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Cancer immunotherapy is at present an active eld of biomedical
research given its relevance in the design of new anti-tumor devices.
Treatments based on activating an anti- tumor immune response would
not only allow removal of the primary tumor, but could also provide
sufcient protection to eliminate neoplastic cells that spread to form distal
metastases [1,2]. There is considerable recent pre-clinical and clinical
evidence showing that manipulation of the innate and adaptive immune
responses to neoplastic cells can control development of tumors, leading
to their elimination [35]. This has led to the concept that immunotherapy
might be a central element in the development of anti-cancer therapies, in
combination with surgery as well as chemo- and radiotherapy [6].
Studies of the anti-tumor immune response indicate that the T
helper type 1 (Th1) lymphocyte-mediated response is much more
effective than that mediated by Th2 lymphocytes [7]. Due to their
Corresponding authors. Barber is to be contacted at Department of Immunology
and Oncology, Centro Nacional de Biotecnologa/CSIC, Darwin 3, Campus de
Cantoblanco, E-28049 Madrid, Spain. Tel.: +34 91 585 5307; fax: +34 91 372 0493.
Morales, Department of Particulate Materials, Instituto de Ciencia de Materiales de
Madrid/CSIC, Sor Juana Ins de la Cruz 3, Campus de Cantoblanco, E-28049 Madrid,
Spain. Tel.: +34 91 334 8995; fax: +34 91 372 0623.
E-mail addresses: puerto@icmm.csic.es (M. del Puerto Morales),
dfbarber@cnb.csic.es (D.F. Barber).
increased cytotoxic activity, Th1 cells have a greater capacity than Th2
cells to eradicate tumors in murine models [7]. Type Th1 responses are
characterized by the production of interferon gamma (IFN-),
interleukin-2 (IL-2) and IL-12, whereas IL-4, IL-5 and IL-10 production
is typical of Th2 responses. Administration of specic cytokines that
promote expansion and/or activation of one type of lymphocyte or
another may thus be useful in cancer immunotherapy. Many studies
point to IFN- as the most effective cytokine in tumor elimination.
Injection of IFN--blocking monoclonal antibodies into experimental
animals suppresses their ability to reject transplanted tumors [8,9].
Compared to normal mice, 129/SvEv mice decient in proteins
involved in IFN- receptor signaling develop primary tumors more
frequently, which grow more rapidly at lower carcinogen doses [10];
results were similar for IFN--decient C57BL/6 mice [11,12]. In models
of genetically induced tumor formation (p53/ mice), lack of IFN- or
of its signaling pathway proteins further enhances tumor development
[10,12]. IFN- acts at two levels; rst, it promotes the differentiation
and expansion of tumor-specic Th1 CD4+ T cells and cytolytic T cells
(CTL), and also activates macrophages [9]. Secondly, IFN- acts directly
on tumor cells, increasing their immunogenicity [8].
Local cytokine administration in the vicinity of neoplastic cells
improves the immune response to solid tumors [13]. The most
commonly used method to increase local cytokine concentration is
tumor cell transduction with cDNA encoding a cytokine of interest,

0168-3659/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2008.05.028
 R. Mejas et al. / Journal of Controlled Release 130 (2008) 168174
although this approach poses serious problems for clinical application
[14]. Synthetic polymers and microspheres generated with gelatin and
chondroitin sulfate have also been tested as controlled cytokine release
systems [1517], although to be effective, the microspheres must reach
the tumor at the appropriate concentration. Nanotechnology provides
valuable new tools that would allow controlled local release of
cytokines. One of the most promising methods for delivery of drugs
to their points of action is by binding them to magnetic particles that
are then targeted using an external magnetic eld. The rst trial of
magnetism to attract a drug to the site of action used intravenously
injected epirubicin-coated magnetic particles. After injection, a
magnetic eld was applied as close to the tumor as possible for
45 min, resulting in efcient drug administration to the site [18].
