merging high doxorubicin loading with pronounced magnetic response and bio-repellent properties in...
TRANSCRIPT
Drug Delivery
Merging High Doxorubicin Loading with Pronounced Magnetic Response and Bio-repellent Properties in Hybrid Drug Nanocarriers Aristides Bakandritsos , * Aristeidis Papagiannopoulos , * Eleni N. Anagnostou , Konstantinos Avgoustakis , * Radek Zboril , Stergios Pispas , Jiri Tucek , Vasyl Ryukhtin , Nikolaos Bouropoulos , Argiris Kolokithas-Ntoukas , Theodore A. Steriotis , Uwe Keiderling , and Frank Winnefeld
2381© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com
DOI: 10.1002/smll.201102525
Hybrid magnetic drug nanocarriers are prepared via a self-assembly process of poly(methacrylic acid)- graft -poly(ethyleneglycol methacrylate) (p(MAA -g- EGMA)) on growing iron oxide nanocrystallites. The nanocarriers successfully merge together bio-repellent properties, pronounced magnetic response, and high loading capacity for the potent anticancer drug doxorubicin (adriamicin), in a manner not observed before in such hybrid colloids. High magnetic responses are accomplished by engineering the size of the magnetic nanocrystallites ( ∼ 13.5 nm) following an aqueous single-ferrous precursor route, and through adjustment of the number of cores in each colloidal assembly. Complementing conventional magnetometry, the magnetic response of the nanocarriers is evaluated by magnetophoretic experiments providing insight into their internal organization and on their response to magnetic manipulation. The structural organization of the graft-copolymer, locked on the surface of the nanocrystallites, is further probed by small-angle neutron scattering on single-core colloids. Analysis showed that the MAA segments selectively populate the area around the magnetic nanocrystallites, while the poly(ethylene glycol)-grafted chains are arranged as protrusions, pointing towards the aqueous environment. These nanocarriers are screened at various pHs and in highly salted media by light scattering and electrokinetic measurements. According to the results, their stability is dramatically enhanced, as compared to uncoated nanocrystallites, owing to the presence of the external protective PEG canopy. The nanocarriers are also endowed with bio-repellent properties, as evidenced by stability assays using human blood plasma as the medium.
Dr. A. Bakandritsos , E. N. Anagnostou , Prof. N. Bouropoulos , A. Kolokithas-Ntoukas Department of Materials ScienceUniversity of Patras, Rion 26504, Greece E-mail: [email protected]; [email protected]
Dr. A. Papagiannopoulos , Dr. S. Pispas Theoretical and Physical Chemistry InstituteNational Hellenic Research FoundationVassileos Constantinou Ave. 48, 11635 Athens, GreeceE-mail: [email protected]
E. N. Anagnostou , Prof. K. Avgoustakis School of Pharmacy University of PatrasRion 26504, GreeceE-mail: [email protected]
Prof. R. Zboril , Dr. J. Tucek Regional Centre of Advanced Technologies and MaterialsDepartments of Physical Chemistry and Experimental PhysicsFaculty of Science, Palacky University783 71 Olomouc, Czech Republic
Dr. V. Ryukhtin , U. Keiderling Helmholtz-Zentrum BerlinHahn-Meitner Platz 1, 14109 Berlin, Germany
Prof. N. Bouropoulos Institute of Chemical Engineering and High Temperature Chemical ProcessesFoundation for Research and Technoloy-Hellas (FORTH)P.O.Box 1414, GR-26504, Patras, Greece
Dr. T. A. Steriotis Institute of Physical ChemistryNational Center for Scientifi c Research “Demokritos”53 10, Athens, Greece
Dr. F. Winnefeld Empa, Swiss Federal Laboratories for Materials Testing and ResearchLaboratory for Concrete/Construction Chemistry8600 Duebendorf, Switzerland
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1. Introduction
Organic/inorganic hybrid colloids are supramolecular assem-
blies, chemically modifi ed in order to overcome the natural
incompatibility between organic and inorganic phases. After
being merged together, the so-formed material may exhibit
a completely different range of physical characteristics as
compared to those originally encoded in the organic and
inorganic components. The chemical design of these nano-
composites usually follows a bottom-up approach. The pro-
cedure requires the careful selection of organic and inorganic
phases, in order to achieve their successful assembly. The
organic part typically contains anchoring groups capable to
interact, more or less strongly, with the surface of the inor-
ganic core. Then, the size and chemical composition of the
inorganic core in combination with the organic capping layer
create ex-novo materials which interact with the environment
and respond to various (bio)chemical and electromagnetic
stimuli in a manner that can be potentially tailored at will.
For these reasons, search for fl exible and functional nanocom-
posites constitutes nowadays a challenging ground for basic
and applied sciences. Some successful exploitation of hybrid
colloidal systems can be found, for example, in drug delivery
and therapeutic agents, [ 1–3 ] in sensing/analysis [ 4 ] and in moni-
toring/imaging technologies. [ 5 ] The beauty of such approach
lies, from the outset, in its simplicity: starting from rather
simple molecular architectures, a variety of novel materials,
encoding a large breadth of physicochemical properties, can
be rendered functional in high-tech applications.
In the fi eld of biomedical nanotechnology and, especially,
in connection to cancer therapy, the hybrid nanocomposites
approach has the potential to express the next generation of
effective and commercially feasible drug-vectors. According
to our present knowledge, there are several factors associ-
ated to pharmacotherapies that gate their success or failure
against cancer. The effectiveness of the active drug is often
hampered by appearance of drug resistance and severe
side effects in normal tissues and cells. [ 6 , 7 ] The effi ciency in
delivering the therapeutic agents and its selectivity form the
basis for effective cancer treatment. Both factors are equally
important, in order to limit the systemic drug distribution, to
minimize undesirable side-effects and to hamper phenomena
linked to drug-poisoning and resistance. [ 8 ] In the ideal sce-
nario, the active drug must survive for quite some time in the
blood stream, in order to reach the target tissue before clear-
ance by the reticuloendothelial system, [ 9 , 10 ] and furthermore
should largely accumulate in the proper target (cancer cells
versus healthy cells). The cell-targeting process can be classi-
fi ed as molecular (i.e., a carrier expressing specifi c antibodies
that bind to tumor cell antigens), physical 2 or may benefi t
from combination of these two factors. The molecular tar-
geting strategy is per se very effective, but can suffer from i)
higher therapeutic cost associated to the molecular/synthetic
assembly of the nanocarriers and ii) the possible induction
of immune responses from the targeting moieties (e.g., anti-
bodies). [ 9 ] The physical approach is usually less selective, and
yet may still act rather effectively. The methodology relies
on the Enhanced Permeability and Retention effect. In this
setting, the statistical concentration increase of “stealth”
82 www.small-journal.com © 2012 Wiley-VCH V
nanocarriers occurs in tumor cells as result of their intrinsic
leaky vasculature. [ 10 ] The magnetic targeting approach offers
an additional physical route to pursue in cancer therapy, [ 11 ]
and utilizes magnetic nanoparticle-based drug delivery sys-
tems (MagDDS). This approach also facilitates cell-imaging
and thus highlights an advantage in the use of MagDDS
within treatment. [ 12 ] Depending on the local absorption of
the nanocarrier and upon exposure to external alternating
magnetic fi elds, MagDDS can also induce localized hyper-
thermia in the tissues. [ 13 , 14 ] The process may be translated
into an increased sensitivity of the cancer cells to the drug
and may as well limit cell proclivity towards development of
drug-resistance. [ 15 ]
Despite a number of advantages associated to the use of
MagDDS, rational design of effective nanocomposites still
poses quite some challenges. The ideal MagDDS should in
fact possess fi nely-tuned magnetic properties, should have
the ability to carry effective and large payloads of the desired
anticancer drugs, and should allow accumulation of the
DDS at the tumor site. The consequent drug release at the
appropriate time and rate would constitute then the ultimate
goal. Thus, it is not surprising that extensive employment of
MagDDS in pharmacotherapies are today still in infancy. In
order to fi nely-tune the magnetic properties of MagDDS it is
necessary to tailor the nanocrystallite’s size [ 16 ] and control the
magnetic material content. So far, clinical trials demonstrated
that increasing the drug-delivery system’s (DDS) response
upon manipulation with external magnetic fi elds represents
a hard reach. The limitations encountered are associated to
the physical (magnetic) characteristics of the nanocompos-
ites, as well as to the strength of the external magnetic fi elds
required to trigger the responsive process. [ 17 , 18 ] Nonetheless,
the synthetic effort pursued by several groups in this fi eld and
examination of several (macro)molecules potentially capable
to decorate magnetic nanocrystallites [ 17 , 19–32 ] led, recently, to
the assembly of MagDDS coated with bio-repellent external
protecting shells. [ 25–32 ] The addition of bio-repellent phases
renders the hybrid systems well suited for use in biological
environments. Furthermore the organic phase can potentially
modulate release of the bioactive compound. These novel
systems were fabricated either by encapsulation of magnetic
iron oxide nanocrystallites (MIONs) in silica, [ 26 ] in micellar
systems, [ 26–28 ] or by direct functionalization of the MION’s
surface with various polymers. [ 29–32 ]
In this work we report the assembly and the characteri-
zation of a novel MagDDS, engineered to function as drug-
nanocarrier. This novel nano-assembly combines in a unique
way bio-repellent properties, high magnetic response and
excellent loading capacity for the anticancer drug doxorubicin
(Dox), among other related materials in the fi eld. The nano-
composite utilizes a graft-type copolymer, poly(methacrylic
acid)- graft -poly(ethyleneglycol methacrylate), (p(MAA -g- EGMA)), that acts as corona for the MIONs. The latter were
grown via an alternative single ferrous precursor hydrolytic
route, which secures optimum size and thus magnetic proper-
ties. The organic shell brings to the MIONs stability in biolog-
ically relevant environments, drug loading and bio-repellent
characteristics. The graft-type confi guration was selected in
the design as alternative to the block-type arrangement. [ 30 ] In
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High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
Figure 1 . Molecular structure (A) and cartoon drawing (B) of the PEG-branched copolymer of poly(methacrylic acid)- graft -poly(ethyleneglycol methacrylate).
