Covalently bonded dendrimer-maghemite nanosystems: Nonviral vectors for in vitro gene magnetofection

Article abstract of DOI:10.1039/c0jm03526b

In this work novel nonviral nanosystems for in vitro gene magnetofection are presented. The multifunctional vectors consist of superparamagnetic iron oxide nanoparticles functionalized with low generations of poly(propyleneimine) dendrimers. The dendrimers are attached to the iron oxide nanoparticles through covalent bonds via a one-pot sol-gel synthetic route. This approach allows a direct dendritic decoration of the iron oxide NPs without any additional surface modification. Furthermore, this strategy avoids the multistep procedures of dendritic growth onto solid surfaces. The core-shell hybrid structures are water soluble as colloidal ferrofluids which are long-term stable at physiological pH. In vitro transfection experiments were assayed with Saos-2 osteoblasts, using as reporter gene a plasmid DNA that codes for the green fluorescent protein. Gene delivery experiments were carried out in the presence and in the absence of a magnetic field. The transfection efficiency strongly depends on the presence of the magnetic field and the dendrimer generation. The covalent bonding between the dendrimers and the magnetic nanoparticles surface ensures the vector integrity throughout storage and application. The nanosystems couple the DNA fragments and safely transport them under magnetic stimulus from the extracellular environment to the interior of the cell.

Full text of DOI:10.1039/c0jm03526b

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links <
C
Journal of
Materials Chemistry
Cite this: J. Mater. Chem., 2011, 21, 4598
www.rsc.org/materials
PAPER
Covalently bonded dendrimer-maghemite nanosystems: nonviral vectors for
in vitro gene magnetofection†
ac
ac
b
Blanca Gonzalez, Eduardo Ruiz-Hernandez, Marıa Jose Feito, Carlos Lopez de Laorden,ac Daniel Arcos,ac
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
b
b
b
ac
*
ꢀ
Cecilia Ramırez-Santillan, Concepcion Matesanz, Marıa Teresa Portoles and Marıa Vallet-Regı
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Received 18th October 2010, Accepted 4th January 2011
DOI: 10.1039/c0jm03526b
In this work novel nonviral nanosystems for in vitro gene magnetofection are presented. The
multifunctional vectors consist of superparamagnetic iron oxide nanoparticles functionalized with low
generations of poly(propyleneimine) dendrimers. The dendrimers are attached to the iron oxide
nanoparticles through covalent bonds via a one-pot sol–gel synthetic route. This approach allows
a direct dendritic decoration of the iron oxide NPs without any additional surface modification.
Furthermore, this strategy avoids the multistep procedures of dendritic growth onto solid surfaces. The
core–shell hybrid structures are water soluble as colloidal ferrofluids which are long-term stable at
physiological pH. In vitro transfection experiments were assayed with Saos-2 osteoblasts, using as
reporter gene a plasmid DNA that codes for the green fluorescent protein. Gene delivery experiments
were carried out in the presence and in the absence of a magnetic field. The transfection efficiency
strongly depends on the presence of the magnetic field and the dendrimer generation. The covalent
bonding between the dendrimers and the magnetic nanoparticles surface ensures the vector integrity
throughout storage and application. The nanosystems couple the DNA fragments and safely transport
them under magnetic stimulus from the extracellular environment to the interior of the cell.
order to improve the efficiency of the gene transfer, a nonviral
vector should fulfill the following requirements:6–8
Introduction
Gene transfection represents an alternative or complementary
therapy in the treatment of cancer and other gene-based
diseases.1,2 This strategy consists of deliberately introducing
nucleic acids into the nucleus of cells. In terms of gene therapy,
the incorporation of nucleic acids is mainly intended to replace
deleterious mutant alleles with functional ones, or even to induce
the malignant cell apoptosis. Moreover, the DNA reprogram-
ming of bacteria and eukaryotic cells opens huge possibilities in
the field of biotechnology.3–5
(i) capability to complex nucleic acids and protect them against
serum nuclease enzymes at the extracellular compartment, (ii)
exhibit positive net electric surface charge at physiological pH to
overcome the negative potential of the cell membrane, since
otherwise the cell membrane impedes the incorporation of
negatively charged phosphate-containing DNA, (iii) possess
a mechanism to protect DNA from the acid environment inside
endosomes and (iv) chemical stability to maintain the integrity
until reaching the nucleus. Furthermore, by means of preparing
multifunctional vectors, transfection can be accelerated thus
hindering the biochemical attack over the gene. In this sense,
magnetofection is an excellent alternative to significantly reduce
the transfection time from several hours to less than 60 min. The
association of superparamagnetic nanoparticles (NPs) with gene
vectors makes possible to assist the transfection into cells by the
application of an external magnetic field, which targets and
reduces the duration of the gene delivery, enhancing the effi-
ciency of the DNA vector.9 In this sense, the coating of super-
paramagnetic iron oxide NPs with polycationic polymers such as
polyethyleneimine makes magnetofection feasible.10–12
The use of nonviral vectors is one of the main strategies for
gene delivery into the target cell nucleus. These nanosystems
induce minimal host immune responses and they have been
increasingly proposed as a safer alternative to viral vectors. In
aDepartamento de Quꢀımica Inorgaꢀnica y Bioinorgaꢀnica, Facultad de
Farmacia, Universidad Complutense de Madrid, Plaza de Ramoꢀn y Cajal
s/n, 28040 Madrid, Spain. E-mail: vallet@farm.ucm.es; Fax: +34 91
3941786; Tel: +34 91 3941843
bDepartamento de Bioquꢀımica, Facultad de Ciencias Quꢀımicas, Universidad
Complutense de Madrid, Avenida Complutense s/n, 28040 Madrid, Spain
cNetworking Research Center on Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Spain
Dendrimers are tree-like highly branched macromolecules that
possess unique three-dimensional architectures. Their features
include a monodisperse polymeric constitution, well-defined
internal cavities, nanometric dimensions, and the presence of
a large number of functionalities on the surface.13 Dendrimers
† Electronic supplementary information (ESI) available: Reagents and
equipment, X-ray powder diffraction pattern of the g-Fe2O3 NPs,
selected characterization data for dendritic precursors (G1–G3), DLS
measurement of G3-MNPs after months of storage. See DOI:
10.1039/c0jm03526b
4598 | J. Mater. Chem., 2011, 21, 4598–4604
This journal is ª The Royal Society of Chemistry 2011

View Article Online
have many biomedical applications, for instance, as contrast
agents in magnetic resonance imaging, in drug delivery and in
neutron capture and gene transfection therapies.14–16 Regarding
using the divergent growth approach, only a small proportion of
the higher generations of dendrimers can be perfect33,34 and
moreover, with the use of a solid support the steric interference
effect would be pronounced.
