Silica-iron oxide magnetic nanoparticles modified for gene delivery: a search for optimum and quantitative criteria.

Abstract

To optimize silica-iron oxide magnetic nanoparticles with surface phosphonate groups decorated with 25-kD branched polyethylenimine (PEI) for gene delivery.
Surface composition, charge, colloidal stabilities, associations with adenovirus, magneto-tranduction efficiencies, cell internalizations, in vitro toxicities and MRI relaxivities were tested for the particles decorated with varying amounts of PEI.
Moderate PEI-decoration of MNPs results in charge reversal and destabilization. Analysis of space and time resolved concentration changes during centrifugation clearly revealed that at >5% PEI loading flocculation gradually decreases and sufficient stabilization is achieved at >10%. The association with adenovirus occurred efficiently at levels over 5% PEI, resulting in the complexes stable in 50% FCS at a PEI-to-iron w/w ratio of ≥7%; the maximum magneto-transduction efficiency was achieved at 9-12% PEI. Primary silica iron oxide nanoparticles and those with 11.5% PEI demonstrated excellent r(2)* relaxivity values (>600 s(-1)(mM Fe)(-1)) for the free and cell-internalized particles.
Surface decoration of the silica-iron oxide nanoparticles with a PEI-to-iron w/w ratio of 10-12% yields stable aqueous suspensions, allows for efficient viral gene delivery and labeled cell detection by MRI.

Full text

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RESEARCH PAPER
Silica-Iron Oxide Magnetic Nanoparticles Modified for Gene
Delivery: A Search for Optimum and Quantitative Criteria
Olga Mykhaylyk & Titus Sobisch & Isabella Almstätter & Yolanda Sanchez-Antequera & Sabine Brandt & Martina Anton &
Markus Döblinger & Dietmar Eberbeck & Marcus Settles & Rickmer Braren & Dietmar Lerche & Christian Plank
Received: 4 August 2011 /Accepted: 19 December 2011
# Springer Science+Business Media, LLC 2012
ABSTRACT
Purpose To optimize silica-iron oxide magnetic nanoparticles
with surface phosphonate groups decorated with 25-kD
branched polyethylenimine (PEI) for gene delivery.
Methods Surface composition, charge, colloidal stabilities,
associations with adenovirus, magneto-tranduction efficiencies,
cell internalizations, in vitro toxicities and MRI relaxivities were
tested for the particles decorated with varying amounts of PEI.
Results Moderate PEI-decoration of MNPs results in charge
reversal and destabilization. Analysis of space and time resolved
concentration changes during centrifugation clearly revealed that
at >5% PEI loading flocculation gradually decreases and sufficient
stabilization is achieved at >10%. The association with adenovi-
rus occurred efficiently at levels over 5% PEI, resulting in the
complexes stable in 50% FCS at a PEI-to-iron w/w ratio of ≥7%;
the maximum magneto-transduction efficiency was achieved at
9–12% PEI. Primarysilicaironoxide nanoparticlesand those with
11.5% PEI demonstrated excellent r2* relaxivity values
(>600 s−1(mM Fe)−1) for the free and cell-internalized particles.
Conclusions Surface decoration of the silica-iron oxide nano-
particles with a PEI-to-iron w/w ratio of 10-12% yields stable
aqueous suspensions, allows for efficient viral gene delivery and
labeled cell detection by MRI.
KEY WORDS colloidalstability.magneticnanoparticles.
MRIrelaxivity.silica-polyethyleniminecoating.transduction
ABBREVIATIONS
ATCC
DMEM
EDTA
FCS
ME-FFE
MNP
mPDAC
MRI
PBS
PEI
SIO-MNP
SiOx
American Type Culture Collection
Dulbecco’s modified Eagle’s medium
Ethylenediaminetetraacetic acid
fetal calf serum
multi-echo gradient echo
magnetic nanoparticles
mouse pancreatic ductal adenocarcinoma
magnetic resonance imaging
Dulbecco’s phosphate buffered saline
polyethylenimine
silica-iron oxide magnetic nanoparticle
silica-like coating of the iron oxide
nanoparticles
Transmission Electron Microscopy
tetraethyl orthosilicate
3-(trihydroxysilyl) propylmethylphosphonate
transducing units
virus particle
X-ray photoelectron spectroscopy
TEM
TEOS
THPMP
TU
VP
XPS
Electronic supplementary material The online version of this article
(doi:10.1007/s11095-011-0661-9) contains supplementary material,
which is available to authorized users.
O. Mykhaylyk (*):Y. Sanchez-Antequera:S. Brandt:M. Anton:
C. Plank
Institute of Experimental Oncology and Therapy Research
Klinikum rechts der Isar der Technischen Universität München
Ismaningerstrasse 22
81675 Munich, Germany
e-mail: olga.mykhaylyk@lrz.tu-muenchen.de
T. Sobisch:D. Lerche
LUM GmbH
Rudower Chaussee 29 (OWZ), 12489 Berlin, Germany
I. Almstätter:M. Settles:R. Braren
Department of Radiology, Klinikum rechts der Isar, Technische
Universität München( Munich, Germany
M. Döblinger
Department of Chemistry, Ludwig-Maximilians-Universität München
Munich 81377, Germany
D. Eberbeck
Physikalisch-Technische Bundesanstalt
Abbestraße 2-12, 10587 Berlin, Germany
Pharm Res
DOI 10.1007/s11095-011-0661-9