this way, the many PEG branches attached to the polymeric
backbone form a motif that resembles the proteoglycan
structure [ 33 , 34 ] seen in the extracellular cell-matrix. [ 35 ] Fur-
thermore, the polymer choice leads to assembly of nanocom-
posites with high PEG density, [ 36 , 37 ] which have been found
to endure long time in the blood stream. [ 38 , 39 ] Our hybrid
system, MagP(MAA -g- EGMA), in fact revealed a pro-
nounced resistance to protein-induced aggregation, even after
long incubation (24 h) with human serum. The results showed
the ability of MagP(MAA -g- EGMA) to withstand well the
unavoidable interference brought by protein binding and/or
interactions with serum proteins. The selected nature of the
polymeric corona should allow incorporation of other type
of drugs as well, such as for example those bearing amino-
groups. The added feature grants to MagP(MAA -g- EGMA)
a certain degree of plasticity in drug-binding, and thus poten-
tially renders this synthon a fl exible drug-carrier.
2. Results and Discussion
2.1. The Assembly Process of Size-Controlled Magnetic Nanocarriers
The synthesis of MagP(MAA -g- EGMA) hybrid system
employed alkaline hydrolytic condensation of a single fer-
rous precursor into MIONs [ 21 , 40 ] in presence of the surface
functionalizing polymer p(MAA -g- EGMA) (see Figure 1
and Table 1 for structural details), which reacted with MIONs
by a simple self-assembly process. [ 41 ] The methacrylic acid
mono mers interact during nanocrystallites’ growth through
the carboxylate groups with the Fe ions situated on the
MION`s surface. Clear evidence of such interactions have
been probed in detail in a previous publication, for the case
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinhe
Table 1 . Structural specifi cations of p(MAA -g- EGMA).
M w a) n b) m b) p b) MAA [wt.%]
PEG [wt.%]
mmol of anionic sites/ g of solid
67 000 77 13 102 12.4 87.6 1.1 × 10 − 3
a) weight-averaged molecular weight; b) number of monomers based on M w .
Figure 2 . Intensity-the hybrid colloids.
small 2012, 8, No. 15, 2381–2393
of another polycarboxylated polymer. [ 21 ]
It would be interesting to comment
here on the assembly mechanism of the
reported nanocarriers and note that
such type of graft- or block-copolymers
tend to self assemble into nanocolloids
when appropriate cations are present [ 42 ]
(i.e., Ca + 2 ), interacting electrostatically
with MAA segments. It could be con-
sidered that in the present case, the self-
assembly directing agent is the inorganic
nanoparticle itself. The favorable interac-
tions with the carboxylate groups of the
copolymer lead to the self-assembly of
the polymeric chains around one, or more
nanoparticles in the case of the clustered
colloids. This is very reminiscent and sim-
ilar to the complexation/micellization by cations, just if the
nanoparticle is considered as an artifi cial atom (cation) or
clustering site, in general. The evidence of this organization
is discussed in the analysis of small-angle neutron scattering
measurements.
Depending on temperature, reaction time employed and
extend of centrifugation steps (see Experimental Section and
Scheme S1 in the Supporting Information), three different
sized nanocarriers were isolated. The mean hydrodynamic
diameters, as probed with dynamic light scattering, were
found to vary signifi cantly, from large, D h = 85 ± 2 nm in the
isolated fraction coded as MagP(MAA -g- EGMA)1, medium
size with D h = 63 ± 2 nm in MagP(MAA -g- EGMA)2, to
rather small with D h = 41 ± 1 nm in MagP(MAA -g- EGMA)3.
The intensity-weighted hydrodynamic diameter distributions
for the nanocarriers are shown in Figure 2 (volume- and
number-weighted are also available in Figure S1 in the Sup-
porting Information). The observed dependence of D h on the
extent of the centrifugation steps, points towards materials
prone to self-accumulate in multiple MION clusters, where
the carboxylate residues of the polymer backbone act as
bridges for the otherwise isolated islands of material.
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weighted hydrodynamic size distribution graphs of
A. Bakandritsos et al.
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Figure 3 . a) Zeta-potential ( ζ p ) response upon pH variation of the solvent (1 mM NaCl in H 2 O), b) hydrodynamic diameter ( D h ) and ζ p (inset) response of MagP(MAA- g -EGMA)3 upon variation of the NaCl concentration in aqueous solutions, c) TGA footprints recorded for the plain polymer and the hybrid colloids. The trend of MagP(MAA -g- EGMA)1 and MagP(MAA -g- EGMA)2 exactly overlapped. d) D h values of the hybrid colloids versus pH variation in H 2 O. Standard deviations on individual measurements are shown by vertical bars. (n = 3). The solid line is a guide for the eye.
2.2. Stability Aspects and Thermogravimetric Analysis
The successful assembly of MION cores with the copolymer
was assessed by deriving the zeta potential response ( ζ p )
versus pH for two systems: uncoated MIONs and coated
particles characterized by smaller size, MagP(MAA -g- EGMA)3, ( Figure 3 a). Uncoated MIONs exhibit isoelectric
point (IEP) at pH ∼ 6.5, as previously reported in litera-
ture. [ 43 ] The IEP of MagP(MAA -g- EGMA)3 was indeed very
different and shifted down to pH ∼ 4.2, indicating changes
in surface chemistry and properties. The modulation of IEP
values towards regions distant from those considered physi-
ologically relevant, is an important parameter to address in
the post-design process, since it defi nes the viability of the
nanocomposite to act as drug-delivery-system. At higher pHs,
ζ p became negative, while at pH lower than 4.2 ζ p turned
positive. The negative ζ p is associated to ionization of the
polymer’s carboxylate groups, occurring at high pH values,
but also to negatively charged–Fe-O − groups present on the
iron oxide surface. The positive ζ p values at pH < 4.2 can
4 www.small-journal.com © 2012 Wiley-VCH
be associated to protonation of the–Fe-OH groups
(–Fe-OH 2 + ). [ 43 ] At this pH and below, the majority of the
carboxylate residues are protonated in the polymer as well,
therefore they do not have charge (–COOH, pKa = 5.6). [ 30 ]
Connecting the present results to previous ones, when MIONs
were coated with 90 kDa sodium carboxymethylcellulose [ 21 ]
(NaCMC) (Figure 3 a, MagCMC), ζ p was found negative
throughout the whole pH range. This, from one hand, may be
attributed to the higher percentage of carboxylates remaining
unprotonated in NaCMC, due to their lower pKa value
(pKa ∼ 3–4 [ 44 ] ). On the other hand, the signifi cantly higher
amount of carboxylates per MIONs and the distance of the
shear-plane (where ζ p is measured) from the MION surface
should be also considered for the case of MagCMC. More
specifi cally, based on calculations from the molecular struc-
ture of the polymers and from the polymer/MION weight
ratios as obtained from thermogravimetric analysis, there are
13 and 30 mmol of carboxylates per 100g of hybrid colloids
for the MagP(MAA -g- EGMA) and MagCMC respectively.