To enable a more extensive study of the IFN- effect on tumor
progression by modulating the Th1 immune response in the tumor
microenvironment, it was necessary to optimize a magnetic nanoparticle-
based system for IFN- delivery. Three different methods were used to
produce uniform magnetic nanoparticles (polydispersity degree b20%)
with different crystallinity and surface characteristics. One was a
conventional method of coprecipitation of Fe salts in water, widely used
for commercial products and for previous studies of biomolecule
immobilization. Another was a new method based on the decomposition
of an organic precursor in organic media, which assures formation of
uniform, highly crystalline particles; nally, we used the laser pyrolysis
technique, a unique method for producing dry powders in a single step
starting from a gaseous precursor [19,20]. We analyzed the adsorption/
release of murine interferon gamma (IFN-) onto these iron oxide
nanoparticles with various coatings. Particle size and crystal structure, as
well as the coating, affect the magnetic properties of the material [19] and
are therefore predicted to have an effect on its adsorption/release capacity.
2. Materials and methods
2.1. Nanoparticle synthesis
Three samples were synthesized using coprecipitation in alkaline
media of iron salts (sample C), as well as thermal decomposition in an
organic solvent (sample O) or by laser pyrolysis (sample L) of a metal-
organic precursor. The particle surface was appropriately modied in
each case to achieve high negative charge, small aggregate size and
stability in biological media.
Sample C consists of dextran-coated magnetite nanoparticles
synthesized in a single step by coprecipitation of iron (II) and (III)
salts in a basic ammonium solution in the presence of dextran [21].
Samples O and L were synthesized in two-step processes. For sample
O (DMSA-coated), hydrophobic magnetite nanoparticles were synthe-
sized by decomposition of iron acetylacetonate in phenyl ether
[22,23]; in a second step, the nanoparticles were surface modied
with DMSA to remove the oleic acid and render a negative charge at
pH 7 [24,25]. Sample L was synthesized by laser pyrolysis of an iron
pentacarbonyl aerosol following a reported procedure [26,27].
Particles were then boiled in nitric acid and iron nitrate to increase
crystallinity [28], and nally coated with one of three molecules,
phosphonoacetic acid (sample LP), citric acid (sample LC) or
carboxydextran (sample LCD) [29,30]. A control sample consisting of
iron oxide particles with a positive surface charge was prepared by
coprecipitation in the presence of aminodextran (control sample).
2.2. Structural and colloidal characterization
Particle size, shape and degree of polydispersity were analyzed by
transmission electron microscopy (TEM) using a 200-KeV JEOL-2000
FXII microscope. TEM samples were prepared by placing one drop of a
dilute suspension of magnetite nanoparticles on a carbon-coated
copper grid and allowing the solvent to evaporate at room tempera-
ture. Average particle size (DTEM) and distribution were evaluated by
169
measuring the largest internal dimension of at least 300 particles.
Data were tted with a log-normal distribution function and the
logarithmic standard deviation was obtained for the samples. The
standard deviation was calculated from the logarithmic standard
deviation as described [31].
The phase of the iron oxide particles was identied by powder X-ray
diffraction. The X-ray patterns were collected between 2070 (2)
degrees in a Bruker D8 Advance diffractometer with CuK radiation.
Crystal size (DXRD) was calculated from the broadening of the (311)
reection of the spinel structure following standard procedures.
Hydrodynamic size and evolution of the zeta potential versus pH was
determined using a Zetasizer Nano-ZS device (Malvern Instruments).
Hydrodynamic size was measured from a dilute suspension of the
sample in water at pH 7 in a standard cuvette. Zeta (Z)-potential was also
measured from dilute samples in water in a Zeta Potential cuvette with
0.01 M KNO3 as support electrolyte and at different pH values.
Thermogravimetric analysis (TG) of samples was carried out in a
Seiko TG/ATD 320 U, SSC 5200 (Seiko Instruments) to evaluate
material weight loss during heat treatment. For this analysis, a heating
rate of 10 C/min in air atmosphere from room temperature to 700 C
was applied to the sample.