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INTRODUCTION
Since the first publications on magnetically enhanced
nucleicaciddelivery,alsoknownasmagnetofection,numerous
studies have confirmed the utility of the technique and
have greatly expanded its scope as a research tool. For
magnetofection,vectorsfornucleicaciddeliveryareassociated
with magnetic nanoparticles and the delivery process is
directed and enhanced by the application of gradient
magnetic fields. This procedure sediments the full applied
vector dose on target cells in culture within minutes and is
suitable to accumulate orretain avectordoseina targettissue
in vivo. Magnetofection leads to enhanced cellular uptake,
improved dose-response profiles and transfection/transduc-
tion kinetics of virtually any vector type (1).
Two types of magnetic nanomaterials are commonly
used to magnetize gene delivery vectors. These include
functionalized polymeric micro- and nanoparticles with
entrapped iron oxide nanoparticles (2,3) and magnetic iron
oxide nanoparticles of the core-shell type (4). The assembly
of magnetic (nano) particles with nucleic acids or viral
particles occurs due to electrostatic and hydrophobic
interactions (4) and specific ligand-ligand interactions
(2,3,5). The first magnetic core-shell nanoparticles that
were designed for gene delivery were transMAGPEIiron
oxide nanoparticles that were stabilized with high molecular
weight polyethylenimine (PEI) of 800 kD (4). These and other
particles that were modified using PEIs of different molecular
weights readily associated with lentiviral, retroviral, adenovi-
ral and measles virus vectors and allowed for efficient in vitro
magnetic transduction (4,6–8). A major limitation in
polyethylenimine-mediated gene delivery is dose-dependent
cytotoxicity (9–11). Therefore, further optimizations in nano-
particle design and formulation are important objectives in
terms of efficiency in gene delivery, cell viability, and in the
case of stem cells for the preservation of pluripotency upon
magnetofection (1,8,12). The nature of the surface coating
components and their arrangement in the surface layer of the
nanoparticles were shown to play a crucial role in the efficacy
of the magnetic vectors (13,14). Recently we have demon-
strated significant effects of nanoparticle coatings on the
activity of the adenovirus-magnetic nanoparticle complexes
in vitro and in vivo (15) as well as on the efficacy of gene delivery
to Jurkat T cells using non-viral vectors associated with
magnetic nanoparticles (16).
Mesoporous silica particles have been developed recently
in nanotechnology for drug/gene delivery and biosensing
(17,18). Silica coatings are known to result in well stabilized,
high quality aqueous suspensions of iron oxide nanoparticles
(19,20) and could improve the chemical stability of the
nanoparticles and reduce their toxicity (21,22).
Composite silica particles with entrapped magnetite
nanocrystals have been engineered (23). The coating of the
magnetite “capped” mesoporous silica nanoparticles with
25-kD polyethylenimine and their subsequent association
with DNA resulted in the effective transfection of H292
human lung mucoepidermoid carcinoma cells with efficien-
cies that were superior to those obtained with the Polymag™
and Lipofectamine 2000 systems (24).
We sought to design magnetic core-shell nanoparticles
with an iron oxide core that combined the positive charac-
teristics of the silica and polyethylene coatings and that were
efficient in gene delivery. We synthesized iron oxide nano-
particles by precipitating Fe(II)/Fe(III) hydroxide in an
aqueous solution for their transformation into magnetic iron
oxide, followed by the hydrolysis and condensation of
tetraethyl orthosilicate (TEOS) to produce siloxane bonds
(Si-O-Si) and to promote condensation into the (SiOx)
silica-like coatings of the iron oxide nanoparticles. The
co-condensation of 3-(trihydroxysilyl) propylmethyl-
phosphonate (THPMP) yielded a silicon oxide layer with
surface phosphonate groups (SiOx/Phosphonate) and
endowed the aqueous suspension with a highly negative
electrokinetic potential of the particles as shown schematically
in Fig. 1. We further modified these particles by the addition
of 5w% of 25-kD branched PEI based on the iron weights of
the particles, resulting in a charge reversal. This material,
which we have described previously (25), demonstrated high
transfection efficiencies in vitro as a component of magnetic
lipoplexes (25). The lentiviral complexes of the PEI-modified
silica-ironoxidemagneticnanoparticlesallowedfortheexcellent
transduction of cultured and primary cells as hematopoietic
stem cells and human mesenchymal cells in a procedure that
we have termed magselectofection (26). Recently, we have
shown that the efficiency of an oncolytic adenovirus can be
dramatically increased in vitro and in vivo when combined with
PEI-decorated silica-iron oxide nanoparticles (15).
The drawback of this system is that the electrokinetic
potentials of the particles titrated with 5w% PEI in the water
suspension and colloidal stabilities decrease during storage,
resulting in a gradual loss of the magnetofection capacity of
the particles. The agglomeration and destabilization of silica
nanoparticle suspensions are known to be serious problems
(27,28), which can be even more pronounced for magnetic
silica-stabilized nanoparticles because of the magnetic
dipole-dipole interparticle interactions. Therefore, we have
been highly motivated to search for the optimal formulation
of PEI-decorated silica iron oxide nanoparticles. Parameters
such as surface charge, colloidal stability, association with
viral vectors, stability of the complexes at high serum con-
centrations and transduction efficiencies were used for the
optimization of the particle/vector assembly. We have also
performed imaging experiments on a clinical 1.5 T MRI
system, to evaluate the MRI contrast relaxivities of the free
and cell internalized primary silica-iron oxide nanoparticles
and the particles that were decorated with PEI.
Mykhaylyk et al.

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MATERIALS AND METHODS
Materials
Dulbecco’s modified Eagle’s medium (DMEM), L-
glutamine, Dulbecco’s phosphate buffered saline (PBS),
minimal essential vitamins, and 0.25% trypsin/0.02%
EDTA solutions were obtained from Biochrom AG
(Berlin, Germany). Sodium
purchased from Amersham Biosciences (New South
Wales, Australia), fetal calf serum (FCS) was purchased
from PAN-Biotech (Aidenbach, Germany), Triton X-100
was purchased from AppliChem (Darmstadt, Germany),
and D-luciferin was purchased from Synchem OHG
(Felsberg, Germany). All other chemicals were of analytical
grade and used without further purification (Sigma-Aldrich,
Steinheim, Germany). Tissue culture plates and flasks were
obtained from Techno Plastic Products (Trasadingen,
Switzerland). A 96-magnet plate with a permanent magnetic
field with a field strength and gradient of 70−250 mT and 50
−130 T/m,respectively, which was generated at the cell layer
location, was supplied by OZ Biosciences (Marseille, France).
125iodide in NaOH was
Synthesis and Modification of the Core-Shell-Type
Silica-Iron Oxide Magnetic Nanoparticles
Core-shell-type silica-iron oxide magnetic nanoparticles
(MNPs) were synthesized as previously described with slight
modifications (25) by precipitation of Fe(II)/Fe(III) hydroxide
from the aqueous solution of the mixture of Fe(II) and Fe(III)
salts followed by transformation into magnetite in an oxygen-
free atmosphere. The surface coating resulted from the
condensation of tetraethyl orthosilicate (TEOS) and 3-
(trihydroxysilyl) propylmethylphosphonate (THPMP),
yielding a silicon oxide layer with surface phosphonate
groups (SiOx/Phosphonate). Briefly, 25 mmol (6.8 g) of
ferric chloride hexahydrate and 12.5 mmol (2.5 g) of
ferrous chloride tetrahydrate in 200 ml ddH2O water
that was filtered using a 0.2-μm filter flask under argon
gas were treated with 15 ml concentrated ammonium
hydroxide to obtain a primary precipitate. The material
was heated to 90°C for 15 min and then stirred at this
temperature for 30 min. To form a coating, 375 μl of
TEOS (1.7 mmol) was added. After 30 min, 750 μl of
42% THPMP solution (1.3 mmol) was added, and the
mixture was further incubated at 90°C for 30 min,
cooled to 25°C, diluted two-fold with ethanol, and incubated
for24hwithcontinuousstirring.Theparticleswereseparated
by exposure to a gradient magnetic field and washed twice
with ethanol and once with water. The product was sonicated
for 10 min using a resonance frequency of approximately
20 kHz at 75 mW with impulses applied at 60 s/30 s intervals
anddialyzedextensivelyagainstwaterusingaSpectra/Por®6
50-kD cut-off dialysis membrane. The MNP suspension was
sterilized using
25 kGy.
The decoration of the surface of the SO-Mag5 particles
with 25-kD branched polyethylenimine (PEI) at a PEI-to-
iron w/w ratio of n% resulted in SO-Mag6-n particles with
SiOx/Phosphonate-PEI coatings. A sterile-filtered (0.2 μm)
PEI solution in water with a pH of 7.3 (HCl) was added to
an equal volume of MNP suspension after vortexing to
60Co gamma-irradiation at a dosage of
Fig. 1 Schematics of silica-iron oxide magnetic nanoparticles modified for gene delivery. Magnetic iron oxide nanoparticles with a coating resulting from
hydrolization and condensation of tetraethyl orthosilicate and 3-(trihydroxysilyl)propylmethyl phosphonate are further “decorated” with 25-kD branched
polyethylenimine at different PEI-to-iron w/w% ratios n yielding the SO-Mag6-n particles suitable for association with gene delivery vectors.
Silica-Iron Oxide Nanoparticles for Gene Delivery