In addition, NaCMC is a linear semi-rigid polyelectrolyte
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High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
with M w = 90 kDa, while P(MAA- g -EGMA) is branched with
M w = 67 kDa with a more streamline and fl exible charged
backbone. Thus, in the latter case, the shear-plane must be sit-
uated much closer to the surface of the MIONs and, in turn,
the positive surface charge of the MIONs at low pH can be
more effectively expressed.
Enhancement of the surface properties of MagP(MAA -g- EGMA)3 emerged more clearly by following the D h
response versus ionic strength of the medium (H 2 O), which
contained NaCl salt in increasing amount (from 0 to 2 M).
Figure 3 b illustrates the results obtained for plain MIONs,
MagCMC and MagP(MAA -g- EGMA)3. Uncoated MIONs
and MagCMC were rather responsive to NaCl, as evidenced
by the fast fl occulation and then precipitation of the sus-
pended materials due to charge screening effects induced
by Na + . On the contrary, MagP(MAA -g- EGMA)3 remained
nearly unaffected up to 2 M NaCl. Only a minor increase
in D h ( ∼ 110 nm) was observed in the range 0.02–2 M, but
without appearance of macroscopic sign such as turbidity of
the solution. The zeta-potential of MagP(MAA -g- EGMA)3,
as shown in the inset of Figure 3 b, moved towards values
closer to zero, as –COO − were increasingly screened by Na +
cations. The stability of the system was also probed in phos-
phate buffer saline (PBS), as alternative medium. In this
environment, MagP(MAA- g -EGMA)3 remained very stable
for at least 48 h, and only after that a minor increase of par-
ticle size was observed, of about 10 ± 2 nm, thus reaching an
approximate dimension of 50 nm. At present, it is unclear
why PBS exerts such small effect on the colloidal suspen-
sion compared to NaCl, however, we do speculate that the
phenomenon could mirror different extend of chain hydra-
tion in response to different salt environments. Adsorption of
phosphate groups from PBS could be another reason of the
stability, as suggested by one of the reviewers.
The hybrids MagP(MAA -g- EGMA)1, 2, and 3, as well
as the parent copolymer p(MAA -g- EGMA) were subjected
to thermogravimetric analysis (TGA), under N 2 stream.
The recorded trends (mass change versus T ) are shown in
Figure 3 c. The copolymer p(MAA -g- EGMA) gave a residual
weight of 8.8 wt.% at 500 ° C, once compared to its initial
mass. Taking into consideration such contribution, an
averaged 11 ± 3 wt.% polymer content was extracted for
MagP(MAA -g- EGMA) samples.
In order to operate as functional drug-carrier, the nano-
system must exhibit a large degree of stability in regions
of pHs close to physiological. The calculated D h responses
of MagP(MAA-co-EGMA)3 towards pH variations of the
medium are illustrated in Figure 3 d. The engineered nano-
particles endured well in a broad range of pHs (5 < pH < 11).
However, down to more acid environment (pH = ∼ 4.2 ± 0.2)
a very sharp increase suddenly appeared, but as soon as
the pH was further lowered, it fl attened back to the values
witnessed at high pHs. It is worth noticing that the sudden
D h increase emerged in the same pH region of the IEP of
the system, around pH ∼ 4. Thus, it is tempting to specu-
late that the two phenomena could be linked to each other.
Other processes may however contribute to the witnessed
upsurge of D h . For example, amplifi cation may arise from an
© 2012 Wiley-VCH Verlag Gmbsmall 2012, 8, No. 15, 2381–2393
increased number of hydrogen bonding interactions among
PEG oxygen atoms and hydrogen residues belonging to
protonated carboxylates in PMAA, [ 45 ] which in fact exhibit
critical pH close to 4. [ 46 ] It is also possible that the system
undergoes to some kind of micellization processes, similar
to what is observed in analogous graft- and block-copoly-
mers, which tend to self-assemble into micelles at low
pH values. [ 46 ]
2.3. Structural Characterization of the Hybrid System by TEM and XRD
TEM analysis of the three isolated preparations of
MagP(MAA- g -EGMA), which differ considerably from
each other in hydrodynamic sizes, allowed to dissect the
size of the magnetic nanocrystallites. It emerged that the
MIONs consistently have a number-mean crystallite size
of 13.5 ± 3 nm. Figure 4 a and b show representative TEM
images for hybrids MagP(MAA- g -EGMA)3 and 1 respec-
tively. All three hybrids displayed similar characteristics in
the TEM images with no aggregates or clustered nanoc-
rystallites. This observation leads to the conclusion that
in the case of the multi-core colloids (i.e., MagP(MAA-
g-EGMA)1 and 2) the nanocrystallites are held together
though polymer chains in a bridging confi guration. The
crystalline nature of the MIONs was evident from the
appearance of the atomic planes in the higher resolution
TEM image shown in Figure 4 c. Our fi ndings indicate that
the synthetic procedure employed and irrespective of the
diverse temperature used, did not affect the fi nal dimen-
sion of the inner core material but rather the extend of
clustering and in turn the hydrodynamic diameter of the
hybrids. For comparison, size estimation was also per-
formed by XRD measurements (Figure 4 e), using the full
width at half maximum (FWHM) of the [311] diffraction
peaks of the spinel structures. It was found that no differ-
ences were present between the FWHM of the peaks among
MagP(MAA- g -EGMA)1, 2 and 3,all being consistent with
∼ 15 ± 1 nm particle sizes, according to Scherrer equation.
The values derived from XRD measurements are slightly
higher than those estimated from TEM images. This is due
to the fact that XRD peak intensities arising from larger
nanocrystallites (i.e., diffraction arising from more atomic
planes) are higher than those originating from smaller
nanocrystallites. The mean particle size estimated from
XRD is therefore closer to a volume- or weight-averaged
mean, while that estimated from TEM is number-weighted.
Additional XRD data have been added for comparison in
Figure 4 e, regarding a sample produced by conventional
co-precipitation procedure (mean particle size = ∼ 6 nm).
The comparison helps to clarify the signifi cant advantage
of the single-ferrous-precursor-route, where MIONs may
have sizes signifi cantly higher than those obtained from
the co-precipitation route, particularly when the latter
process takes place in presence of polymeric ligands such
as PMAA/PEG block- or co-polymers, [ 30 , 47 , 48 ] and other
polycarboxylate/PEG based polymers. [ 29 , 49–51 ]
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Figure 4 . a,b) Typical TEM images from the hybrids MagP(MAA -g- EGMA)3 (a) and 1 (b). c) High-resolution image showing that even the smaller particles are crystalline. d) Representative size distribution diagram and the log-normal fi t as obtained from the size of ∼ 1000 nanocrystallites from several micrographs. e) XRD patterns focused on the 311 diffraction peak from the MIONs spinel structure.
2.4. Structural Characterization of the Hybrid System by SANSPOL
The overall structural organization of MagP(PMAA- g -
EGMA)3 was further studied with polarized small-angle
neutron scattering (SANSPOL). The SANS experiments
gave additional information about particle sizes and internal
organization of the nanocarriers. Figure 5 a shows fi ts of
I − ( q ⊥ H ) (blue circles) and I + ( q ⊥ H ) (red circles) data sets
based on a model made by one inner core and two shells of
lower scattering length densities. In addition, inset of Figure 5 a
shows the scattering length density profi le of the spherical
nanoparticles in function of the distance ( r ) from the centre.
The two shells correspond to non-magnetic materials (i.e.,
MAA- g -pEGMA coating) and thus magnetic scattering is
not expected from them. The fi tting parameters are collected
together in Table 2 .
The core diameter was estimated at 12.4 nm ± 0.2, while
the inner shell had a thickness of 6.7 ± 0.3 nm and the second
shell (the outer layer), composed of 90% v/v water, had a
thickness of 11 ± 2 nm. The resulting diameter of the hybrid
colloid, based on this model is estimated at 48 ± 2 nm. The
results derived from TEM provided an averaged 13.5 nm for
the MIONs diameter, while from DLS measurements the
colloid’s diameter was estimated at ∼ 41 nm (Figure 2 ), thus
SANS results corroborate fairly well with the other tech-
niques. The recorded relatively high scattering length den-
sity (SLD) of the inner shell ( η = ∼ 1 × 10 10 cm − 2 ) suggests
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a layer rich in polycarboxylate segments, which reside near
the surface of the inorganic core. The outer layer with a very
low SLD ( − 0.45 10 10 cm − 2 ), but higher than that of water, can
be attributed to a shell rich in PEG segments. Although with
caution and based on the approximate volume fractions of
the materials occupying each shell, the obtained hybrids can
be schematically described by the model shown in Figure 5 b.