Fourier transform infrared spectra (IR) of the iron oxide nanopar-
ticles were recorded between 3600 and 400 cm 1 in a Nicolet 20 SXC
FTIR to conrm the iron oxide phase, the nature of the coating and its
bonding to the surface. Samples were prepared by diluting iron oxide
powder in KBr at 2% by weight and pressing into a pellet.
Iron concentration of the samples was analyzed in a plasma
emission spectrometer (ICP; Perkin Elmer Optima 2100 DV) after
sample digestion in nitric and hydrochloric acids.
2.3. Cytokine adsorption/release
For analysis of the adsorption process, nanoparticles were added in
excess to the medium with 2 ng/ml murine IFN- (Peprotech) and
incubated (2 h, room temperature (RT), with shaking). The recombi-
nant murine IFN- is a homodimeric protein. Each subunit has 134
amino acids and a molecular weight of 15.65 kDa, and the chemical
formula is C699H1109N195O205S4. Nanoparticles were separated magne-
tically from supernatants and the supernatant (unbound IFN-) was
collected for analysis. Nanoparticles were resuspended in appropriate
medium with 1% bovine serum albumin (BSA; Calbiochem), washed
(30 min, RT, with shaking) and separated from supernatant (washed
IFN-). To determine the best conditions for the IFN- desorption
after nanoparticle resuspension, the pH of the medium was modied
between 5.5 and 9 at 0.5 unit intervals, using HCl or NaOH in 0.5% NaCl.
Finally nanoparticles were separated magnetically from the suspen-
sion and supernatants again collected for analysis (released IFN-).
Other experimental parameters were optimized for samples C and
O, including magnetic separation time (to avoid interference by
remaining magnetic nanoparticles in the medium), ionic strength,
adsorption pH, and adsorption and release times. The effect of particle
coatings on adsorption/release was then studied under the optimized
experimental conditions.
2.3.1. Effect of ionic strength of the medium on IFN- adsorption
To test the effect of the ionic strength of the medium, we used
magnetic nanoparticles, one with a weak (sample C) and one with a
strong negative surface charge (sample O). Both were incubated in
excess with 2 ng/ml IFN- in phosphate-buffered saline (PBS; Roche)
0.9% NaCl, or 0.5% NaCl (2 h, RT, with shaking), after which
nanoparticles were separated using magnets and supernatants were
collected for analysis.
2.3.2. Effect of pH of the medium on IFN- release
After incubation of dextran (C)-, and DMSA (O)-coated particles in
0.5% NaCl, pH 7 with IFN- (1 ng/ml; 2 h, RT, with shaking), particles
170 R. Mejas et al. / Journal of Controlled Release 130 (2008) 168174

Table 1
Magnetite nanoparticles prepared by various methods and coated with different molecules: structural properties of the powders and colloidal properties of the corresponding
suspensions

Sample Preparation DTEM Coating % coating Hydrodynamic Z-potential

method (nm) diameter (nm) (mV) pH 7
Control Coprecipitation 6 Aminodextran 22 160 +26
C 10 Dextran 14 100 17
O Decomposition 4.5 DMSA 24 110 34
in solution
LP Laser pyrolysis 10 Phosphonoacetic acid 35 80 45
LC 10 Citric acid 18 80 38
LCD 10 Carboxydextran 64 100 46

were separated magnetically from supernatants (1 ml aliquots,
30 min, RT). Supernatants were collected and nanoparticles resus-
pended in 0.5% NaCl with 1% BSA, and then washed (30 min, RT, with
shaking), after which nanoparticles were re-isolated and supernatants
obtained. To release IFN- from particles, washed IFN--coated
nanoparticles were resuspended in 0.5% NaCl in a pH range from 5.5
to 9 (0.5-unit intervals) and incubated (2 h, RT, with shaking), followed
by magnetic isolation of particles and supernatant collection.