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obtain the required PEI-to-iron ratio. The reagents were
mixed in opposite order (MNP suspension into PEI solution)
only if indicated.
Physico-Chemical Characterization of the MNPs
Iron Analysis
Iron concentration per unit weight of dry nanomaterials and
MNP concentration in the suspensions as related to iron
concentration per unit volume were determined spectropho-
tometrically by forming complexes with 1,10-phenanthro-
line as described previously (29). The concentrations of the
shell components were indirectly calculated from the iron
concentrations and accounting for the X-ray diffraction
data from the phase composition of the core.
Transmission Electron Microscopy
To activate the grid and to remove hydrocarbon con-
tamination, a formvar/carbon coated 400 mesh copper
grid was treated by a hydrogen/oxygen plasma of
50 mW for 30 s (Solarus Model 950, Gatan GmbH). A
5-μL drop of the SO-Mag5 nanoparticle suspension con-
taining 25 μg Fe/ml was placed onto the grid and
incubated for 10 min. Samples were rinsed carefully with
two drops of double-distilled water and air-dried prior to
imaging at an accelerating voltage of 80 kV using a
TITAN 80–300 S/TEM (FEI Company).
X-Ray Diffraction
X-ray diffraction patterns were obtained for the powdered
sample using a Burevestnik DRON-4-07 diffractometer
(St.-Petersburg, Russia) with nickel-filtered CuKαirradiation.
The mean crystallite size 〈d〉 was calculated from the
broadening of the X-ray diffraction (XRD) peaks using
the Scherer formula.
Magnetic Structure of the SO-Mag5 Nanoparticles
The static magnetic properties of the SO-Mag5 particles
were evaluated by measuring the quasistatic magnetization
in the applied DC-fields or M(H) by using the MPMS
commercial susceptometer (Quantum Design, USA). The
dynamic magnetic properties of the particles were studied
by the magnetorelaxometry (MRX) method using a device
that was described previously (30). In short, the magnetic
moments of the MNPs in a sample are aligned in an
external magnetic field of approximately 2 kA/m. After
switching off the field, the decay of the magnetization
within approximately 500 μs is measured by a low-Tc
SQUID at T0295 K.
The relaxation of the non-interacting immobilized MNPs
is governed by the Néel relaxation time τN0τ0exp{KV/kBT}
(kBbeing the Boltzmann constant), which is determined by
the effective anisotropy energy that is represented by the
equation EA0K V. The observed relaxation of the magneti-
zation is the superposition of the relaxation of the particles
with various anisotropy energies. The moment superposi-
tion model (MSM) (31) considers the anisotropy constant K
to be independent of the particle size and superimposes the
relaxation contributions of each size. By fitting the median μ
and distribution parameter σ of the lognormal size distribu-
tion to the relaxation curve using the MSM, the size distri-
bution can be reconstructed. Note that this size distribution
considers the effective magnetic sizes to be equal to the core
sizes for proper single domain MNPs that lack magnetic
correlations among each other.
Using a similar MSM for the description of the M(H)-
data of noninteracting MNPs (32);
MðHÞ ¼ fMS
1
V
Z
0
1
f ðμ;σ;dÞp
6d3Lðd;MS;T;HÞdd þ APMP
ð1Þ
where L, T, and H denote the Langevin function, temperature,
andexternalfieldstrength,respectively,andusingd0(6V/π)1/3,
we fitted μ and σ of the distribution f(μ,σ,d). Here, we
added a paramagnetic signal MPwith the arbitrary amplitude
AP(additional fit parameter), representing the magnetization
behavior of a non-ferrimagnetic portion of the MNP,
which is often denoted as a magnetic “dead layer”.
Then, the mean diameter corresponding to the volume
V of the magnetic portion of a core particle, dmV; i.e., the
size of its effective magnetic domain, was estimated. The
volume fraction ϕ01/3 cFeMMρM−1was estimated from
the density ρMand molar mass MMof the bulk magnetite
and iron concentration (cFe) of the sample, which was
determined by Prussian Blue Staining in combination
with a light absorption measurement at 690 nm.
Dynamic Light Scattering
The mean hydrodynamic diameter (Dh) and electrokinetic
potential (ξ) of the coated MNPs were measured by photon
correlation spectroscopy (PCS) using a Malvern Zetasizer
Nano Series 3000 HS (Malvern Instruments GmbH,
Herrenberg, Germany).
X-Ray Photoelectron Spectra
X-ray photoelectron spectra (XPS) were recorded using a
Kratos analytical electron spectrometer (Manchester, UK)
that employs monochromated AlKα excitation (1486.6 eV)
Mykhaylyk et al.

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andanalyzestheelementalcompositionofthesurfacecoating.
The samples were allowed to dry on aluminium foil and
measured in the electrostatic-mode. An instrument vacuum
of at least 10−9mbarwas maintained during the analysis. The
relative elemental surface composition was calculated based
on the Fe2p peak.
Evaluation of the Stability of MNP Suspensions
by STEP-Technology Combined with Multisample
Analytical Centrifugation
The time- and space-resolved detection of light transmission
(STEP-Technology) combined with multisample analytical
centrifugation (LUMiSizer, LUM, Germany (33,34)) was
applied for the analysis of the colloidal stability and
determination of the particle size distributions of the
MNP suspensions (35). The shapes and progressions of
the transmission profiles of the MNP suspensions that
were measured during centrifugation along the optical
cuvette contained the information regarding the state of
a dispersion, like flocculation and particle properties. The
transmission profiles are representative of the distribution of
the particle concentrations over the entire sample length.
Based on this, sedimentation and clarification kinetics and
the velocity and particle size distributions can be quantified.
Particle-Virus Interaction
Viral Vectors
The third generation, self-inactivating lentivirus vector
LVSFeGFPexpressingeGFPundercontroloftheconstitutive
spleen focus-forming virus LTR promoter was produced fol-
lowing a protocol that was established by Wübbenhorst et al.
(36). The transfer vector pHIV-7SFeGFP was packaged by
the transient calcium phosphate co-transfection of 293T cells
using a mixture of HIV-1-derived pMD.GP (Gag-Pol),
pRSV-rev and the VSV-pseudotyping pMD.G packaging
constructs. The harvest containing the viral particles was
filtered through a 0.45-μm filter, and the resulting virus stock,
with an infectivity of 8.1×106TU/ml, was stored at −80°C
in aliquots. The biological titer of the eGFP lentivirus
(infectious particles/ml or transducing units/ml0TU/ml)
was determined in CMS5 cells as described by Barry et al. (37)
with modifications. Similarly, the third generation, self-
inactivating lentivirus vector LVSFeGFPLuc expressing a
fusion protein that consisted of eGFP and firefly luciferase
reporters was produced. The stock of the lentivirus contained
1.0×109vector particles (VP/ml) with an infectivity of
1.2×106TU/ml.
An adenoviral vector expressing the chimeric green
fluorescent protein fused to HSV1 thymidine kinase
(AdV-(TK/GFP)(fus)) that was driven by the mCMV
promoterwasconstructedandkindlyprovidedbyDr.Rodolfo
Goya (38) and will further be referred to as the AdV vector.
The AdV vector was expanded in 293 cells and purified by
double cesium chloride gradient centrifugation according to
Hitt et al. (39) with modifications, resulting in a virus stock
containing 4.3×1012VP/ml and 2.6×1011TU/ml. To
determine the virus particle titer (40), the aliquot of the
virus stock was diluted 1 to 20 in PBS that contained
0.1% sodium dodecyl sulphate, mixed thoroughly for
2 min and centrifuged at 8000 g for 5 min. The optical
density at 260 nm was measured, and the physical virus
titer was calculated, taking into account that an OD of 1
corresponds to 1.1×1012VP/ml. Aliquots of the stock
were stored at −80°C.
Radioactive Labeling of the Adenovirus
TheAdVadenoviruswaslabeledwithradioactive125iodideas
previously described (41). Briefly, 100 μl of the virus stock
containing 9.8×1010infectious units per ml and 4.4×
1012VP/ml was mixed with 4 μl of sodium
(1 mCi in 10 μl, Amersham Biosciences) in a iodogene tube
(Thermo Scientific Pierce Protein Research Products) and
incubated with gentle agitation for 40 min. After adjusting
the volume to 500 μL with PBS, the labeled virus was sepa-
rated from unbound label by gel filtration using a PD-10
column (GE Healthcare) that was preequilibrated with PBS.
The product fraction containing 1.23×1011VP/ml with a
radioactivity of 2,833 kBq/ml (determined using a Wallac
1480 Wizard 3 automatic gamma counter, Finland) was used
to further quantify the virus association with the MNPs.
125iodide
Adenovirus Association and Magnetic Sedimentation
with Magnetic Nanoparticles and Stability of the Complexes
in the Presence of FCS
For the binding studies, 50 μl of a 2-to-3 dilution series of
the MNPs in ddH2O (0−60 μg of Fe) were mixed with
250 μl of125I-labeled AdV suspended in PBS (1.2 VP/ml,
5×104CPM/ml) in a U-bottom 96-well plate at virus con-
centrations of 1.0×109VP/ml at the time of assembly and
incubated for 20 min at room temperature (RT) to form
magnetic virus complexes at MNP-to-virus ratios of 0−200
fg of Fe/VP. One hundred fifty microliters of the resulting
complexes were transferred into the wells of a new U-bottom
96-well plate containing 150 μl of FCS, followed by gentle
mixing and a 30 min incubation. The U-bottom plate was
positioned on a 96-magnet plate for 30 min prior to the
collection of 50 μl of each supernatant for gamma counting
using the gamma counter. The percentages of adenovirus
particles that associated and magnetically sedimented
with the MNPs in PBS or 50% FCS were calculated as
described in (41).
Silica-Iron Oxide Nanoparticles for Gene Delivery