2.5. Magnetic Characterization of the Nanocarriers
The static magnetic properties of the hybrid colloids,
MagP(PMAA- g -EGMA)1, 2 and 3, were assessed with room-
temperature hysteresis loops, as shown in Figure 6 a. At T =
300 K, they did not show hysteresis, as evidenced by zero values
of the coercivity and remnant magnetization. Thus, all investi-
gated samples behaved as superparamagnets at room temper-
ature, under the characteristic measuring time of the SQUID
technique ( ∼ 10 s). The maximum magnetization ( M max ) of the
MagDDS, derived from the corresponding hysteresis loops at
7 T, was found to be ∼ 74, ∼ 72, and ∼ 67 A m 2 kg − 1 (or emu g − 1 )
for the MagP(MAA- g -EGMA)1, 2, and 3, respectively.
In fact, 95% of the M max value is attained at ∼ 0.5 T, which
is a value comparable with the induction of the magnetic
fi eld produced by a common laboratory Nd-Fe-B magnet.
Considering that the portion of the magnetic inorganic com-
ponent of the hybrid colloid is 89 ± 3 wt.%, then the room-
temperature saturation magnetization ( M S ) value expressed
with regard to the magnetic material’s mass is ∼ 82, ∼ 80, and
erlag GmbH & Co. KGaA, Weinheim small 2012, 8, No. 15, 2381–2393
High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
Figure 5 . a) SANSPOL scattering intensity from the hybrid colloids together with data fi t (solid lines). The inset shows the scattering length density profi le. b) The estimated model of MagP(PMAA- g -EGMA)3 colloid based on SANS fi tting results.
Figure 6 . a) Room-temperature hysteresis loops of the studied MagDDS and b) magnetophoretic response of the MagDDS showing the effect of clustering on their velocity.
∼ 74 Am 2 kg − 1 in the case of MagP(MAA- g -EGMA)1, 2, and
3, respectively. This value is somewhat lower than the value
reported for macroscopic magnetic iron oxides (i.e., ∼ 85 and
∼ 95 A m 2 kg − 1 for bulk γ -Fe 2 O 3 and Fe 3 O 4 , respectively). This
reduction in M S is observed in nanoparticle systems and is
mainly ascribed to the presence of a magnetically disordered
surface cell of the MIONs. Nevertheless, all studied hybrid
colloids exhibit a strong magnetic response at low applied
magnetic fi elds, comparable with that observed for bulk iron
oxide phases. In addition, the presence of coating agents may
signifi cantly reduce the possibility of evolution of magnetic
interparticle interactions, causing aggregation of MIONs and,
thus, degradation of their magnetic properties.
© 2012 Wiley-VCH Verlag Gmb
Table 2. The relevant physical parameters obtained by fi tting the SANS d
Core radius R c [nm]
Thickness of inner shell d 1 [nm]
SLD a) of inner shell ρ 1 [10 10 cm − 2 ]
Thicknessd
6.2 ± 0.1 6.7 ± 0.3 0.99 ± 0.08 1
a) scattering length density.
small 2012, 8, No. 15, 2381–2393
The magnetic response of the hybrid systems was fur-
ther evaluated with magnetophoretic experiments. Despite
the similar M S values, the results from magnetophoresis
(Figure 6 b) evidenced that the cluster size of the nanocar-
riers (indexed by D h ) signifi cantly affects the velocity of
the particles in the magnetic fi eld gradient. The decrease in
absorbance is connected to the reduction in concentration
of the magnetic material in the cuvette. The steeper is the
reduction, the higher the velocity of the particles attracted by
2387www.small-journal.comH & Co. KGaA, Weinheim
ata using the core–shell–shell model.
of outer shell 2 [nm]
SLD of outer shell ρ 2 [10 10 cm − 2 ]
Number density of scattering particles N p [10 15 cm − 3 ]
1 ± 2 − 0.45 ± 0.03 2.6 ± 0.2
A. Bakandritsos et al.
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Figure 7 . D h changes of uncoated or polymer-coated MIONs before and after incubation with human blood plasma.
the magnetic fi eld. Clearly, as the hydrodynamic size reduces,
and thus the number of magnetic cores in the colloidal clus-
ters, the nanocarrier’s velocity is reduced, despite the fact that
a smaller hydrodynamic diameter should produce a smaller
viscous drug on the particle ( F drag = 3 π η D h u , where η is the
viscosity and u the velocity). In support to this interpretation
of the results, and in order to lift any ambiguities due to small
variations of the M S of the hybrids, multi-core clusters with
D h = 130 nm composed from 6 nm nanocrystallites were also
subjected to the same evaluation. Undoubtedly, 6 nm parti-
cles feature signifi cantly smaller M S than particles larger than
10 nm, [ 16 ] as it has been also experimentally proven in our lab
from the data shown in Figure 6 a. Nevertheless, these multi-
core clusters displayed higher magnetophoretic mobility than
MagP(PMAA -g- EGMA)3, a system composed from 13.5 nm
particles, but with signifi cantly smaller D h = 41 nm, thus
smaller amount of MIONs per colloidal entity. Riffl e and co-
workers [ 50 ] noted that the use of magnetic fi eld gradients for
particle manipulation is more effi cient for clusters rather than
for single nanoparticles. The Hatton’s group observed that
clustered colloids based on polymer functionalized MIONs
were attracted more effi ciently by magnetic fi elds generated
on a magnetic separation column. [ 47 , 52 ] They noticed that the
difference between the two fractions of the magnetic col-
loids after the magnetic-column separation process was not
only due to cluster size, but also due to the mean size of the
primary magnetic nanocrystallites forming each cluster. The
present results, referring to clusters of different sizes but with
almost identical mean size of the primary magnetic nano-
crystallites, to our opinion, unequivocally underpin the impor-
tance of the number of MIONs participating in each colloidal
entity, which is a matter of considerable research. [ 53–55 ] Fur-
ther discussion on the magnetophoresis results is available in
Supporting Information.
2.6. The Interactions of MagP(PMAA- g -EGMA) Hybrids with Human Plasma and Doxorubicin
The bio-repellent properties of the hybrid colloids were
addressed by monitoring alterations of D h after interac-
tion with human plasma, in the time-window of 24 h. Any
modifi cations observed in the D h were attributed to inter-
actions occurring between plasma proteins and the poly-
meric corona of the hybrids. Such interactions may induce
D h increase either in response to protein binding and/
or due to aggregation processes. The screening results are
www.small-journal.com © 2012 Wiley-VCH V
shown in Figure 7 . Additional measurements performed on
uncoated MIONs and MagCMC coated nanocomposites
(prepared as previously described [ 21 , 56 ] ) have been added in
the fi gure for comparison. Uncoated MIONs strongly inter-
acted with serum proteins, resulting in formation of large
aggregates (Figure 7 a). Blood serum induced D h increase
on MagCMC as well, but contrary to uncoated MIONs, the
particle size distribution remained monomodal (Figure 7 b).
The MagP(PMAA- g -EGMA)3 hybrid instead, revealed a
pronounced resistance to protein-induced size increase (or
aggregation), and only marginal increase of D h was recorded.
This observation indicates the ability of MagP(PMAA- g -
EGMA)3 to withstand well interactions with serum proteins,
for prolonged time, and thus highlights the nanoparticles
potential to act in vivo as carriers.
MagP(PMAA- g -EGMA)3 was then screened against
binding of the drug doxorubicin (Dox). Dox is a well-known
potent anticancer drug, which acts by interacting with the
cell DNA by intercalation, stabilizing the topoisomerase II
complex and preventing the replication processes of cells.
The development and validation of a standard protocol for
Dox loading is described in detail in the Experimental Sec-
tion. The hybrids MagP(PMAA- g -EGMA)1 and 3 were
incubated with increasing concentration of Dox solutions,
which are the salt-forms commonly used in pharmacotherapy
for the intravenous administration of this drug. The graphs
shown in Figure 8 indicate the high drug loading ability of
MagP(PMAA- g -EGMA)1 and 3, which was determined at
22 ± 2 and 15 ± 2 wt.% respectively. The recorded values are
particularly high compared to the majority of MagDDS found
in literature. In particular, Guo and co-workers, [ 30 ] directly
functionalized the surface of the MIONs, similarly to us,
but using different methods and coating materials (a block-
copolymer followed by co-precipitation route). Loading of
Dox was reported in that case as high as 20 wt.%, but the
nanoparticles featured rather low M S value ( ∼ 8 A m 2 kg − 1 ).