2.4. IFN- detection
Unbound, washed and released IFN- were detected by enzyme-
linked immunosorbent assay (ELISA) using the Mouse IFN- ELISA Set
(BD Biosciences). Some samples were further analyzed by Western
blot with a rabbit anti-murine IFN- polyclonal antibody (0.2 g/ml,
Peprotech) and a horseradish peroxidase-coupled goat anti-rabbit
secondary antibody (0.15 g/ml, Dako).
2.5. Functional IFN- assay after adsorption/release
Murine macrophages were obtained from C57BL/6 mice by
peritoneal injection of casein (9% w/v in PBS). After 24 h, macrophages
were collected from the peritoneum by washing with ice-cold PBS in
sterile conditions. After centrifugation (350 g, 5 min) cells were
resuspended in complete RPMI medium (10% FCS, L-glutamine,
penicillin/streptomycin, Na-pyruvate, non-essential amino acids and
-mercaptoethanol), cultured in 6-well plates (2 106 cells/well in
3 ml) and incubated for 20 h in standard cell culture conditions (37 C,
5% CO2 in humidied air). Cells were washed with PBS and treated
appropriately with 10 ng/ml IFN- released from nanoparticles, IFN-
bound to DMSA-coated nanoparticles or free IFN- as positive control;
an untreated well was used as a control. After 24 h incubation, treated
cells were washed with PBS, collected and stained with anti-CD40-PE
and anti-CD11b-FITC (both from BD Pharmingen) antibodies and
analyzed by ow cytometry. All procedures using animals were
carried out with the approval of the CNB Ethics Committee following
EU guidelines.
3. Results and discussion
3.1. Synthesis and characterization of the nanoparticles
Magnetic nanoparticles were prepared by coprecipitation and by
decomposition of a metal-organic precursor, either in solution or from
an aerosol. These methods were optimized to obtain uniform particles,
with a polydispersity degree b20% and thus within the monodispersed
range (Table 1); TEM images of the iron oxide nanoparticles are shown
in Fig.1. Particles prepared by decomposition and laser pyrolysis had an
average diameter of 4.5 and 10 nm, respectively, and standard
deviations in the monodispersed range (b0.20). Iron oxide nanopar-
ticles synthesized by coprecipitation in the presence of dextran
(sample C) had a 10 nm diameter and a standard deviation of 35%.
The presence of dextran reduces and regulates particle growth, but for
sample C, particle size distribution (a consequence of the mechanism
of particle formation [19,20]) was still wider than that for the
decomposition synthesis methods (samples O, L).
We characterized the nature of the iron oxide nanoparticles and their
surface coating by X-ray diffraction and IR spectroscopy (Figs. 2, 3). All
diffraction peaks corresponded to spinel structure similar to magnetite
or maghemite, and the crystal size calculated from the peak broadening
(sample C = 10 nm, sample O = 5 nm, sample L = 9 nm) concurred with
mean TEM size (Table 1 and Fig. 2). In the low frequency IR area, two
strong absorption bands were assigned at 390 and 600 cm 1 to the FeO
stretching of iron oxide (Fig. 3). The shape and position of these bands in
samples C and O suggest that the majority of iron oxide is magnetite,
although some oxidation has taken place and maghemite is also present
[32]. The appearance of new shoulders at higher frequency in this IR
region indicated that laser pyrolysis samples L and LP consist mainly of

Fig. 1. TEM images of magnetite nanoparticles prepared by coprecipitation (sample C) and by decomposition of a metal-organic precursor, either in solution (sample O) or by laser
pyrolysis of an aerosol (sample L).
 R. Mejas et al. / Journal of Controlled Release 130 (2008) 168174
Fig. 2. X-ray diffraction patterns for magnetite nanoparticles prepared by different
methods (see Table 1 for sample description). Peaks are labeled according to the
magnetite spinel structure.
maghemite rather than magnetite; this is due to the oxidant conditions
of the recrystallization process.