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Cell Culture
SH-SY5Y human neuroblastoma cells that were used for
the in vitro experiments (further referred to as SH-SY5Y
cells) were grown in DMEM supplemented with 15% FCS
(Gibco), 2 mM L-glutamine and 1% non-essential amino
acids. McA-RH7777 rat hepatocellular carcinoma (HCC)
cells obtained from the ATCC (LGC Standards GmbH,
Wesel, Germany), CMS5, 293 and 293T cells were cultured
in Dulbecco’s modified Eagle medium (DMEM) (Gibco)
that was supplemented with 10% heat-inactivated FCS
(Gibco), 1% non-essential amino acids (PAA), 100 U ml−1
penicillin (PAA), and 100 μg ml−1streptomycin (PAA). This
medium will hereafter be referred to as the cell culture
medium. Mouse pancreatic ductal adenocarcinoma cells
were extracted as primary cells from a CKp53lox mouse
(Ptf1awt/Cre; Kraswt/LSL-G12D; p53fl/fl) by Marija Trajkovic-
Arsic, II. Med. Clinic, Gastroenterology, Klinikum rechts
der Isar, Technische Universität München (referred to as
511181 cells) and were cultured in the same cell culture
medium. The cells were grown at 37°C in a humidified
atmosphere containing 5% CO2.
Magnetotransduction Efficiency with Lentiviral
Magnetic Vectors of the Silica-Iron Oxide MNPs
Decorated with PEI
Magnetotransduction In Vitro
SH-SY5Y cells were seeded into a 96-well plate at a density
of 20,000 cells per well. For the lentiviral magnetotransduc-
tion that was conducted 24 h later, 50 μl of 2-fold serial
dilutions of the magnetic complexes of the lentiviral vector
LVSFeGFPLuc were added per well. To prepare the
SO-Mag6-n/LVSFeGFPLuc magnetic complexes, 72 μl
of the virus stock were mixed with 6 μl of the SO-Mag6-n
stock (100 μg Fe/ml) and kept at RT for 20 min to allow for
complex assembly followed by a dilution to 360 μl with
DMEM (without additives), resulting in MNP:VP ratios of
10 fg Fe/VP, 6×107VP/ml and 7.2×104TU/ml. The
complexes were freshly prepared before use. A magnetic
field was applied by positioning the cell culture plate into
the 96-well magnetic plate for 30 min.
Evaluation of the Reporter Gene Expression
To evaluate the transfection efficiency in terms of the per-
centage of eGFP-positive cells, we washed the cells twice
with PBS supplemented with 1% FCS (hereafter referred to
as FACS buffer) at 48 h post-transfection, fixed the cells
using the Cytofix™ Fixation Buffer (Becton Dickinson) and
resuspended the cells in 0.5 ml of FACS buffer for the FACS
analysis using the FACSVantage™ device (Becton
Dickinson, with an argon laser excitation maximum at
488 nm; fluorescence was measured using a 530/30-nm
bandpass filter). At minimum, 20,000 events per sample
were analyzed. The luciferase expression assay on the cell
lysate was performed as described elsewhere (29).
Infectivity of the Magnetic Lentiviral Complexes over the Course
of Storage
To assess the changes that occurred in the infectivity of the
magnetic lentiviral complexes during storage, the magnetic
complexes of the SO-Mag6-10 particles with the self-
inactivating lentivirus vector LVSFeGFP in addition to
the complexes with polybrene (PB) were prepared and
stored in 250 μl aliquots at −4°C or −80°C until the
titer determination in the CMS5 cells, which was carried
out at days 2, 37, 72, 149 of storage. The complexes will
be further referred to as SO-Mag6-10/LV and PB/LV.
Complexes that were freshly prepared each time just
prior to infection (SO-Mag6-10/LV and PB/LV) were
used as references. To prepare the SO-Mag6-10/LV
magnetic complexes, 2,000 μl of the LVSFeGFP stock
(8.1×106TU/ml and 7.8×109VP/ml) were mixed with
16 μl of the SO-Mag6-10 stock (5 mg Fe/ml) and kept at
RT for 20 min to allow for complex assembly and
divided into 250 μl aliquots to be stored or immediately
used for cell transduction. Similarly, 2,000 μl of the
LVSFeGFP stock were mixed with 20 μl of PB stock
solution in water containing 800 μg PB/ml.
For the titer determination, the CMS5 cells were seeded
into a 12-well plate at a density of 100,000 cells per well.
After 24 h, the medium was removed, and 250 μl/well of
serial dilutions of the SO-Mag6-10/LV complexes with the
cell culture medium or of the PB/LV complexes in cell
culture medium containing 8 μg PB/ml were applied. After
2 h, 1 ml cell culture medium per well was added, and the
cells were further incubated for 48 h until sampling for the
FACS analysis. The percentage of eGFP-positive cells was
plotted against the log10of the applied volume of the virus
stock per cell. The dose-response curves were fitted with
logistic functions using the OriginPro 8G software. The
virustiterwascalculatedassumingthatthevirusdoseresulting
in 50% reporter-positive cells corresponded to 0.5 TU/cell.
Particle-Cell Interaction
Cell Labeling with MNPs
SHSY5Y human neuroblastoma cells were seeded at a
density of 700,000 cells per well in a 6-well plate. HCC
and 511181 cells were seeded at a density of 500,000 and
200,000 cells per well, respectively. At 24 h post cell seeding,
the cell culture medium of each well was changed to a MNP
Mykhaylyk et al.