Lee and co-workers [ 29 ] demonstrated that direct function-
alization of the MIONs surface with polymers (poly(3-
(trimethoxysilyl)propyl methacrylate- r -PEG methyl ether
methacrylate- r - N -acryloxysuccinimide), led to appreciable
retention of the magnetic properties of the hybrid system,
which unfortunately exhibited low drug loading capacity
( M S = 45 A m 2 kg − 1 , 2 wt.% loading in Dox). Other methods
have been well explored in literature, and were based
on encapsulation of the magnetic nanoparticles in other
media (for example silica, [ 25 ] polymeric micelles [ 26 , 28 ] or
liposomes [ 57 ] ), but usually resulted into very low magnetic
erlag GmbH & Co. KGaA, Weinheim small 2012, 8, No. 15, 2381–2393
High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
Figure 8 . Doxorubicin loading on the hybrid nanocarriers a) MagP(MAA -g- EGMA)1 and b) MagP(MAA -g- EGMA)3. ( n = 5) Insets depict cartoons of the nanocarriers’ possible structure.
Figure 9 . Doxorubicin release kinetics from the nanocarrier (MagP(MAA -g- EGMA)1) in various release media.
material contents and thus limited magnetic responses.
Recently, Quan and co-workers [ 58 ] succeeded in assembling
one hybrid system made by Dox/Fe 3 O 4 (IONP)/HAS (Human
Serum Albumin), which showed high drug loading capacity
with respect to IONP (1/2/20, w/w/w, i.e., 4.3 wt.% in Dox
and 8.7 wt.% in magnetic material) and MRI ability in vivo.
Although analysis of the magnetic properties of the nano-
particles were not carried out, the M S of this system would
be limited below 10 A m 2 kg − 1 , due to the magnetic mate-
rial content. Therefore, based on the pertinent results in this
research fi eld, the diffi culty in merging successfully high drug
content and magnetization is quite evident. However, our
system harmoniously combined three important properties:
i) limited interactions with human blood plasma proteins,
ii) high drug loading ability and iii) high magnetic response and
M S . Even though delivery experiments on cancer cell lines for
the present MagDDS have not yet been performed, in a pre-
vious work we have demonstrated that Dox loaded MagDDS
based on polycarboxylate-coated MIONs did not affect
drug’s activity against HT29 cancer cells. [ 21 ] Regarding the
higher loading capacity of MagP(PMAA- g -EGMA)1, that
© 2012 Wiley-VCH Verlag Gmbsmall 2012, 8, No. 15, 2381–2393
is the mutli-core hybrids, at the moment, it could be hypoth-
esized that the local interconecting polymer network (practi-
cally similar to a nanogel) is able to host higher amounts of
Dox. But this requires further investigation in order to better
conclude on the participating mechanisms.
Finally, the extent and rate of the drug release from the
MagP(MAA -g- EGMA)1 system was evaluated in distilled
water, phosphate buffer saline (PBS) and bovine serum
albumin (3% w/v) dissolved in PBS. A slow and partial
release of the drug is observed in all cases ( Figure 9 ). The sig-
nifi cant increase in the release from water to PBS is evidence
of the electrostatic interactions between the drug and the
carrier (cations in PBS antagonize doxorubicin for the ani-
onic carboxylate sites of the carrier). A further slight increase
in albumin (known for its amphiphilic nature) suggests that
the drug interacts with the nanocarrier via hydrophobic
inter actions as well. Drug release mechanisms will be dis-
cussed in detail in a follow-up publication.
3. Conclusion
The derivatization of magnetic iron oxide nanocrystal-
lites with the graft copolymer poly(methacrylic acid)- graft -poly(ethyleneglycol methacrylate) of M w = 67 kDa and with
PEG weight content of ∼ 88% and M w = 4.5 kDa, appears
to be an effective route in the assembly of magnetic drug
delivery systems for doxorubicin, where large drug-loading
(22 ± 2 wt.%) and high response to magnetic drug forces
are well expressed. Slow drug release is also another key
attribute observed. Despite the random grafting of PEG
chains on the poly(methacrylic acid) backbone, neutron
scattering experiments indicated that the polymer is organ-
ized around the magnetic core in a non-random way. With
appropriate folding, the methacrylic acid segments are pop-
ulating the area around the magnetic nanocrystallite, while
the PEG grafted chains are protruding towards the aqueous
2389www.small-journal.comH & Co. KGaA, Weinheim
A. Bakandritsos et al.
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full papers
environment forming a protective canopy. The ability of thepolymer to assemble in such a fashion is probably the cause
for their high stability at high concentration of salt, as well
as for the observed bio-repellent properties. Displaying such
properties while retaining low polymer content ( ∼ 11 wt.%) is
another key feature for attaining enhanced magnetic proper-
ties. From the present study it is also deduced that improve-
ment on the magnetic properties of such systems can be
realized by adopting simple aqueous synthetic routes that
can lead to magnetic nanocrystallites whose sizes are closer
to their superparamagnetic limit. We have also shown that
magnetic response can be also increased upon formation of
nanoassemblies made by multi-core colloids (clusters of mag-
netic nanocrystallites), as compared to single-core colloids.
4. Experimental Section
Materials : FeSO 4 · 7H 2 O (99 + %, Chem Lab NV), NH 4 OH solu-tion (30%, for analysis, Carlo Erba). Water used in the experiments was of ∼ 1 μ S cm − 1 conductivity. Human blood plasma was a kind gift from the University Hospital of Patras. Doxorubicin hydrochlo-ride was supplied by LC laboratories (USA). Random copolymers of poly(methacrylic acid)-graft-poly(ethyleneglycol methacrylate) were synthesized as previously described. [ 59 ]
Synthesis of Magnetic Hybrid Colloids : FeSO 4 · 7H 2 O (0.72 g) was dissolved in H 2 O (10 mL) where HCl was added (37%, 0.1 mL). P(MAA- g -EGMA) (0.15 g) was dissolved in H 2 O (30 mL) and the two solutions were mixed in a spherical fl ask under magnetic stir-ring. In this mixture a solution of NH 4 OH (30%) was added (2 mL), and left under refl ux for 24 h (MagP(MAA- g -EGMA)1). In a sepa-rate preparation, the temperature was gradually raised to 50 ° C within a period of 30 min, and then the mixture was kept at this temperature for further 50 min (MagP(MAA- g -EGMA)2). After this time, the reaction fl ask was removed from the hot plate and left to cool down to room temperature. The colloids were then centrifuged (Heraeus, Biofuge pico, RCF = 11 000 g or 13 000 rpm) for 2 h in order to separate the functionalized nanocrystallites from the free polymer and ions (sulfate and ammonium), which were left in the supernatant (supernatant conductivity ∼ 15 mS cm − 1 ). The pellets were re-suspended in H 2 O (24 mL), stirred (30 min) and sonicated (10 min). A second centrifugation and supernatant decanting was performed and the fi nal pellets were resuspended in H 2 O (24 mL) and transferred into a conical glass fl ask, stirred (30 min), soni-cated (10 min) and centrifuged mildly (20 min, ∼ 2100 g ) to remove larger aggregates which were collected in the pellet. After centrifu-gation, the supernatants (MagP(MAA- g -EGMA)1 & 2) were collected and stored at 7 ° C for further use (conductivity ∼ 0.2 mS cm − 1 ). Another part of the MagP(MAA- g -EGMA)2 product was centrifuged at higher gravitational force (3200 g , 20 min) in order to isolate another fraction of the product (MagP(MAA- g -EGMA)3) and com-pare it with the fi rst one. The experimental procedure is schemati-cally illustrated in Scheme 1 in the Supporting Information. The fi ne dispersibility of the hybrid colloids was macroscopically mani-fested by the absence of turbidity (the turbidity test was performed after appropriate dilution ( < 0.05% w/v) due to the very dark colori-zation of the suspension).