the
latter,
polyamidoamine
(PAMAM)
and
poly-
(propyleneimine) (PPI) dendrimers, have been thoroughly
studied recently as nonviral gene transfection agents,6,17,18 espe-
cially for cancer therapy, overcoming the safety disadvantages of
viral vector systems. These dendrimers form compact poly-
cations under physiological conditions able to complex, trans-
port and protect DNA.
In this work we present a novel multifunctional nanosystem
for in vitro gene transfection that consists of poly-
(propyleneimine) dendrimers covalently bonded to maghemite
nanoparticles, as depicted in Scheme 1. On the one hand, PPI
dendrimers can act as nonviral gene transfection agents6,35 and
provide complexation sites for effective DNA binding in the
proposed nanovector. Moreover, the presence of tertiary amines
protects the genes from the acid attack in the endosomal
compartment. On the other hand, superparamagnetic iron oxide
NPs would make magnetofection feasible, speeding up the gene
transfection. It is noteworthy that the covalent bonding between
the dendrimers and the magnetic nanoparticle ensures the vector
integrity during the transfection process.
It has been reported that mesoporous silica nanoparticles and
gold nanoparticles modified with low generation –NH2 termi-
nated dendrimers, such as the PAMAM and PPI dendrimers,
display an effective gene transfection. Since a generation
dependent cytotoxicity has been observed, this approach has the
benefit of using non-toxic generations. Moreover, the drawback
of the limited surface charges of low generation dendrimers is
overcome via the attachment of several dendritic molecules to the
same nanoparticle, which leads to an efficient DNA complexa-
tion.19,20 A similar observation has been reported for systems that
use different sizes of polyethyleneimine polymers attached to
mesoporous silica nanoparticles.21
Results and discussion
A novel approach for the covalent assembly of PPI dendrimers to
maghemite NPs
Different synthetic approaches for the functionalization of
iron oxide nanoparticles with dendritic macromolecules have
been developed for a variety of applications. For intracellular
uptake studies iron oxide NPs have been stabilized with
carboxylated poly(amidoamine) dendrimers through electro-
static self assembly onto the iron oxide surfaces.22–24 Regarding
catalytic applications, attempts to grow dendrimers directly on
magnetic nanoparticles usually fail due to coagulation of the
particles and solubility problems in organic media. To solve these
problems the magnetic nanoparticles can be coated with silica.25
Nevertheless, a stepwise growth of dendritic PAMAM wedges on
the surface of magnetite NPs has been reported for gene
delivery.26 Another relevant approach is the modification of the
iron oxide nanoparticles by combining the electrostatic layer by
layer self assembly technique with dendrimer chemistry. Posi-
tively charged iron oxide NPs were modified with a bilayer
composed of a negatively charged polyelectrolyte and a genera-
tion 5 PAMAM dendrimer.27 In this sense, a more stable shell
coating was obtained when iron oxide NPs were assembled with
multilayers of polyelectrolytes and an outer layer of PAMAM
dendrimers to finally carry out a chemical crosslinking of the
shells.28 By using dendrimers that bear folic acid as the targeting
moiety these multifunctional iron oxide NPs were able to be used
for targeting and imaging of cancer cells.
The magnetic NPs were prepared as maghemite ferrofluid
according to the Massart method.36,37 Controlled alkaline
coprecipitation of FeII and FeIII ions yields nanometric magnetite
(Fe3O4), which is subsequently oxidized to maghemite (g-Fe2O3).
In this fashion continuous variation of the iron oxide cores from
magnetite to maghemite is avoided.