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suspension. The MNPs were diluted in cell culture medium
to achieve the desired applied iron dose per cell. A range of
2–60 μg Fe ml−1was used for the SHSY5Y cells, resulting in
an iron dose of approximately 7.5 to 240 pg Fe/cell. A
range of 12.5–100 μg Fe ml−1was used for the hepatic cells
and 5–80 μg Fe ml−1for the 511181 cells, resulting in an
applied iron dose of approximately 25 to 400 pg Fe per cell
in a 1 to 2 dilution series. After a 24 h incubation, the cells
were washed with PBS and incubated with 100 units ml−1of
a heparin solution that was added to the cell culture medi-
um for 20 min at 37°C to dissociate any loosely bound
nanoparticles. Next, the cells were washed with PBS and
trypsinized using a 0.25% trypsin/0.02% EDTA solution to
analyze the associated/internalized iron.
Based on the results of the saturation curves (Fig. 9b),
cells were labeled for the preparation of MRI phantoms.
HCC cells were seeded at a density of 80,000 cells per cm2
in a 75 cm2dish, while 511181 cells were seeded at a density
of 46,667 cells per cm2in a 75 cm2dish. At 24 h post-cell
seeding, the SO-Mag6-11.5 particle suspension in the cell
culture medium was applied at a dose of 100 pg Fe cell−1.
The MNPs were incubated with the cells for 24 h, washed
twice with PBS to dissociate any loosely bound nanoparticles
and trypsinized. After washing with PBS, the cells were fixed
with BD Cytofix™ (BD Biosciences), washed three times with
PBS and stored at 4°C in PBS/0.5% NaN3until their use in
further analyses. The cells that were used to prepare the
calibration phantoms for MRI imaging were loaded with 39
and 27pgFe/cellforthe HCCand 511181cells,respectively,
for the analysis of exogenic non-heme iron, which was
performed as described below.
Analysis of Cell-Associated/Internalized Iron
For the analysis of the exogenic non-heme iron from the
cell-associated/internalized MNPs, the analysis of the
non-heme iron concentrations of the MNP-labeled cells was
performed as described elsewhere (42). Briefly, approximately
200,000 trypsinized cells were washed with PBS and then
spun down bycentrifugation. The supernatant was discarded,
and the cell pellet was resuspended in 250–500 μl of an acid
mixture containing 3 M HCl and 0.6 M trichloracetic acid.
After an overnight incubation at 65°C, the samples were
centrifuged, and 50 μl of the clear supernatant was analyzed
for its iron concentration by a colorimetric method with
1,10-phenanthroline as previously described (29). Basal
non-heme iron levels that were determined in non-labeled
cells were used as references.
Cytotoxicity Evaluation
The MTT assay, based on the reduction of the MTT
reagent into formazan by superoxide anion radicals that
are produced in the mitochondrial respiratory chain (43),
was carried out as described previously (29) to assess the
cytotoxicity of the MNPs. Briefly, SHSY5Y cells were
seeded at a density of 20,000 cells per well in a 96-well
plate. At 24 h post cell seeding, the cell culture medium
of each well was changed to a MNP suspension. After
48 h incubation with MNPs, cells were washed once with
PBS, the supernatant was discarded, and the cells were
incubated for 1–2 h in 100 μl of 1 mg/ml MTT solution
that was prepared in Hank’s balanced salt solution with
5 mg/ml glucose. Afterwards, 100 μl solubilization solu-
tion (10% Triton X-100 and 0.1 N HCl in anhydrous
isopropanol) was added and incubated at 37°C overnight
with shaking to dissolve the formazan. The optical density
was measured at 590 nm. Untreated cells were used as
references.
Evaluation of MRI Contrast Efficiency of MNPs
Calibration Phantoms for MR Imaging
Calibration phantoms of the MNPs and MNP-labeled cells
for MR imaging were prepared in 24-well plates. Sample
and background wells were arranged in an alternating
order to avoid signal interference of different samples. In
addition spaces in-between wells were filled to avoid
signal interference from air. The background wells and
spaces between the wells were filled with an agarose gel of a
tissue-mimicking composition. According to Christoffersson
et al. (44), tissue-mimicking phantom material can be
prepared by using different concentrations of nickel
and agarose. Increasing the nickel (II) ions concentration
shifts the T1 values to longer relaxation times, and
increasing agarose concentrations results in shorter T2
relaxation times (44). The gel phantom-mimicking relax-
ivity of the rat liver tissue (T10550 ms and T2048 ms)
was prepared with 198 mM Ni(NO3)2, 2.45% agarose
and 0.5% sodium azide. The gel phantom mimicking
the mouse pancreatic ductal adenocarcinoma (mPDAC)
tissue with relaxation times of T101200 ms and T20
115 ms was prepared with 0.53 mM Ni(NO3)2, 1.01%
agarose and 0.5% sodium azide. The 12 background
wells and cavities between the wells on the upside and
bottom were filled with the described Ni-doped agarose
gel.
For the 12 sample wells, a gel containing a 1.5-fold
concentration of nickel and agarose was prepared, which is
further referred to as the internal agarose gel. A 2 to 3
dilution series in water, beginning with 72 μg Fe/ml
(1.3 μM Fe) as the highest iron concentration, was prepared
for both the particles and labeled cells.
Themaximumfinalcellconcentrationoftheliverphantoms
was 6.2×105cells ml−1, and was 8.6×105cells ml−1for the
Silica-Iron Oxide Nanoparticles for Gene Delivery