Dynamic Light Scattering (DLS) and Electrokinetic Measure-ments : DLS was performed on aqueous dispersions of ∼ 0.08%
390 www.small-journal.com © 2012 Wiley-VCH V
w/v in magnetic material (expressed as Fe 2 O 3 ), where the pH and NaCl concentration were pre-adjusted at the desired values. The samples were left to age for 24 h before measurements for equilibration, unless otherwise specifi ed. Electrokinetic measure-ments for the determination of mobility and zeta-potential values of the suspensions were performed with a Malvern Instrument ZetaSizer Nano equipped with a variable temperature compart-ment and with a 4 mW He-Ne laser, operating at a wavelength of 633 nm and having an avalanche photodiode as a detector. Data were acquired with laser Doppler velocimetry and with the phase analysis light scattering mode (PALS), after equilibration at 25 ° C. Reported zeta-potential values are the sum of 50 runs. Dynamic light scattering was performed using the same instrument, where scattered light was collected at a fi xed angle of 173 ° . The hydro-dynamic diameters reported are the mean of 4 measurements and each measurement was the sum of 12 correlograms and fi t-ting procedures. The cumulants analysis was applied and the zero-average values are reported as the intensity-weighted mean hydrodynamic diameter ( D h ). Additional light scattering measure-ments at different angles (30–150 ° ) were made on an ALV/CGS-3 Compact Goniometer System (ALV GmbH, Germany), using a JDS Uniphase 22 mW He-Ne laser, operating at 632.8 nm, and an Avalanche photodiode detector. The system was interfaced with a ALV-5000/EPP multi-tau digital correlator with 288 channels and a ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. Temperature control of the meas-uring cell was achieved by the use of a model 9102 circulator from Polyscience, USA. Autocorrelation functions were measured at least fi ve times and analyzed by the cumulants method and the CONTIN routine.
Magnetophoresis : The magnetophoretic experiments were per-formed using a Hitach Digilab U-2800 spectrophotometer and by inserting next to the cuvette holder a cylindrical Nd-Fe-B magnet (dimensions: diameter = 20 mm, thickness = 10 mm).
SANSPOL Measurements and Analysis : Small-angle neu-tron scattering with polarized neutrons (SANSPOL) experiments were performed on the V4 instrument at the BERII reactor of HZB in Berlin. Scattering profi les were collected at 1 − , 4 − and 12-m sample–detector distances, whose combination provides a scat-tering wave vector range between 4 × 10 − 2 and 3 nm − 1 . The neutron wavelength was selected at λ = 0.605 nm. The raw data were cor-rected for background noise, transmission, and detector effi ciency, and calibrated to an absolute scale with water as a standard, using the BerSANS data reduction software. [ 60 ] A horizontal magnetic fi eld of about 1 T was applied horizontally at the sample position and oriented perpendicular to the incoming neutron beam.
In SANSPOL [ 61 , 62 ] the sample is placed in a polarized neutron beam (i.e., neutrons with spins aligned in the same direction). A magnetic fi eld H is applied to the sample perpendicular to the beam. Using a spin-fl ipper, the spins of the incident neutrons can be aligned either parallel or antiparallel to the fi eld on the sample. The scattering intensity ( I ) as a function of scattering wave vector ( q ), depends on the neutron spin orientation and it is denoted as I − ( q ) and I + ( q ) for parallel and antiparallel confi guration, respectively.
For perfect alignment of neutron spin the scattering intensity is generally written as: [ 61 ]
(−,+ ) (q ⊥ H) = Np [FN (q) ± FM (q)]2 · SI (q)
erlag GmbH & Co. KGaA, Weinheim small 2012, 8, No. 15, 2381–2393
High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
for collection perpendicular to the magnetic fi eld. Where N p is the number of scattering particles per unit volume, F N ( q ) and F M ( q ) are the nuclear and magnetic form factors of the particles, respec-tively, and S ( q ) is the interparticle structure factor. For the hybrid nanoparticles of this study, we have employed a spherically sym-metric model, consisting of a core of Fe 3 O 4 which scatters neutrons both via magnetic and nuclear scattering, surrounded by a poly-meric corona which scatters only via nuclear scattering.
For the form factor of spherically sym-metric particles [ 63 ] we used the general formula FN,M (q ) = 4π
∞∫
0(ρ N , M ( r ) − ρ N,M,solvent ) r 2 sin qr
qrdr . Where ρ N , M ( r )
is the (nuclear or magnetic) scattering length density as a func-tion of the distance r from the centre of the spherical particle and ρ N ,solvent = − 0.56 × 10 10 cm − 2 for H 2 O which is used as a solvent ( ρ M ,solvent = 0). No interparticle contributions to the scattering profi les were taken into account ( S ( q ) ≈ 1) for the dilute disper-sions under study. The calculated SANSPOL profi les were convo-luted with the instrumental resolution [ 63 ] function and a constant value has been added, in order to account for the incoherent scat-tering. [ 63 ] A scattering length density profi le of a F 3 O 4 core and two concentric shells of constant scattering length density provides us the best fi t with the minimum number of fi tting parameters to our SANSPOL data. The fi tting parameters were: the core radius ( R c ), the thickness and nuclear scattering length density of the inner shell ( d 1 , ρ 1 ), the thickness and nuclear scattering length density of the outer shell ( d 2 , ρ 2 ) and the number of particles per unit volume ( N p ). For the magnetite core the following parameters have been used, ρM ,Fe3O4 = 1.46 ·1010cm−2 and ρN,Fe3O4 = 6.97 ·10 10cm −2 . The I − ( q H ) and I + ( q H ) data sets were fi tted simultaneously, employing the SIMPLEX least square minimization algorithm as done in previous works. [ 64 ]
SQUID Magnetization Measurements : A superconducting quantum interference device (SQUID, MPMS XL-7, Quantum Design) has been used for the magnetic measurements. The hys-teresis loops of all studied samples were collected at a tempera-ture of 300 K in external magnetic fi elds from –7 to + 7 T.
Spectrophotometric Determination of Fe Ions : The Fe 2 O 3 content in the magnetic colloids was determined spectrophoto-metrically with the 1,10-phenanthroline method. [ 65 ] The colloid (100 μ L) was mixed in a microcentrifuge tube with concentrated HCl (200 μ L, 37%) in order to dissolve completely MIONs into Fe ions. After 30 min the sample turned yellow, and then was diluted (to 10 mL) with water, using a volumetric fl ask. A portion of this sample (1 mL) was transferred into another volumetric fl ask (10 mL), where hydroxylamine hydrochloride (NH 2 OH · HCl) was added (0.1 mL, 1g L − 1 ) in order to reduce Fe + 3 to Fe + 2 . 1,10-phen-anthroline (1 mL, 100 g L − 1 ) and CH 3 COONa (0.8 mL, 1.2 M ) were then added and the solution fi nally was diluted up to 10 mL. After 10 min the absorbance at 510 nm was recorded and the concen-tration (ppm) was calculated based on a previously prepared cali-bration curve covering the range 0.1 to 3.5 ppm. The method was further validated with standard anhydrous FeCl 2 (anhydrous beads 80 mesh, 99.9%), as well as using commercial Fe 3 O 4 nanopowder, both from Aldrich. The error of the determination was estimated below 2.5%.
Thermal Analysis : The polymer content in the hybrid material was determined with thermogravimetric measurements (TA instru-ments, Q500). Measurements were performed under N 2 fl ow. Neat P(MAA- g -EGMA) polymer was fi rst studied in order to determine
© 2012 Wiley-VCH Verlag Gmbsmall 2012, 8, No. 15, 2381–2393
the residue of the polymer. Based on these results, the polymer content in the hybrids was fi nally determined. Measurements were also performed in dry air, but polymer oxidative decomposition in the presence of iron oxide nanoparticles was found to be different from the polymer’s decomposition under the same conditions but in the absence of iron oxide nanoparticles. Therefore N 2 atmos-phere was preferred.
Electron Microscopy (TEM) : Samples for TEM were prepared by casting a droplet of a dilute aqueous suspension (0.01% w/v in Fe 2 O 3 ) of the hybrids on copper grids coated by Formvar carbon fi lm. Micrographs were obtained by a JEOL, JEM-2100 instrument operating at 200 kV.
X-ray Diffraction : X-ray diffraction was performed on a D-800 Siemens diffractometer, with Ni-fi ltered CuK α radiation. Samples were dried on a glass plate, then collected and ground and fi nally spread with the aid of ethanol on the Bruker sample holder with a Si wafer (holder appropriate for low sample volumes).
Interactions with Human Blood Plasma : Colloids of magnetic nanocariers (50 μ g in hybrids) were incubated with human blood plasma in centrifuge tubes (15 mL) for 24 h under mild shaking. Following, the magnetic material was isolated by a magnet. The pellet was washed once with water and then reconstituted in water. The size of the reconstituted colloid was determined with DLS.