The synthetic route followed for the covalent grafting of
dendrimers to the g-Fe2O3 NPs is shown in Scheme 1. Our
approach is based on the previous synthesis of the partially
silylated dendritic precursors (Chart 1).38 Reaction conditions of
this step are optimized to get the monosubstituted dendrimers as
the major product. Accordingly, one of the peripheral amino
groups of the dendrimers, as an average, is used to introduce
a reactive functional group, such as –Si(OEt)3, that allows
covalent bonding to the Fe–OH groups on the surface of the
magnetic nanoparticles (MNPs) in
synthesis.
a subsequent step of
Herein, we describe a process to covalently graft PPI den-
drimers onto the naked surface of maghemite nanoparticles
(g-Fe2O3 NPs). The dendrimers are strongly anchored onto the
iron oxide surface in one synthetic step, by exploiting the sol–gel
approach to attach functional alkoxysilane derivatives to iron
oxide NPs.29–32 This method represents a new strategy to
synthesize dendrimer functionalized magnetic nanoparticles that
form stable colloidal solutions in water and facilitates potential
biomedical applications of these magnetic nanoparticles. Our
attractive methodology is less time consuming and keeps intact
the structure of the dendrimer, versus the use of multistep
procedures that lead to dendritic wedges or dendrons or, to
a certain extent, to hyperbranched polymers. Statistically, when
Scheme 1 Dendritic functionalization of iron oxide nanoparticles
through sol–gel chemistry with alkoxysilane PPI dendrimers. Magnetic
NPs functionalized with APTS (as a model), G1, G2 and G3 were
prepared. Schematic structure of G3-MNPs material.
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 4598–4604 | 4599

View Article Online
carried out under identical experimental conditions, failed to
yield stable and water soluble MNPs. The obtained sample was
a non-homogeneous dispersion with aggregates and a high
polydispersity, and behaved neither as the bare g-Fe2O3 NPs
ferrofluid nor as the MNP-G3 particles. Thermogravimetric and
chemical analyses gave a low organic content, indicating the loss
of the unattached dendrimer with the magnetic washings
(Table 1).
Surface charge measurements
The surface modification of the g-Fe2O3 NPs was confirmed with
the zeta (z)-potential measurements of the functionalized MNPs
colloidal aqueous solutions. The attachment of APTS or the PPI
dendrimers to the g-Fe2O3 NPs surface provides an amino group
density that decisively defines the MNPs surface charge along the
pH range, i.e., their zeta potential (Fig. 1.A). The bare iron oxide
NPs have the isoelectric point (IEP) at neutral pH and it is shifted
towards higher pH values after functionalization (Table 1).
As the generation of the grafted dendrimer increases there is an
exponential increase in primary and tertiary amines of the den-
drimer framework. This is reflected by the progressive shift in the
IEP values, rising to ca. 11 for the third dendrimer generation. It
is noteworthy that the z-potential distributions at a given pH are
monomodal for all materials, indicating single component
samples and homogeneous surface coverage of the MNPs
(Fig. 2.A). Furthermore, the z-potential return titration curves of
Chart 1 3-Aminopropyltriethoxysilane and schematic representation of
dendritic precursors G1–G3.
The surface modification of g-Fe2O3 NPs was performed
following a sol–gel based wet-chemistry synthetic route, under
basic pH conditions.39 In this way, the silanization reaction leads
to the formation of core–shell hybrid structures, although under
these conditions the self-condensation of alkoxysilane dendritic
precursors in the presence of water cannot be avoided. Then,
alkoxysilane auto condensation is produced, but it is hampered
in the higher dendrimer generations due to steric hindrance.
Nanosystems prepared with G3 dendrimers exhibit experimental
organic amounts closer to theoretical values, when compared
with G2, G1 or even with only APTS, as demonstrated by
thermogravimetric and elemental analyses (Table 1). This means
that the ratio of interdendrimer condensation versus condensa-
tion with the inorganic surface decreases as the dendrimer
generation increases. This fact is explained in terms of steric
hindrance between G3 dendrimers, avoiding the self-condensa-
tion of alkoxysilane dendritic precursors, thus favouring
condensation with the maghemite nanoparticles.
In order to confirm the covalent anchoring of the dendrimers
through the alkoxysilane moiety with the Fe–OH groups, we
performed a parallel attempt to functionalize the g-Fe2O3 NPs
with the third generation dendrimer lacking the triethoxysilane
group. Moreover, this test was also useful to discard the coating
of the MNPs through electrostatic interactions. The experiment,
Table 1 Percentage of maghemite, experimental and theoretical organic
material content, isoelectric point and hydrodynamic particle size of the
magnetic nanoparticles
% Org.
Matter
Theoretical
% g-
Fe2O3
% Org.
Matter
Particle
Size (nm)a
Material
IEP
7.1
g-Fe2O3
NPs
APTS-
100
0
0
8
99.87
0.13
3.66
9.0
16
MNPs
G1-MNPs
G2-MNPs
G3-MNPs
G3-MNPs*b
98.46
90.10
72.66
94.27
1.54
9.90
27.34
5.73
20.77
35.92
53.66
52.45
10.1
10.6
11.0
N/Ac
18
21
39
N/Ac
Fig. 1 (A) Evolution of z-potential curves as a function of pH for bare
g-Fe2O3 NPs and APTS and dendrimer functionalized MNPs. Inset:
magnification. (B) Evolution of z-potential return titration curves as
a function of pH for the third generation dendrimer functionalized
MNPs.
a
Maximum of the size distribution, measured by DLS in water (1–5 mg
b
mLꢀ1) at pH 3 (HNO3). g-Fe2O3 NPs with G3 dendrimer lacking the
triethoxysilane group.
polydispersity.
c
Could not be measured due to high
4600 | J. Mater. Chem., 2011, 21, 4598–4604
This journal is ª The Royal Society of Chemistry 2011

View Article Online
since z-potential at pH 7.4 still maintains a high positive value
(28 mV). A lower particle size, equal to 34 nm, can be attributed
to a backfolding of the end groups of PPI dendrimers that takes
place at high salt concentration, giving rise to a dense core
dendritic structure.40 The shift to lower z-potential values (5 mV)
of the nanosystem in cell culture media supplemented with serum
indicates serum protein adsorption onto the nanoparticles.