Page 8

mPDAC phantoms, accounting for the MNP loading of 39
and 28 pg Fe/cell for the McA-RH7777 rat hepatocellular
carcinoma and 511181 mouse PDAC cells, respectively.
The dilutions of the MNPs or MNP-labeled cells and
the internal agarose gel were pre-warmed at 65°C.
Then, 1.5 ml MNP dilution were vortex-mixed with
3 ml internal agarose gel in a 15-ml falcon tube to
distribute the material homogenously and avoid air
bubbles and transferred into the according well. The
plate was allowed to cool down slowly and was sealed
with paraffin to avoid the evaporation of water during
storage at 4°C.
MR Imaging Experiments
All of the imaging experiments were performed using a
clinical 1.5 T MRI system (1.5 T Achieva, Philips Medical
System, Best, The Netherlands) with the 8-channel SENSE
head coil for signal reception. T2and T2*maps of the
calibration phantoms were measured using the following
sequences: for T2, a multi spin echo sequence with TR0
2,000 ms, TE0n * 4.9 ms (n01…30), flip angle090°,
FOV0160×88, resolution01×1×3 mm3, 3 slices of
3 mm thickness with no gap and a total scan time of
6:04 min, and for T2*, a multi-echo gradient echo sequence
(FFE) with TR01,000 ms, TE02.1+n * 3.2 ms (n00…15),
flip angle090°, FOV0160×92, resolution01×1×3 mm3,
and 3 slices of 3-mm thickness with no gap and a total scan
time of 4:40 min.
T2maps were calculated from the multi spin-echo data
using the standard MR scanner mono-exponential fitting
routine. For the T2*maps, the complex data of the multi-
echo gradient-echo sequence were analyzed using the
RelaxMapsTool from the Philips PRIDE data evaluation
software package. This tool calculates B0maps for all slices
and, as a first order deviation from a mono-exponential
signal decay, takes into account the sink-shaped oscillation
of the multi echo signal that is induced by the through plane
B0gradient (45).
The rectangular agarose phantom plates were centrically
positioned on the head cushion of the coil. For the analysis,
circular regions of interest (ROIs) were manually chosen
for each well, and the mean (± standard deviation) R2
and R2*values were calculated from the T2 ad T2*
values. The mean R2*values were plotted against the MNP
concentrations and/or the iron concentration to determine
the correspondingtransverserelaxivities (r2*,s-1(mMFe)−1)by
linear regression.
Statistical Analysis
The results are expressed as the mean ± standard deviation
(SD).
RESULTS
Physico-Chemical Characteristics of Silica-Iron Oxide
MNPs
The iron concentration of the silica iron oxide SO-Mag5
MNPs was 0.52 g iron per g dry weight, suggesting,
accounting for the phase composition of the core described
below, that the coating concentration was 0.28 g matter per g
dry weight. The hydrodynamic diameter that was measured
intheaqueoussuspensionwas(40±14)nm.Theelectrokinetic
potential was highly negative at −38.0±2.0 mV.
A representative TEM image of the particles is shown in
Fig. 2a. The evaluation of the TEM image reveals that the
iron oxide core of the particles has an average diameter
of (6.7±1.5) nm (Fig. 2b). This is similar to the average
crystallite size of 6.8 nm, which was determined from X-ray
powder diffraction patterns using the Scherrer formula
(Fig. 2d). All of the reflections in the electron diffraction
patterns(Fig.2c)andXRDpatternscanbeindexedaccording
to cubic magnetite.
Data on quasistatic magnetization, M(H), and magneto-
relaxometry (MRX) data for the SO-Mag5 nanomaterial
are given in Fig. 2e and f. By fitting (1) to the measured M
(H) data (Fig. 2e), we have obtained the mean magnetic
volume diameter dmV0(7.3±0.6) nm and distribution
parameter σ00.44±0.01 for the effective magnetic sizes
that represent the mean core size of a single MNP
domain. The saturation magnetization MS0(362±10)
kA/m049 Am2/kg094 Am2/kg(Fe) (estimated from the
M(H) curve using Eq. 1) refers to the whole particle core
volume andislowerthan thesaturationmagnetizationofbulk
magnetite (MS,bulk0480 kA/m092 Am2/kg0127 Am2/kg
(Fe) (46)). A reduction in the magnetization compared to
the bulk material MS,bulk-MSmay be due to the partial
oxidation of the core material into maghemite, for which
MS,bulk0(340–390) kA/m (47). However, it may also result
from the so called magnetically “dead layer” at the surface of
the nanoparticles that can be thought of as a transition layer
betweenacoating materialandthe magneticallyactiveregion
oftheparticle core(48). The magneticallydeadlayercould be
chemisorption induced (49) or result from a surface spin
disorder (50), which is typical for magnetic nanoparticles
that are smaller than the critical diameter and have high
surface-to-volume ratios (51). This is supported by the
poor saturation behavior of M(H) in Fig. 2e.
The fitting of the MRX curve (Fig. 2f, freeze dried
sample) resulted in a core magnetic diameter of (6.4±1)
nm and σ00.35±0.04, where an anisotropy constant of
K0(6±3) kJ/m3was found. The hydrodynamic size dVC
that was evaluated from the MRX curve for the liquid
suspension (Fig. 2f) was (95±14) nm with the related distri-
bution parameters σ00.57±0.04.
Mykhaylyk et al.

Page 9

XPS is an excellent analytical method for characterizing
surfaces. High-resolution X-ray photoelectron spectra
provide the elemental surface composition and resolve
the oxidation states of individual elements. Fragments
of the XPS spectra of the SO-Mag5 nanoparticles are
shown in Figure S1, and data on the relative elemental
compositions of the surface are given in the Figure table.
The phosphor-to-silicon elemental ratio of 1.23 to 4.96,
accounting for a phosphor-to-silicon ratio of 1 to 1 in a
structural formula of THSMP (C4H12NaO6PSi), allows for
the assignment of 1.23 atomic percent Si to the product of the
THSMP condensation. The remaining 3.73% silicon belongs
to the product of the TEOS condensation in the surface layer
or SiOx. Taking into account the molecular weight of
THSMP (238.18), the average number of phosphonate
groups was estimated to be ∼1.5 x1021groups per g coating
weight.
For a 6.7 nm magnetite particle core, the average weights
of the particle and coating are 6.2×10−19g Fe and 3.4×
10−19g of coating material per particle. Therefore, there is
on average approximately 500 phosphonate groups per
particle. Assuming that the density of the silica coating
material is 2 g/cm3, the volume of the silica coating on the
surface is approximately 170 nm3, which gives a thickness of
a
c
d
b
ef
468 10
0
10
20
30
40
d=(6.7±1.5) nm
n=255
Particle number
d (nm)
20406080
1000
2000
3000
4000
I [rel. units]
Fe3O4
<d>=6.8 nm
Fig. 2 Morphology, phase
composition and magnetic
structure of the core of the
SO-Mag5 nanoparticles. The X-
ray photoelectron spectrocopy
data were moved to the supple-
mentary materials (Figure s2)
during the review process. (a)
Transmission electron microscopy
image and (b) derived data on the
diameter of the core, (c) electron
diffraction and (d) X-ray
diffraction patterns. (e) M(H)
curve and (f) MRX-data of fluid
aqueous suspension and of a
suspension after freeze drying in
10% mannitol (preserving the
overall volume of the sample).
Silica-Iron Oxide Nanoparticles for Gene Delivery

