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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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http://doi.wiley.com/10.1002/smll.201102525

A. Bakandritsos et al.

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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”

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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

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 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


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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

Verlag GmbH & Co. KGaA, Weinheim small 2012, 8, No. 15, 2381–2393


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

www.small-journal.com © 2012 Wiley-VCH V

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



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


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


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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



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


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



2

full papers
environment forming a protective canopy. The ability of the
polymer 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)



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


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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Received: November 30, 2011 Revised: February 15, 2012Published online: May 2, 2012


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