Accordingly, bigger aggregates of ca. 53 nm are formed.11,21
Magnetic measurements
The magnetic properties of the ferrofluid containing the MNPs
functionalized with the third generation dendrimer were inves-
tigated by means of vibrating sample magnetometry. The
magnetization curve normalized to the mass of g-Fe2O3 is dis-
played in Fig. 3, together with the measurement of the bare
g-Fe2O3 NPs ferrofluid. The MNPs preserve their super-
paramagnetic properties after the covalent anchorage of the G3
dendrimer. This characteristic superparamagnetic behaviour is
indicated by the absence of a hysteresis loop on the magnetiza-
tion curve, i.e., zero coercivity and zero remanence, measured at
room temperature. Moreover, the initial slopes show a rapid
approach to saturation, suggesting a high magnetic suscepti-
bility. The saturation magnetization values are 58.44 and 50.93
emu gꢀ1 for the bare g-Fe2O3 NPs and the MNP-G3, respec-
tively. These values are lower than that of bulk maghemite
(76 emu gꢀ1), but comparable to results reported for similar size
Fig. 2 (A) z-potential distributions at pH 7.1 (IEP of g-Fe2O3 NPs) for
MNPs before and after functionalization with G3 dendrimer. (B)
Hydrodynamic diameter distribution obtained by DLS for the bare g-
Fe2O3 NPs and G3 dendrimer functionalized MNPs nanoparticles.
the dendrimer functionalized MNPs do not show any hysteresis,
which indicates a high chemical stability of the dendrimer–iron
oxide linkage through the siloxane linker and is consistent with
covalently bonded dendrimers (Fig. 1.B). The IEP values,
ranging from ca. 10 to 11, as well as the z-potential value at pH
7.4, ca. +37 mV, for the dendrimer-MNPs represent a high
positive charge at physiological pH. Therefore, aggregation or
flocculation of the MNPs is avoided at pH 7.4 since the elec-
trostatic stability of the colloidal ferrofluids is ensured.
iron oxide NPs, ranging from 30 to 60 emu gꢀ1 41,42
The
.
Hydrodynamic diameter measurements
anchorage of the dendrimer to the iron oxide MNPs surface
results in a decrease in the saturation magnetization. This fact is
attributed to the covalent grafting of the dendrimers through
siloxane bonds, which directly affects the crystallo-chemical
properties of the MNPs surface and leads to an increase in the
magnetically disordered surface layer.43
The hydrodynamic mean diameter distributions of the func-
tionalized MNPs obtained by dynamic light scattering (DLS) are
monomodal and narrow for all materials (Fig. 2.B). It is evi-
denced by a sequential higher value, with the dendrimer gener-
ation grafted, that goes from 8 nm for the uncoated MNPs up to
39 nm for the third generation dendrimer functionalized MNP
(Table 1). The existence of dendrimers with more than one sily-
lated branch in the functionalizing solution is expected from the
synthetic methodology and it has been characterized (see Elec-
tronic Supplementary Information†).38 In such a case, conden-
sation of the same dendritic molecule with a number of MNPs
cannot be overruled and contributes to some extent to a MNPs
aggregation process. This is shown by the asymmetry of the size
distribution, which is shifted towards higher values for a low
number of particles after dendrimer attachment (Fig. 2.B).
The dendrimer-MNPs showed excellent long-term colloidal
stability in water solution, remaining indefinitely stable in water
at pH ¼ 7.4. The hydrodynamic diameter was monitored by DLS
at different periods of time, remaining practically unchanged for
months (ESI†). Such colloidal stability can be attributed to both
steric and electrostatic repulsive forces, as expected from DLS
and z-potential measurements. Importantly, the covalent
attachment of the dendrimer shell to the particle surface ensures
that the dendritic surface function remains on the nanoparticle
throughout storage time as well as during application.
Gene transfection experiments of DNA/G(1,2,3)-MNPs
complexes
In vitro transfection efficiency of the materials was evaluated as
a function of parameters such as dendrimer generation, different
Since dendrimer-MNPs nanosystems were designed to
conduct biological experimentation, surface charge and hydro-
dynamic diameter of the G3-MNPs were also measured in cell
culture media. Data show that G3-MNPs colloidal stability is
preserved in media with a higher ionic strength such as DMEM,
Fig. 3 Magnetization curves at room temperature of the g-Fe2O3 MNPs
and G3-MNPs ferrofluids. The insets show an enlarged view near the
origin.
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 4598–4604 | 4601

View Article Online
plasmid/dendrimer-MNP weight ratios, incubation time and the
presence or absence of the magnetic field. A plasmid DNA
(pEGFP-N3) that codes for an enhanced green fluorescent
protein (EGFP) was used as reporter gene. The highest trans-
fection efficiency (ca. 12%), without cytotoxic reactions, was
obtained with the third generation dendrimer (DNA/G3-MNPs)
using a weight ratio of 1/5 and under magnetic stimulus. No
improvement of the transfection efficiency was found for the
DNA/G3-MNPs complex at weight ratios of 1/10 and 1/20.