Page 10

approximately 1 nm for the coating layer at the surface of
the 6.7-nm iron oxide nanoparticles. This value is consistent
withtheTEMdatashowninFig.2a.Thetotaldiameterofthe
particle (core plus coating) is ∼8.7 nm, and thus the average
outer surface of the particle is ∼60 nm2(or 97 m2/(g Fe)), and
the surface density is ∼8.4 phosphonate groups per nm2
(∼13.3 μmol m−2or 1.3 mmol/(g Fe). This high surface
density of the phosphonate groups ensures a rather high
electrokinetic potential of the particles (−38.0±2.0 mV) when
measured in aqueous suspension.
Decoration of Silica-Iron Oxide Nanoparticles
with Polyethylenimine and Electrokinetic Potentials
and Stabilities of Aqueous Suspensions
as Evaluated by STEP-Technology
The decoration of the surface of the SO-Mag5 particles
with PEI at a PEI-to-iron w/w ratio ranging from 1 to
12 w% resulted in SO-Mag6-n particle series with SiOx/
Phosphonate-PEI coatings. We investigated the relationship
betweenthe amountofPEI thatisadded tothenanoparticles,
the electrokinetic potential, the particle size distribution, the
dispersion stability and the sedimentation behavior.
Increasing the amounts of PEI that were loaded onto
particles caused a gradual shift of the surface charge to
occur, which changed from a highly negative electrokinetic
potential of (−38.0±2.0) mV for primary particles in the
aqueous suspension (n00) to almost neutral particles at 1-2%
PEI with increasing positive potentials for higher n-values up
toabout+40mVatPEI loading levels of n05; a saturation
at this potential of up to n012 was observed (Fig. 3a).
Neutralization of the surface charges resulted in the
aggregation/flocculation of the particles with increased
effective hydrodynamic diameters, according to the DSL
measurements (Fig. 3b). At PEI loading levels of higher
than 5w%, no visual differences in the appearance of the
suspensions were observed with the naked eye.
The dispersion stability was quantified by multisample
analytical centrifugation with photometric detection. This
approach allows for the in situ recording of space- and time-
resolved extinction (concentration) profiles for the entire
sample, measuring from the bottom to the top of the cuvette
during centrifugation. Figure 3c shows the transmission
profiles for the SO-Mag5 and SO-Mag6-n samples that
were obtained during centrifugation for 43 min at 36g. As
can be observed from the time course of the transmission
profiles in Fig. 3c-1, at the beginning of the separation
process, particles and aggregates (small flocs) have enough
space to separate individually (polydisperse sedimentation).
Later, a sharp front of sedimenting particles is formed.
Inside a particle network, all of the particles are moving
with the same velocity (zone sedimentation). The distance
between consecutive profiles narrows because the resistance
against further compaction increases with particle concen-
tration inside the network. Behind the sharp front, a gradual
increase in transmission is observed, which is related to
slowly settling individual fines that are not bound to the
particle network; i.e., the dispersion contains larger flocs and
fines (primary particles and/or small aggregates). As
expected, the characteristics of the transmission profiles
change with the PEI-to-iron ratios.
For the undecorated sample (SO-Mag5), only minor
changes were observed (Fig. 3c-0). A small degree of
polydisperse sedimentation can be traced to just below
the filling height (meniscus) because the average particle
sizeismuchsmaller thanthatofSO-Mag6-1.By doublingthe
amount of PEI (SO-Mag6-1→SO-Mag6-2, Fig. 3c-2), the
separation mechanism changes to purely zone sedimentation.
The flocs settle rapidly, forming a flocculated particle
network. There is no residual turbidity, indicating that
there is no sign of the separate settling of a fine fraction.
In other words, near the IEP, all of the particles are
incorporated into the flocs/network.
Further doubling of the PEI concentration (SO-Mag6-
2→SO-Mag6-4) again changes the separation mechanism.
As shown in Fig. 3c-4, the polydispersity sedimentation of
fines behind the sharp front is again obvious. Compared to
SO-Mag6-1 (Fig. 3c-1), the sedimentation of the fines is
much slower. Further increases in the PEI-to-iron ratios
lead to continuous increases in the fraction of the fines and
consequently in the turbidity of the supernatant, until the
flocculated fraction disappears and zone sedimentation is no
longer traceable (Fig. 3c-10 and c-12).
The appearance of the samples after centrifugation is
documented in Fig. 4c. The dependence of the sediment
height on the PEI-to-iron ratio is shown in Fig. 4a, and the
related change in turbidity of the supernatant is quantified
by the transmission, which is averaged in the middle part of
the sample cell (118–120 mm, Fig. 4b). The sediment height
is largest for SO-Mag6-2 particles, and the transmission is
highest for SO-Mag6-3 particles (highest degree of floccula-
tion, no fines remaining). With greater PEI concentrations,
the sediment height decreases, and the turbidity in the
supernatant increases. The same is obvious from the trans-
mission profiles and can be quantified accordingly. The
effect of the surface decoration of SO-Mag5 on the size
distribution for the varying PEI-to-iron ratios is summarized
in Fig. 4d. The primary material has 95% of particles that
are smaller than 40 nm. Loading with 1% PEI per mass of
iron results in a coarse fraction between 2 and 5 μm;
however, this also contains approximately 10% of a
broadly distributed fine fraction between 100 nm and
2 μm. At 2% or 3% PEI (a similar curve), the amount of the
fine fraction is reduced. After this point, a pronounced
bimodality is observed that contains a mix of aggregates
that are smaller than 100 nm and larger than 2 μm.
Mykhaylyk et al.

Page 11

ab
0
0
3
3
6
6
9
9
12
12
-40
-40
-20
-20
0
0
20
20
40
40
Electrokinetic potential [mV]
PEI-to-Iron w/w ratio [%]
water, pH 7.3 (HCl)
water, pH 7.3 (HCl)
DMEM+10% FCS
DMEM+10% FCS
0033669912 12
1010
100100
10001000
1000010000
Average Dh [nm]
Electrokinetic potential [mV]
Average Dh [nm]
c
Position [mm]Position [mm]
Transmission [%]
001122
100100
8080
6060
4040
2020
00
100100
8080
6060
4040
2020
00
100100
8080
60 60
40 40
20 20
00
334455
77
1010 12 12
Filling hightFilling hight
Gradual
clarificationclarification
First profile First profile
Last profileLast profile
Sediment Sediment
Zone Zone
sedimentationsedimentation
Polydisperse
sedimentationsedimentation
Position of cell bottomPosition of cell bottom
Zone
sedimentationsedimentation
Polydisperse
sedimentation
of finesof fines
Zone
sedimentationsedimentation
Polydisperse
sedimentation
of finesof fines
Zone
sedimentationsedimentation
Filling hight Filling hight
105115125105115125105 115125
Transmission [%]
Gradual
Polydisperse
Zone
Polydisperse
sedimentation
Zone
Polydisperse
sedimentation
Zone
Fig. 3 Characteristics of the aqueous suspensions of silica-iron oxide nanoparticles decorated with polyethylenimine at different PEI-to-iron w/w ratios. (a)
Surface charges and (b) hydrodynamic diameter as measured in water suspension or after dilution in DMEM medium containing 10% FCS. (c) Stability as
evaluated by the time- and space-resolved detection of light transmission (STEP-Technology) combined with multisample analytical centrifugation. The
evolution of the transmission profiles as registered in multiple points along the cuvette is displayed for different time points during the course of centrifugation
of aqueous suspensions of the SO-Mag5 particles and the SO-Mag6-n particles that were titrated with PEI at different PEI-to-iron w/w% ratios n (n values are
shown in the grey quadrates at the right bottom corner of the figure); centrifugation was conducted for 43 min at 20°C and 500 rpm (36g at the bottom of
the cuvette). The position of the filling height is approximately 107.5 mm, and the position of the cuvette bottom is at 129.5 mm.
Silica-Iron Oxide Nanoparticles for Gene Delivery