In the intermediate IR region, we identied surface coating
molecules as well as synthesis-derived impurities, i.e., the sharp
band at 1380 cm 1 due to nitrate impurities remaining from acid
recrystallization (samples L, LP) (Fig. 3). From 1150 cm 1 to 1000 cm 1,
multiple strong absorption bands appear in the C and LP spectra due to
the CO single bond stretching modes of the dextran and phospho-
noacetic acid, respectively. Sample LP showed broad absorption bands
in this region, with bands at 1050 and 1010 cm 1 corresponding to the
split triply degenerate 3 (PO) vibration and at 980 cm, correspond-
ing to the 1 (PO). This assignment is commonly associated with a
monodentate bond between particle and phosphonate [33]. The small
differences in band intensity and position between this sample and
those previously reported are due mainly to pH and to phosphonate
concentration.
In the high energy range, the two absorption bands near 3000 cm 1
were due to CH stretching (anti-symmetric and symmetric). These
bands were partially masked for samples C and LP, because of the
intense OH stretching modes of dextran hydroxyl groups and water
molecules. At 1630 cm 1, we observed a weak absorption band in all
spectra due to adsorbed water. This peak was more intense in sample O
because of the overlapping of the DMSA C = O stretching mode.
Thermogravimetric analyses of the powder samples were used to
quantitate the coating (Table 1). In the case of the phosphate-coated
sample (LP), phosphate is not lost after heating up to 1000 C; analysis
of Fe content was therefore carried out by elemental analysis by ICP.
The weight percentage of coating molecules adhered to the nano-
particle surface increased from 14% for dextran-coated sample C to
24% for DMSA-coated sample O and 64% for the carboxydextran-
coated sample (LCD). This LCD N O N C coating percentage trend did not
correlate as predicted with the increase in particle size (O N C N LCD),
Fig. 3. Infrared spectra for magnetite nanoparticles coated with distinct carboxyl
molecules (see Table 1 for sample description).
171
Fig. 4. Evaluation of the Z-potential versus pH for magnetite nanoparticles coated with
different molecules (see Table 1 for sample description).
indicating that surface nature and negative charge are the principal
factors that affect the amount of coating adsorbed on the nanoparticle
surface.
3.2. Characterization of the suspensions
All negatively charged samples had similar hydrodynamic sizes
(100 nm), which is essential for comparison of the adsorption/
release process between IFN- and the coated nanoparticles (Table 1);
the amount of IFN- bound might otherwise vary as a function of
aggregate size, interfering with sample comparison. The hydrody-
namic size values were stable for months and sedimentation was not
observed.
Nanoparticle dispersion stability depends on charge and surface
chemistry, which give rise to both steric and electrostatic repulsion
[34,35]. Conventionally, a zeta potential higher than 30 mV would be
considered stable, i.e., the dispersion will resist aggregation [35].
The evolution of the Z-potential vs. pH for samples coated by dif-
ferent molecules is shown in Fig. 4. In the case of DMSA-, phos-
phonoacetic- and citric acid-coated samples (O, LP and LC), the
ionizable carboxylate groups on their surfaces govern the pH-
dependent evolution of the Z-potential, whose high values resemble
those reported in similar cases [35]. A pure polymer-coated sample,
such as dextran sample C, shows lower electrostatic stabilization
(lower Z-potential), due to the lack of ionizable surface groups; the
steric effect helps to maintain a stable dispersion, independently of
the pH. For the carboxydextran-coated sample (LCD), the charged
polymer on the surface provides both stability factors. The electro-
static effect is reected in the Z-potential values, higher than those for
sample C and similar to samples LC and LP. The pH dependence of the
Z-potential thus showed intermediate variation between sample C
and samples LC and LP. Finally, the uncoated pyrolysis sample (L)
shows the typical Z-potential values reported for bare iron oxide
nanoparticles, which is 0 at pH 7 [36].
3.3. Optimization of IFN- adsorption/release on magnetic nanoparticles
3.3.1. Magnetic particles interfere with IFN- detection in ELISA
The presence of a small quantity of magnetic nanoparticles in the
medium interfered severely with the IFN- detection assay (Fig. 5A),
underlining the need to apply magnetic separation for a time period
sufcient for complete removal of nanoparticles from the medium.