Considering that nonviral transfection on Saos-2 cells have been
rarely reported and that studies with several commercial nonviral
vectors showed, as the best result after optimization, comparable
transfection efficiencies (ca. 13%),44 this nanosystem can be
considered an important contribution to the field.
Fig. 5 (A) Cell viability and apoptosis of osteoblasts after magneto-
fection with the DNA/G3-MNPs complex (1/5 weight ratio). (B) Fluo-
rescence confocal micrographs of osteoblasts transfected by
magnetofection with the DNA/G3-MNPs complex.
Studies with DNA/G1-MNPs and DNA/G2-MNPs complexes
demonstrate higher values of cell viability (98%) and lower values
of apoptosis (1%). However very low transfection efficiency
(<1%) was obtained with these complexes in the same conditions
(Fig. 4.B).
cells (25%) in comparison with control, but very low levels of
apoptosis were detected (4%), thus indicating the absence of
cytotoxicity of this material. Fig. 5.B shows the significant EGFP
expression after osteoblast magnetofection with the DNA/G3-
MNPs complex observed by confocal microscopy.
These results could be explained in terms of the increasing
amounts of primary and tertiary amine groups, insofar the
dendrimer generation increases. At pH ¼ 7.4 the peripheral
primary amines (pKa 9–11) are positively charged ammonium
groups, complexing the DNA fragment through ionic interaction
with negatively charged phosphate groups of nucleic acids. The
entry into the cell via endocytosis is optimized since protonated
residues on the resulting compact complexes favours the binding
to the negatively charged cell surface. Thereafter, the tertiary
amines (pKa 5–8) of the dendrimer framework are responsible for
a ‘‘proton sponge’’ behavior at the intracellular level. Their
protonation protects the DNA from degradation in an endo-
somal compartment (pH 5–6). Both, DNA complexation for the
endocytosis process and proton sponge behaviour, would explain
the efficiency found with the third generation system.6,17,35
Comparing the three dendritic generations attached to the
MNPs, it is observed that gene transfection occurs when the
amine content is higher than a threshold amine value, which is
the case for the third generation nanosystem (see organic matter
percentage, Table 1). The magnetic field was essential for the
gene delivery in short periods of time. Exposure to the magnet for
up to 20 min produced significant transfection efficiency, and no
transfection was observed for the same delivery time in the
absence of the magnetic field (Fig. 4).
Experimental
Preparation of magnetic nanoparticles
Magnetic nanoparticles (MNPs) were prepared by controlled
coprecipitation of FeII and FeIII ions and subsequent oxidation,
resulting in a nanometric maghemite (g-Fe2O3) containing-fer-
rofluid.29,36,37 Briefly, NH4OH (75 mL, 30% wt.) was added under
vigorous stirring to an aqueous solution of ferric chloride
(875 mL, 21.51 g FeCl3$6H2O) and ferrous chloride (42.5 mL
HCl 1.5 M, 7.83 g FeCl2$4H2O) to obtain a black precipitate.
The precipitate was magnetically decanted and the particles were
oxidized to maghemite (g-Fe2O3) by the addition of a solution of
iron nitrate (150 mL, 20.41 g Fe(NO3)3$9H2O) and heating at
90 ꢁC for 30 min. After that, the product was washed with
acetone several times, and finally dispersed in water/HNO3 at pH
3 to a concentration of 311 mg mLꢀ1. The ferrofluid so-obtained
was composed of magnetic nanoparticles with an average
diameter of 8 nm at pH 3, as measured by dynamic light scat-
tering. The isoelectric point of the resulting ferrofluid occurs at
neutral pH (pH ¼ 7.1), as derived from z-potential results.
Magnetic measurements from this ferrofluid are characteristic of
a superparamagnetic material since no hysteresis loop is dis-
played, and saturation magnetization is equal to 58 emu gꢀ1
(62 emu gꢀ1 for the dry powdered nanoparticles). Specific surface
area of the dry powdered sample measured by nitrogen adsorp-
The biocompatibility of DNA/G3-MNPs complex was evalu-
ated by the analysis of cell viability and apoptosis by flow
cytometry after magnetofection (Fig. 5.A). The results demon-
strated that the treatment produced a slight decrease in viable
tion analysis was 140 m2 gꢀ1
.
Synthesis of silylated dendrimers G1–G3
Alkoxysilane partial functionalization of the first, second and
third generation of poly(propyleneimine) dendrimers,
DAB(NH2)x (x ¼ 4, 8, 16), was carried out.38 As an example, the
synthesis of G3 is described: Under an inert atmosphere of N2
a solution of 3-isocyanatopropyltriethoxysilane (0.0809 g, 82 mL,
0.327 mmol) in dry CH2Cl2 (45 mL) was added dropwise (over
a 3 h period of time) to a rapidly stirred solution of DAB-3-
(NH2)16 (0.5516 g, 0.327 mmol) in dry CH2Cl2 (45 mL). The
reaction mixture was stirred overnight at room temperature and
Fig. 4 Transfection efficiencies for the DNA/G(1,2,3)-MNPs complexes
(1/5 weight ratio) for 20 min of incubation time (A) without magnetic
stimulus and (B) under magnetic stimulus.