Doxorubicin Loading : A dispersion of MagP(MAA- g -EGMA)3 ( ∼ 45 μ L or ∼ 120 μ g of hybrids) were mixed with i) 40, ii) 100, iii) 150, iv) 200, and v) 300 μ L of doxorubicin hydrochloride solu-tion in water (200 ppm). The fi nal volume was adjusted in each case to 1.2 mL. The vials were shaken overnight at 25 ° C and then centrifuged (15 000 g for 1 h). The non-absorbed Dox in the supernatant (non-entrapped drug fraction) was determined based on a previously prepared calibration curve with a fl uorescence spectrophotometer Hitachi F2500 by measuring the residual fl uo-rescence of the solution at 550 nm. The reference Dox solutions were prepared in the following way: Dox of known concentration was dissolved in H 2 O and concentrated HCl was added, in order to obtain a fi nal concentration of 1.5 M to secure full protona-tion of the amino group of the drug and the highest possible drug solubility. After 24 h the fl uorescence spectrum was recorded. HCl also served on the Dox determination from the processed samples (after MIONs separation) because it would dissolve any ultrasmall MIONs remaining in the supernatant, since they can interfere with Dox analysis. MIONs absorb light below 550 nm, which is in the same region where Dox emits. The wt.% drug’s loading is fi nally expressed as μg(Dox)
μg(Dox) + μg(carrier) × 100 .
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements
The laboratory of Prof. P. Koutsoukos from the Department of Chemical Engineering, University of Patras is acknowledged for the
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[ 1 ] D. A. Giljohann , D. S. Seferos , L. D. Weston , M. D. Massich , C. P. Pinal , C. A. Mirkin , Angew. Chem. Int. Ed. 2010 , 49 , 3280 .
[ 2 ] A. S. Lübbe , C. Alexiou , C. Bergemann , J. Surg. Res. 2001 , 95 , 200 – 206 .
[ 3 ] P. Kyeongsoon , S. Lee , E. Kang , K. Kim , K. Choi , I. C. Kwon , Adv. Funct. Mater. 2009 , 19 , 1553 .
[ 4 ] G. Mistlberger , K. Koren , E. Scheucher , D. Aigner , S. M. Borisov , A. Zankel , P. Pölt , I. Klimant , Adv. Funct. Mater. 2010 , 20 , 1842 .
[ 5 ] C. Corot , P. Robert , J. M. Idée , M.Port , Adv. Drug Deliver. Rev. 2006 , 58 , 1471 .
[ 6 ] J. Kaiser , Science 2009 , 326 , 218 . [ 7 ] T. Tsuruo , M. Naito , A. Tomida , N. Fujita , T. Mashima , H. Sakamoto ,
N. Haga , Cancer Sci. 2003 , 94 , 15 . [ 8 ] R. Duncan , Nat. Rev. Cancer 2006 , 6 , 688 . [ 9 ] M. Shokeen , E. D. Pressly , A. Hagooly , A. Zheleznyak , N. Ramos ,
A. L. Fiamengo , M. J. Welch , C. J. Hawker , C. J Anderson , ACS Nano 2011 , 5 , 738 .
[ 10 ] V. P. Torchilin , in Handbook of Experimental Pharmacology , Vol 197 (Eds: M. Schäfer-Korting ) Springer , 2004 , p13 and p27 .
[ 11 ] Scientifi c and Clinical Applications of Magnetic Carriers , (Eds: U. Häfeli , W. Schütt , J. Teller , M. Zborowski ) Plenum Press , New York 1997 .
[ 12 ] a) R. C. Semelka , T. K. Helmberger , Radiology 2001 , 218 , 27 ; b) M. G. Harisinghani , J. Barentsz , P. F. Hahn , W. M. Deserno , S. Tabatabaei , C. H. Van de Kaa , J. de la Rosette , R. Weissleder , New Engl. J. Med. 2003 , 348 , 2491 ; c) W.S. Enochs , G. Harsh , F. Hochberg , R. Weissleder , J. Magn. Reson. Imaging 1999 , 9 , 228 .
[ 13 ] S. Müller , Nanomed.-Nanotechnol. 2009 , 5 , 387 . [ 14 ] F. K. H. van Landeghem , K. Maier-Hauff , A. Jordan , K. T. Hoffmann ,
U. Gneveckow , R. Scholz , B. Thiesen , W. Brück , A. von Deimling , Biomaterials 2009 , 30 , 52 .
[ 15 ] B. Hildebrandt , P. Wust , O. Ahlers , A. Dieing , G. Sreenivasa , T. Kerner , R. Felix , H. Riess , Crit. Rev. Oncol. Hemat. 2002 , 43 , 33 .
[ 16 ] Y. W. Jun , J. H. Lee , J. Cheon , Angew. Chem. Int. Ed. 2008 , 47 , 5122 .
[ 17 ] C. Alexiou , W. Arnold , R. J. Klein , F. G. Parak , P. Hulin , C. Bergemann , W. Erhardt , S. Wagenpfeil , A. S. Lübbe , Cancer Res. 2000 , 60 , 6641 .
provision of the X-ray diffractometer. Dr. Maria Kollia from the Labo-ratory of Microscopy and Microanalysis of the University of Patras is acknowledged for assistance in transmission electron microscopy. Finally, we deeply thank Dr. G. Zoppellaro (Palacky University) for the manuscript editing. This research was in part supported by the European Union (European Social Fund–ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) - Research Funding Program: Thales. Investing in knowledge society through the European Social Fund. This work was also partly sup-ported by the Operational Program Research and Development for Innovations–European Social Fund (CZ.1.05/2.1.00/03.0058), grants from the Ministry of Education of the Czech Republic (1M6198959201 and MSM6198959218), and a grant from the Academy of Sciences of the Czech Republic (KAN115600801). Partial support by the European Commission under the 7th Frame-work Programme through “Research Infrastructures” action of the “Capacities” Programme contract number CP-CSA_INFRA-2008-1.1.1. Number 226 507-NMI3, is also acknowledged.
92 www.small-journal.com © 2012 Wiley-VCH
[ 18 ] A. S. Lübbe , C. Bergemann , H. Riess , F. Schriever , P. Reichardt , K. Possinger , M. Matthias , B. Dörken , F. Herrmann , R. Gurtler , P. Hohenberger , N. Haas , R. Sohr , B. Sander , A. J. Lemke , D. Ohlendorf , W. Huhnt , D. Huhn , Cancer Res. 1996 , 56 , 4686 .
[ 19 ] M. Liong , J. Lu , M. Kovochich , T. Xia , S. G. Ruehm , A. E. Nel , F. Tamanoi , J. I. Zink , ACS Nano 2008 , 2 , 889 .
[ 20 ] F. Dilnawaz , A. Singh , C. Mohanty , S. K. Sahoo , Biomaterials 2010 , 31 , 3694 .
[ 21 ] A. Bakandritsos , G. Mattheolabakis , G. Chatzikyriakos , T. Szabo , V. Tzitzios , D. Kouzoudis , St. Couris , K. Avgoustakis , Adv. Funct. Mater. 2011 , 21 , 1465 .
[ 22 ] Z. Li , L. Wei , M. Gao , H. Lei , Adv. Mater. 2005 , 17 , 1001 . [ 23 ] C. Fang , N. Bhattarai , C. Sun , M. Zhang , Small 2009 , 5 ,
1637 . [ 24 ] C. Munish , S. Jahn , R. Georgieva , J. F. Lutz , H. Bäumler , D. Wang ,
Chem. Mater. 2009 , 21 , 1906 . [ 25 ] F. H. Chen , L. M. Zhang , Q. T. Chen , Y. Zhang , Z. J. Zhang , Chem.
Commun. 2010 , 46 , 8633 . [ 26 ] N. Nasongkla , E. Bey , J. Ren , H. Ai , C. Khemtong , J. S. Guthi ,
S. F. Chin , A. D. Sherry , D. A. Boothman , J. Gao , Nano Lett. 2006 , 6 , 2427 .
[ 27 ] J. H. Park , G. Von Maltzahn , E. Ruoslahti , S. N. Bhatia , M. J. Sailor , Angew. Chem. Int. Ed. 2008 , 47 , 7284 .
[ 28 ] M. M. Yallapu , S. P. Foy , T. K. Jain , V. Labhasetwar , Pharm. Res. 2010 , 27 , 1 .