Several experiments indicated an optimal separation time of 30 min
for this study, allowing particle removal while avoiding IFN-
degradation (not shown).
3.3.2. Effect of ionic strength of the medium on adsorption
The ionic strength of the medium was predicted to have a strong
negative effect on the adsorption process. As nanoparticle/IFN-
binding is based on attraction between opposite surface charges, the
172 R. Mejas et al. / Journal of Controlled Release 130 (2008) 168174
Fig. 5. Optimization of IFN- adsorption/release from nanoparticles. (A) The presence
of nanoparticles in the medium interfered with ELISA assay for IFN- detection
(O, DMSA- coated; C, dextran-coated; LP, phosphonoacetic acid-coated; LC, citrate-coated;
LCD, carboxydextran-coated; Control, aminodextran-coated). (B) ELISA using two types of
nanoparticles showed that the amount of IFN- lost during the adsorption process was
dependent on ionic strength. (C) ELISA analysis of IFN- release from nanoparticles as a
function of pH. (D) Analysis of IFN- release as a function of time. AD show mean SD for
triplicate samples from a representative experiment of three performed.
ionic cloud that forms around the particle could interfere with
adsorption. We observed greater adsorption in low ionic strength
medium (Fig. 5B), and thus used a 0.5% NaCl solution in further
experiments. The amount of IFN- lost in the adsorption process was
greater when we used weakly charged nanoparticles, especially in
high ionic strength media (Fig. 5B), conrming the electrostatic nature
of the nanoparticle:IFN- interaction.
3.3.3. Effect of pH on IFN- release
We subsequently established appropriate pH conditions for IFN-
release from the nanoparticles. The largest amount of cytokine
was released at pH 8 (Fig. 5C). This supported the concept that
interaction is due mainly to attraction between opposite surface
charges, since the pH for release is near the theoretical isoelectric
point (Pi) for murine IFN-. As predicted, positively charged control
nanoparticles adsorbed only negligible amounts of IFN- (see
below). We observed a second release peak at low pH (5.5), which
was not used in the experiments, as it is far from physiological
conditions (pH 7.4).
Fig. 6. IFN- adsorption/release from nanoparticles. (A) In ELISA, unbound IFN- in
binding medium and washes varied according to nanoparticle surface coating. (B) ELISA
showing IFN- release at pH 8. (C) ELISA showing IFN- release from O nanoparticles at
RT or at 37 C. A, B, and C show mean SD for triplicate samples from a representative
experiment of three performed. (D) Western blot detection using sample O and control to
analyze IFN- adsorption/release. +, commercial IFN-; O-IFN-, washed IFN--adsorbed
sample O nanoparticles; O, unadsorbed sample O nanoparticles; Control-IFN-, washed
IFN--adsorbed sample control nanoparticles.
 R. Mejas et al. / Journal of Controlled Release 130 (2008) 168174
3.3.4. Effect of incubation time on IFN- release
To determine incubation times that would allow maximum IFN-
release with minimum degradation, we used IFN- bound to samples C
and O. Particles were resuspended in 0.5% NaCl, pH 8, and incubated (1, 2,
3, 5 or 20 h, RT with shaking). After magnetic removal of nanoparticles,
supernatants were analyzed by ELISA; IFN- release was most efcient at
2 h, with no notable increase thereafter (Fig. 5D). The amount of IFN-
decreased at longer incubation times (320 h), probably due to
degradation.
3.4. IFN- adsorption/release as a function of nanoparticle surface charge
The IFN- adsorption/release process was studied for several types
of negatively charged magnetic nanoparticles, using positively charged
particles (aminodextran-coated) as a control. We measured IFN- loss
as unbound IFN- in binding medium and washes. We found no IFN-
binding to positively charged nanoparticles, whereas it bound to
negatively charged nanoparticles to a greater or lesser extent,
depending on the surface coating (Fig. 6A). Attractive electrostatic
interaction was thus observed between negatively charged nanopar-
ticles and IFN- at pH 7, with DMSA (sample O)- and carboxydextran
(LCD)-coated nanoparticles showing the most efcient IFN- binding.