4602 | J. Mater. Chem., 2011, 21, 4598–4604
This journal is ª The Royal Society of Chemistry 2011

View Article Online
then filtered off (under an inert atmosphere). To this solution
EtOH was added and CH2Cl2 removal under vacuum afforded
the dendritic precursor dissolved in 100 mL of EtOH, ready to be
immediately used in the next step.
isolated DNA was resuspended in Tris-EDTA (pH 8.0) at
a concentration of 1 mg mLꢀ1
.
Human Saos-2 osteoblasts were used for magnetofection test.
Saos-2 were seeded on 6 well culture plates (CULTEK S.L.U.,
Madrid, Spain, reference 153516, growth surface of 11 cm2/well),
at a density of 105 cells/well, in 2 mL growth medium at 37 ꢁC for
24 h prior to the experiment. Cells were approximately 50%
confluent at the time of transfection.
Attachment of dendrimers onto the iron oxide nanoparticles
surface
The amount of alkoxysilane derivative required to functionalize
0.50 g of MNPs was calculated to achieve a maximum surface
coverage, i.e., a 100% nominal degree of surface functionaliza-
tion. Therefore, it was taken into account the specific surface
area of the isolated powder materials, an average surface
concentration of Fe–OH groups in g-Fe2O3 of 14 mmol –OH per
The complexes were prepared by gently mixing 1 mg of pEGFP
and G1-MNPs, G2-MNPs and G3-MNPs materials at weight
ratios of 1/2.5, 1/5, 1/10 and 1/20 in Tris-EDTA buffer. The
complex mixtures were incubated 30 min at RT. An equal
volume of OptiMEM-1 medium (CE, Invitrogen S A, Spain) was
added to these DNA/G1-MNPs, DNA/G2-MNPs and DNA/
G3-MNPs complex solutions which were incubated for another
30 min at RT. Cells were washed three times with Phosphate
Buffer Saline (PBS) and 1.4 mL OptiMEM-1 were added by well.
Then, 100 ml of each DNA/G(1,2,3)-MNPs complex solution
were added to the cultures resulting in a final volume of 1.5 mL.
In a typical magnetofection experiment, the cell culture plate
was placed upon the magnetic plate for 20 min. After that, the
transfection mixture containing residual DNA/G(1,2,3)-MNPs
complexes was removed and the transfected cells were cultured
for 2 days with DMEM supplemented with 10% calf serum and
antibiotics in order to evaluate the enhanced green fluorescent
protein (EGFP) expression by flow cytometry and confocal
microscopy. Controls either in the absence or presence of
G(1,2,3)-MNPs materials or pEGFP plasmid were carried out.
Also, control transfection experiments with Escort IV Trans-
fection Reagent (Sigma, non-magnetic vector) were carried out
obtaining 17% transfection efficiency after 6 h of delivery time.
29
m2 and the stoichiometry of three Fe–OH groups with one
dendritic molecule or –Si(OEt)3.
The surface modification of g-Fe2O3 NPs was performed
following a sol–gel based wet-chemistry synthetic route, under
basic pH conditions.30,39 The functionalization is performed at
pH ¼ 11, far enough off the IEP of the bare g-Fe2O3, so the
surface chemical reactions are performed on isolated NPs not on
flocculates.
A solution of the calculated amount of alkoxysilane derivative
(APTS, G1, G2 or G3) in 100 mL of EtOH was added under
stirring to 100 mL of the maghemite ferrofluid containing 0.5 g of
MNPs. The pH value was set to 11.0 with ammonia solution 30%
wt and the reaction was then placed in an ultrasonic bath for 5 h
ꢁ
(T ¼ ca. 50 C). The resulting MNPs were thoroughly washed
with EtOH–H2O and water, assisted by magnetic separation, to
remove any non-covalently bound and unbound organic mole-
cules (silylated or non-silylated dendrimers), and finally left in
aqueous media (pH ¼ 3, HNO3) as colloidal solutions. Prior to
the in vitro transfection experiments the pH of the colloidal
dendrimer-MNPs solutions was adjusted to 7.4, and the iron
content of the resulting ferrofluids was determined by atomic
emission spectroscopy.
Analysis of biocompatibility and transfection efficiency of
G(1,2,3)-MNPs by flow cytometry
Dendrimer-MNPs colloidal solutions were characterized by
means of atomic emission spectroscopy, magnetic measurements,
dynamic light scattering and zeta-potential measurements. Dry
samples for solid state characterization (XRD, TGA, DTA,
chemical microanalysis and FTIR) were prepared by extensive
dialysis against water and subsequently dried and grounded.
Remarkably, G3 functionalized MNPs show excellent water
solubility after drying.
In order to evaluate the percentage of transfected osteoblasts and
the material biocompatibility, the medium was aspirated after
magnetofection and the cells were washed with PBS and har-
vested using 0.25% trypsin-EDTA solution. After 15 min, the
reaction was stopped with culture medium and the cells were then
centrifuged at 310 ꢂ g for 10 min and resuspended in fresh
medium for the analysis of EGFP expression, cell viability and
apoptosis by flow cytometry. The number of transfected cells was
obtained by the EGFP fluorescence analysis in a FacScalibur
Becton Dickinson flow cytometer. Cell viability was determined
by addition of propidium iodide (PI; 0.005% in PBS, Sigma-
Aldrich Corporation, St. Louis, MO, USA) to stain the DNA of
dead cells. The SubG1 fraction of the cell cycle, used as indicative
of apoptosis, was evaluated by incubation of cells with Hoechst
33258 (PolySciences, Inc., Warrington, PA, USA) (Hoechst 5 mg
mLꢀ1, ethanol 30%, and BSA 1% in PBS) for 30 min at room
temperature in darkness. The fluorescence of Hoechst was
excited at 350 nm and the emitted fluorescence was measured at
450 nm in an LSR Becton Dickinson flow cytometer. Each
experiment was carried out three times and single representative
experiments are displayed. For statistical significance, at least
10 000 cells were analyzed in each sample and the mean of the
fluorescences emitted by these single cells was used.