[ 29 ] a) M. K. Yu , Y. Y. Jeong , J. Park , S. Park , W. J. Kim , J. J. Min , K. Kim , S. Jon , Angew. Chem. Int. Ed. 2008 , 47 , 5362 ; b) H. Lee , K. Y. Mi , S. Park , S. Moon , J. Jung , M. Y. J. Yong , H. W. Kang , S. Jon , J. Am. Chem. Soc. 2007 , 129 , 12739 .
[ 30 ] a) M. Guo , Y. Yan , H. Zhang , H. Yan , Y. Cao , K. Liu , S. Wan , J. Huang , W. Yue , J. Mater. Chem. 2008 , 18 , 5104 ; b) M. Guo , C. Que , C. Wang , X. Liu , H. Yan , K. Liu , Biomaterials 2011 , 32 , 185 .
[ 31 ] F. M. Kievit , O. Veiseh , N. Bhattarai , C. Fang , J. W. Gunn , D. Lee , R. G. Ellenbogen , J. M. Olson , M. Zhang , Adv. Funct. Mater. 2009 , 19 , 2244 .
[ 32 ] G. R. Reddy , M. S. Bhojani , P. McConville , J. Moody , B. A. Moffat , D. E. Hall , G. Kim , Y.- E. L. Koo , M. J. Woolliscroft , J. V. Sugai , T. D. Johnson , M. A. Philbert , R. Kopelman , A. Rehemtulla , B. D. Ross , Clin. Cancer Res. 2006 , 12 , 6677 .
[ 33 ] R. A. Dwek , Chem.Rev. 1996 , 96 , 683 . [ 34 ] N. B. Holland , Y. Qiu , M. Ruegsegger , R. E. Marchant , Nature
1998 , 392 , 799 . [ 35 ] G. M. Lee , B. Johnstone , K. Jacobson , B. Caterson , J. Cell Biol.
1993 , 123 , 899 . [ 36 ] J.-F. Lutz , J. Polym. Sci. Pol. Chem. 2008 , 46 , 3459 . [ 37 ] H. Ma , M. Wells , T. P. Beebe , A. Chilkoti , Adv. Funct. Mater. 2006 ,
16 , 640 . [ 38 ] G. Prencipe , S. M. Tabakman , K. Welsher , Z. Liu , A. P. Goodwin ,
L. Zhang , J. Henry , H. Dai , J. Am. Chem. Soc. 2009 131 , 4783 .
[ 39 ] S. Park , H.-S. Yang , D. Kim , K. Jo , S. Jon , Chem. Commun. 2008 , 25 , 2876 .
[ 40 ] A. Bakandritsos , N. Bouropoulos , R. Zboril , K. Iliopoulos , N. Boukos , G. Chatzikyriakos , S. Couris , Adv. Funct. Mater. 2008 , 18 , 1694 .
[ 41 ] R. Toomey , M. Tirrell , Annu. Rev. Phys. Chem. 2008 , 59 , 493 .
[ 42 ] a) Y. Li , Y.-K. Gong , K. Nakashima , Y. Murata , Langmuir 2002 , 18 , 6727 ; b) H. R. Sondjaja , T. A. Hatton , K. C. Tam , Langmuir 2008 , 24 , 8501 .
[ 43 ] A. Bakandritsos , G. C. Psarras , N. Boukos , Langmuir 2008 , 24 , 11489 .
[ 44 ] C. W. Hoogendam , A. de Keizer , M. A. Cohen Stuart , B. H. Bijsterbosch , J. A. M. Smit , J. A. P. P. van Dijk , P. M. van der Horst , J. G. Batelaan , Macromolecules 1998 , 31 , 6297 .
Verlag GmbH & Co. KGaA, Weinheim small 2012, 8, No. 15, 2381–2393
High Doxorubicin Loading, Magnetic Response, and Bio-repellent Properties in Drug Nanocarriers
[ 45 ] a) S. C. Lee , K. J. Kim , Y.- C. Jeong , J. H. Chang , J. Choi , Macromole-cules 2005 , 38 , 9291 ; b) H. Liu , C. Li , H. Liu , S. Liu , Langmuir 2009 , 25 , 4724 .
[ 46 ] A. M. Mathur , B. Drescher , A. B. Scranton , J. Klier , Nature 1998 , 392 , 367 .
[ 47 ] G. D. Moeser , K. A. Roach , W. H. Green , P. E. Laibinis , T. A. Hatton , Ind. Eng. Chem. Res. 2002 , 41 , 4739 .
[ 48 ] J.-F. Lutz , S. Stiller , A. Hoth , L. Kaufner , U. Pison , R. Cartier , Biomacromolecules 2006 , 7 , 3132 .
[ 49 ] U. O. Häfeli , J. S. Riffl e , L. Harris-Shekhawat , A. Carmichael-Baranauskas , F. Mark , J. P. Dailey , D. Bardenstein , Mol. Pharm. 2009 , 6 , 1417 .
[ 50 ] Q. Zhang , M. S. Thompson , A. Y. Carmichael-Baranauskas , B. L. Caba , M. A. Zalich , Y.-N. Lin , O. T. Mefford , R. M. Davis , J. S. Riffl e , Langmuir 2007 , 23 , 6927 .
[ 51 ] P. Papaphilippou , L. Loizou , N. C. Popa , A. Han , L. Vekas , A. Odysseos , T. Krasia-Christoforou , Biomacromolecules 2009 , 10 , 2662 .
[ 52 ] R. Sondjaja , T. A. Hatton , M. K. C. Tam , J. Magn. Magn. Mater. 2009 , 321 , 2393 .
[ 53 ] J. Ge , Y. Hu , M. Biasini , W. P. Beyermann , Y. Yin , Angew. Chem. Int. Edit. 2007 , 46 , 4342 .
[ 54 ] V. Schaller , G. Wahnström , A. Sanz-Velasco , P. Enoksson , C. Johansson , J. Magn. Magn. Mater. 2009 , 321 , 1400 .
[ 55 ] a) C. Paquet , H. W. De Haan , D. M. Leek , H.-Y. Lin , B. Xiang , G. Tian , A. Kell , B. Simard , ACS Nano 2011 , 5 , 3104 ; b) P. Dames , B. Gleich , A. Flemmer , K. Hajek , N. Seidl , F. Wiekhorst , D. Eberbeck , I. Bittmann , C. Bergemann , T. Weyh , L. Trahms ,
© 2012 Wiley-VCH Verlag Gmbsmall 2012, 8, No. 15, 2381–2393
J. Rosenecker , C. Rudolph , Nat. Nanotechnol. 2007 , 2 , 495 ; c) J. Ge , Y. Hu , M. Biasini , W. P. Beyermann , Y. Yin , Angew. Chem. 2007 , 119 , 4420 .
[ 56 ] The plot from uncoated MIONs has been previously pub-lished, but it is also made here available for comparison; A. Bakandritsos , G. Mattheolabakis , R. Zboril , N. Bouropoulos , J. Tucek , D. G. Fatouros , K. Avgoustakis , Nanoscale 2010 , 2 , 564 .
[ 57 ] C. Sangregorio , J. K. Wiemann , C. J. O’Connor , Z. A. Rosenzweig , J. Appl. Phys. 1999 , 85 , 5699 .
[ 58 ] Q. Quan , J. Xie , H Gao , M. Yang , F. Zhang , G. Liu , X. Lin , A. Wang , H. S. Eden , S. Lee , G. Zhang , X. Chen , Mol. Pharmaceutics 2011 , 8 , 1669 .
[ 59 ] F. Winnefeld , S. Becker , J. Pakush , T. Gotz , Cement Concrete Comp. 2007 , 29 , 251 .
[ 60 ] U. Keiderling , Appl. Phys. A74 , 2002 , S1455 . [ 61 ] M. Bonini , A. Wiedenmann , P. Baglioni , J. Appl. Crystallogr. 2007 ,
40 , s254 . [ 62 ] A. Wiedenmann , J. Appl. Crystallogr. 2000 , 33 , 428 . [ 63 ] A. Papagiannopoulos , M. Karayianni , G. Mountrichas , S. Pispas ,
A. Radulescu , J. Phys. Chem. B 2010 , 114 , 7482 . [ 64 ] A. Papagiannopoulos , C. M. Fernyhough , T. A. Waigh ,
A. Radulescu , Macromol. Chem. Phys. 2008 , 209 , 2475 . [ 65 ] O. Mykhaylyk , Y. S. Antequera , D. Vlaskou , C. Plank , Nat. Protoc.
2007 , 2 , 2391 .
Received: November 30, 2011 Revised: February 15, 2012Published online: May 2, 2012
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