We tested all coated nanoparticles in the conditions described above to
determine which released the most IFN-. Both DMSA (sample O)- and
carboxydextran (LCD)-coated nanoparticles released approximately 75% of
the initial IFN- bound; both IFN- binding and release were thus efcient
using these nanoparticles (Fig. 6B). Other negatively charged nanoparticles
showed lower IFN- release values, whereas positively charged amino-
dextran particles did not release protein, as they were unable to bind it.
Considering potential in vivo applications, we tested IFN- released by
sample O at 37 C, (standard human body temperature and thus, that of cell
culture). Results were similar to those obtained at RT (Fig. 6C).
We also analyzed IFN- adsorption/release on DMSA-coated
nanoparticles by immunoblotting, using positively charged amino-
dextran-coated control particles. The presence of nanoparticles did
not interfere with IFN- detection by Western blot. Results were
similar to those obtained in ELISA (Fig. 6D).
3.5. IFN- function following adsorption/release from nanoparticles
IFN- activates macrophages, leading to increased production and
secretion of several cytokines, as well as of certain membrane proteins,
Fig. 7. IFN- function after adsorption/release. Cytometric analysis of CD40 expression
on primary murine macrophages after treatment with commercial IFN- (IFN-
control), washed IFN--adsorbed sample O (O-IFN-) nanoparticles, or IFN- released
from sample O nanoparticles (released IFN- Untreated ( ) and treated
() macrophages.
173
including CD40 [37,38]. To determine IFN- function, we used nanopar-
ticle-released IFN- to activate primary murine macrophages in vitro and
analyzed cell CD40 expression by ow cytometry. The nanoparticle
adsorption/release process did not modify IFN- function, as the
percentage of activated macrophages expressing CD40 and mean
uorescence levels were similar for IFN--adsorbed sample O nanopar-
ticles, IFN- released from sample O nanoparticles, and for the commercial
IFN- control (Fig. 7).
4. Conclusions
We successfully prepared and characterized a series of uniform
magnetic nanoparticles with different coatings and hydrodynamic sizes
around 100 nm. Various synthesis methods were developed and
adjusted to meet the requirements for biomedical applications. Changes
in synthesis method and particle coating not only altered the crystalline
nature of the particles, but also modied their capacity to adsorb and
release cytokines. We measured the IFN- adsorption/release capacity of
the magnetic nanoparticles and evaluated the possibilities for drug
delivery in biomedical applications. Particles prepared by decomposi-
tion in organic media and further modied with dimercaptosuccinic
acid showed the most efcient adsorption/release capacity. This
preparation method is one of the most promising routes for the
synthesis of nanoparticles with narrow particle size distribution and
high crystalline character. IFN- released from the particles was
functional, as it activated macrophages in vitro. These results support
the design of further experiments to test the in vivo potential of these
delivery systems for cytokine targeting to a tumor site.
Acknowledgements
We thank S. Prez-Yage for excellent technical assistance, and C.
Mark for editorial assistance. R. Mejas receives a pre-doctoral
fellowship from the Spanish Ministry of Education and Science
(MEC) FPU program; D.F. Barber held a Contract-in-Aid from the
Fundacin Cientca de la Asociacin Espaola Contra el Cancer. This
work is supported by grants by the MEC (NAN2004-08805-C04-04 to
SM and DFB) and Fundaci La Marat (053132 to SM). The Advanced
Colloidal Group at the ICMM (CSIC) is supported by the MEC
(NAN2004-08805-C04-01 and MAT2005-03179 to CJS), by the Madrid
regional government (S-0505/MAT/0194 to MPM) and by the EU
(LSHB-CT-2006-037639 to SVV). The Department of Immunology and
Oncology was founded and is supported by the Spanish National
Research Council (CSIC) and by Pzer.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jconrel.2008.05.028.
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