Cell culture for magnetofection studies
Human Saos-2 osteoblasts were maintained in T75 flasks using
DMEM (Dulbecco’s modified Eagle’s medium) supplemented
with 10% heat-inactivated fetal bovine serum (FBS, Gibco,
BRL), 1 mM L-glutamine (BioWhittaker Europe, Belgium),
penicillin (200 mg mLꢀ1, BioWhittaker Europe, Belgium), and
streptomycin (200 mg mLꢀ1, BioWhittaker Europe, Belgium),
ꢁ
under a CO2 (5%) atmosphere at 37 C. All cells were passaged
every 3 days.
A red-shifted variant of GFP expressed from pEGFP-N3
(Clontech, Palo Alto, CA) was used as reporter protein. All
plasmids were amplified in the E. coli strain DH5a and purified
according to the manufacturer’s protocol (Qiagen, USA). The
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 4598–4604 | 4603

View Article Online
13 Dendrimer Chemistry: Concepts, Synthesis, Properties, Applications,
Detection of transfected cells by confocal microscopy
€
ed. F. Vogtle, G. Richardt and N. Werner, Wiley-VCH, Weinheim,
Germany, 2009.
In order to observe the transfected osteoblasts after magneto-
fection, cells were examined by a Biorad MC1025 Confocal Laser
Scanning Microscope. The fluorescence of EGFP was excited at
488 nm and the emitted fluorescence was measured at 530/30 nm.
Cell viability was determined by addition of propidium iodide
(PI; 0.005% in PBS, Sigma-Aldrich Corporation, St. Louis, MO,
USA) to stain the DNA of dead cells.
14 D. Astruc, E. Boisselier and C. Ornelas, Chem. Rev., 2010, 110, 1857–
1959.
15 R. K. Tekade, P. V. Kumar and N. K. Jain, Chem. Rev., 2009, 109,
49–87.
16 S. Svenson and D. A. Tomalia, Adv. Drug Delivery Rev., 2005, 57,
2106–2129.
ꢁ
€
17 C. Dufes, I. F. Uchegbu and A. G. Schatzlein, Adv. Drug Delivery
Rev., 2005, 57, 2177–2202.
18 S.-F. Peng, C.-J. Su, M.-C. Wei, C.-Y. Chen, Z.-X. Liao, P.-W. Lee,
H.-L. Chen and H.-W. Sung, Biomaterials, 2010, 31, 5660–5670.
19 D. R. Radu, C. Y. Lai, K. Jeftinija, E. W. Rowe, S. Jeftinija and V. S.-
Y. Lin, J. Am. Chem. Soc., 2004, 126, 13216–13217.
20 A. M. Chen, O. Taratula, D. Wei, H.-I. Yen, T. Thomas,
T. J. Thomas, T. Minko and H. He, ACS Nano, 2010, 4, 3679–
3688.
21 T. Xia, M. Kovochich, M. Liong, H. Meng, S. Kabehie, S. George,
J. I. Zink and A. E. Nel, ACS Nano, 2009, 3, 3273–3286.
22 E. Strable, J. W. M. Bulte, B. Moskowitz, K. Vivekanandan, M. Allen
and T. Douglas, Chem. Mater., 2001, 13, 2201–2209.
23 J. W. M. Bulte, T. Douglas, B. Witwer, S.-C. Zhang, E. Strable,
B. K. Lewis, H. Zywicke, B. Miller, P. van Gelderen,
B. M. Moskowitz, I. D. Duncan and J. A. Frank, Nat. Biotechnol.,
2001, 19, 1141–1147.
Conclusions
In this article we present a novel nonviral vector designed for
DNA magnetofection based on PPI dendrimers and magnetic
nanoparticles. The dendrimers are attached to the nanoparticles
in one synthetic step, without any additional modification of the
iron oxide surface. The nanosystem is chemically designed with
a covalent bond between both components, thus ensuring the
vector stability from the extracellular environment up to the cell
interior. This strategy provides an important advantage, espe-
cially as a genetic engineering tool since DNA can be delivered in
a short time while keeping the structure integrity. The trans-
fection efficiency depends on the capability of the dendrimer
generation to protect and transport the new gene into the
nucleus.
24 X. Shi, T. P. Thomas, L. A. Myc, A. Kotlyar and J. R. Baker Jr.,
Phys. Chem. Chem. Phys., 2007, 9, 5712–5720.
25 R. Abu-Reziq, H. Alper, D. Wang and M. L. Post, J. Am. Chem. Soc.,
2006, 128, 5279–5282.
26 B. Pan, D. Cui, Y. Sheng, C. Ozkan, F. Gao, R. He, Q. Li, P. Xu and
T. Huang, Cancer Res., 2007, 67, 8156–8163.
27 S. H. Wang, X. Shi, M. Van Antwerp, Z. Cao, S. D. Swanson, X. Bi
and J. R. Baker Jr., Adv. Funct. Mater., 2007, 17, 3043–3050.
28 X. Shi, S. H. Wang, S. D. Swanson, S. Ge, Z. Cao, M. E. Van
Antwerp, K. J. Landmark and J. R. Baker Jr., Adv. Mater., 2008,
20, 1671–1678.
29 C. Flesch, M. Joubert, E. Bourgeat-Lami, S. Mornet, E. Duguet,
C. Delaite and P. Dumas, Colloids Surf., A, 2005, 262, 150–157.
30 S. Campelj, D. Makovec and M. Drofenik, J. Magn. Magn. Mater.,
2009, 321, 1346–1350.
Acknowledgements
This work was supported by the Spanish CICYT through the
project MAT2008-00736 and by the CAM through project
S2009/MAT-1472. We thank C. Aroca and M. Maicas (ISOM,
ꢀ
Universidad Politecnica de Madrid) for their technical assistance
in the magnetic measurements.
31 B. Srinivasan and X. Huang, Chirality, 2008, 20, 265–277.
ꢀ
32 A. Lopez-Cruz, C. Barrera, V. L. Calero-DdelC and C. Rinaldi, J.
Mater. Chem., 2009, 19, 6870–6876.
Notes and references
33 D. A. Tomalia, A. M. Naylor and W. A. Goddard III, Angew. Chem.,
Int. Ed. Engl., 1990, 29, 138–175.
34 A. W. Bosman, H. M. Janssen and E. W. Meijer, Chem. Rev., 1999,
99, 1665–1688.
1 A. L. Feldman and S. K. Libutti, Cancer, 2000, 89, 1181–1194.
2 K. Mancuso, W. W. Hauswirth, Q. Li, T. B. Connor,
J. A. Kuchenbecker, M. C. Mauck, J. Neitz and M. Neitz, Nature,
2009, 461, 784–787.
35 B. H. Zinselmeyer, S. P. Mackay, A. G. Schatzlein and I. F. Uchegbu,
Pharm. Res., 2002, 19, 960–967.
36 R. Massart, IEEE Trans. Magn., 1981, 17, 1247–1248.
3 N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. R. Lytton-Jean,
M. S. Han and C. A. Mirkin, Science, 2006, 312, 1027–1030.
€
4 M. Selbach, B. Schwanhausser, N. Thierfelder, Z. Fang, R. Khanin
and N. Rajewsky, Nature, 2008, 455, 58–63.
ꢀ
37 E. Ruiz-Hernandez, A. Lopez-Noriega, D. Arcos, I. Izquierdo-Barba,
O. Terasaki and M. Vallet-Regı, Chem. Mater., 2007, 19, 3455–
ꢀ
ꢀ
5 J. Dennig and E. Duncan, Rev. Mol. Biotechnol., 2002, 90, 339–347.
6 M. A. Mintzer and E. E. Simanek, Chem. Rev., 2009, 109, 259–302.
7 V. Sokolova and M. Epple, Angew. Chem., Int. Ed., 2008, 47, 1382–
1395.
3463.
38 B. Gonzalez, M. Colilla, C. Lopez de Laorden and M. Vallet-Regı, J.
ꢀ
Mater. Chem., 2009, 19, 9012–9024.
ꢀ
ꢀ
€
39 W. Stober, A. Fink and E. Bohn, J. Colloid Interface Sci., 1968, 26,
62–69.
8 D. Lechardeur, A. S. Verkman and G. L. Lukacs, Adv. Drug Delivery
Rev., 2005, 57, 755–767.
9 F. Scherer, M. Anton, U. Schillinger, J. Henke, C. Bergemann,
40 P. Welch and M. Muthukumar, Macromolecules, 1998, 31, 5892–
5897.
41 F. Montagne, O. Mondain-Monval, C. Pichot, H. Mozzanega and
€
€
A. Kruger, B. Gansbacher and C. Plank, Gene Ther., 2002, 9, 102–
109.
€
A. Elaıssari, J. Magn. Magn. Mater., 2002, 250, 302–312.
42 A. Millan, A. Urtizberea, N. J. O. Silva, F. Palacio, V. S. Amaral,
E. Snoeck and V. Serin, J. Magn. Magn. Mater., 2007, 312, L5–L9.
10 B. Steitz, H. Hofmann, S. W. Kamau, P. O. Hassa, M. O. Hottiger,
B. von Rechenberg, M. Hofmann-Amtenbrink and A. Petri-Fink, J.
Magn. Magn. Mater., 2007, 311, 300–305.
€
43 A.-H. Lu, E. L. Salabas and F. Schuth, Angew. Chem., Int. Ed., 2007,
11 M. Arsianti, M. Lim, C. P. Marquis and R. Amal, Langmuir, 2010, 26,
7314–7326.
12 R. Namgung, K. Singha, M. K. Yu, S. Jon, Y. S. Kim, Y. Ahn, I.-
K. Park and W. J. Kim, Biomaterials, 2010, 31, 4204–4213.
46, 1222–1244.
44 P. Orth, A. Weimer, G. Kaul, D. Kohn, M. Cucchiarini and
H. Madry, Mol. Biotechnol., 2008, 38, 137–144.
4604 | J. Mater. Chem., 2011, 21, 4598–4604
This journal is ª The Royal Society of Chemistry 2011