Page 12

Beyond SO-Mag6-7 and at higher PEI loading concentra-
tions,the distributionsare shifted towardsthose ofthe original
undecorated sample. Samples loaded with 10–12% PEI kept
highly positive electrokinetic potential, colloidal stability and
magnetofection potential during storage. Thus, loading
with 10% PEI resulted in an average particle hydrated
diameterof(89±48)nmandzetapotentialof(40.2±0.6)mV.
After 12 months storage at 4°C, an average particle hydrated
diameter of (69±33) nm and zeta potential of (49.5±0.3) mV
were measured for the same SO-Mag6-10 nanoparticles.
When the order of reagent mixing is changed from the
addition of PEI into the SO-Mag5 dispersion to the addition
of the primary SO-Mag5 particles into the PEI solution, the
PEI-to-iron ratio at which there is maximum flocculation
does not change; however, the dispersion properties at
higher concentrations of PEI are modified. Fig. S2c docu-
ments the appearance of the samples after centrifugation for
this case. Turbidity in the supernatant is markedly reduced
(Fig. S2b), and the sediment is higher (Fig. S2a); i.e., a
higher degree of flocculation is preserved compared to
024681012
0
20
40
60
80
100
Residual transmission [%]
ba
c
d
02468 1012
0
2
4
6
8
10
Sediment hight [mm]
PEI-to-iron w/w ratio [%]
10 1001000
0
20
40
60
80
100
PEI-to-iron w/w ratio (%):
0 1 2 5 7 10
Volume weighted
cumulative size distribution Q3 [%]
Hydrodynamic diameter [nm]
n=01234567891012
Fig. 4 Parameters of stability
for the aqueous suspensions
of silica-iron oxide nanoparticles
decorated with polyethylenimine.
(a) Sediment height and
(b) average transmission in the
supernatant (118–120 mm) for
aqueous suspensions of the
SO-Mag5 particles and the
particles that were titrated with
PEI at different PEI-to-iron w/w%
ratios n; centrifugation was
conducted for 43 min at 20°C
and 500 rpm (36g at the cuvette
bottom). (c) Visual appearances
of the samples after centrifugation.
(d) Volume-weighted cumulative
hydrodynamic diameter
distributions derived from the
transmission profiles shown
in Fig. 3c.
Mykhaylyk et al.

Page 13

that which occurs when PEI is added to the suspension
of the undecorated sample (Fig. 4a and b), despite thefact
that the PEI concentrations are the same.
Magnetic Sedimentation of Adenovirus Associated
with MNPs and Stability of Resulting Complexes
As shown in Fig. 5a, the amount of adenovirus that
associated and magnetically sedimented with the MNPs
in PBS increased with increasing MNP-to-VP ratios in
terms of iron weight per VP. A maximum adenovirus
binding and sedimentation rate of approximately 90% of
the initial virus particles was observed at MNP-to-virus
ratios higher or equal 5 fg of Fe/VP for SO-Mag6-n
particles, n05–12%. At lower PEI-to-Iron w/w ratio of
3% a maximum adenovirus binding and sedimentation
rate of about 75% was observed only at high MNP-to-
virus ratios >100 fg Fe/VP. The complexes of AdV that
were formed with the SO-Mag6-n particles, n07 and
12%, at MNP-to-virus ratios of 3–20 fg Fe/VP remained
stable after 30 min of incubation in the presence of 50%
FCS followed by 30 min of sedimentation on the magnetic
plate (Fig. 5b). When the complexes formed with the particles
decorated with 5 and 3 w% PEI were incubated in 50% FCS
for 30 min, partial destabilization of the adenovirus−MNP
complexes occurred (Fig. 5b, right). It is of note that at
high serum protein concentrations, up to 75% of the
adenovirus was still magnetically sedimented with SO-Mag6-
5 nanoparticles at MNP-to-virus ratios >5 fg of Fe/VP,
whereas only about 40% of the adenovirus was magnetically
sedimented with SO-Mag6-3 complexes even at ratios of
higher than 50 fg of Fe/VP.
The complexes of AdV that were formed with the
SO-Mag6-n particles, n07 and 12%, at MNP-to-virus
ratios of 10–20 fg Fe/VP (Fig. 5c, left) and 3–5 fg Fe/VP
(Fig. 5c, right) remained stable for at least 30 min of incuba-
tion followed by 30 min of sedimentation on the magnetic
plate both in PBS and 50% FCS. At lower PEI-to-iron ratios
050100150
MNP-to-VP ratio [fg Fe/VP]
200
0
50
100
PEI-to-iron w/w ratio (%): 3 5 7 12
Magnetically sedimented virus [%]
a
0369 12
0
20
40
60
80
100
PBS 50% FCS
10 fg Fe/VP
20 fg Fe/VP
Magnetically sedimented virus [%]
b
c
036912
PBS
5 fg Fe/VP
3 fg Fe/VP
50% FCS
PEI-to-iron w/w ratio [%]
050100150200
50% FCS
Fig. 5 Particle-Virus interactions.
Magnetic sedimentation of the
AdV adenovirus associated with
the silica-iron oxide nanoparticles
decorated with polyethylenimine
and the stability of the resulting
complexes in 50% FCS.
(a and b) I125-labeled AdV virus
and silica-iron oxide nanoparticles
decorated with PEI at different
PEI-to-iron w/w % ratios n
(SO-Mag6-n nanoparticles,
n03, 5, 7, and 12) were mixed
with PBS at various nanoparticle-
to-virus particle ratios in relation
to the fg of Fe/VP and incubated
for 20 min for the complex
formation. The resulting
complexes were diluted 1 to 1
with PBS (a) or FCS (b) and then
incubated for 30 min before
being positioned on the
96-magnet plate for 30 min for
the magnetic sedimentation.
The I125radioactivity of the
supernatants was measured to
quantify the percentage of virus
that associated and magnetically
sedimented with the MNPs.
(c) The data from Figures a and b
for fixed nanoparticle-to-virus
particle ratios of 3.4, 5, 10 and
20 fg of Fe/VP are plotted against
the PEI-to-iron w/w % ratios of
the nanoparticles.
Silica-Iron Oxide Nanoparticles for Gene Delivery

Page 14

of 3 and 5% even an increase in MNP-to-VP ratios till 200 fg
Fe/VP did not ensured sufficient association and magnetic
sedimentation of viral particles and stability of the complexes
in the presence of high serum concentration (Fig. 5a and b).
Therefore, the range of loading of the SO-Mag5 nano-
particles with PEI (7–12%) combined with adequate
MNP-to-VP ratios (preferably 10–20 fg Fe/VP) that enabled
the adenoviruses to be highly associated with the MNPs and
the stability of the complexes in 50% FCS allow for the
construction of potentially efficient magnetic complexes for
in vitro and in vivo infections. In concordance with these results,
the adenoviral magnetic vectors with SO-Mag6-12.5
nanoparticles that were formulated at 5 and 10 fg Fe/VP
at MOI of 2.5 and 5 showed transduction efficiencies in the
HUVECs that were more than two orders of magnitude
higher in terms of luciferase reporter expression (ng luciferase/
μg total protein) compared to the non-magnetic vectors (52).
Magnetotransduction Efficiency with Lentiviral
Magnetic Vectors of Silica-Iron Oxide MNPs
Decorated with PEI
Previously, we have found that lentiviral particles are also
efficiently bound and magnetically sedimented with silica
iron oxide magnetic nanoparticles that are titrated with 5%
PEI, and the resulting complexes that are constructed at
2–20 fg Fe/VP efficiently transduce primary hematopoietic
and mesenchymal stem cells (26). For example, at a lentiviral
MOI of 5 and low cell density of 1.5×105cells/mL, 21% of
hCB-CD34+cells were transduced with an eGFP reporter
and maintained their progenitor cell phenotype after
magselectofection compared to only 0.15% transduction
using standard infection protocol at the same cell density.
Lentiviral magselectofection with low MOIs (≤3) yielded
up to 50% transduction of mouse Lin−Sca1+cells, which
persistently reconstituted T and B cells in Il2rg−/−mice,
compared to less than 10% transduction with higher
MOIs using a standard transduction protocol for Lin−bone
marrow cells.
To identify the PEI loading of the nanoparticles that
enables the maximal transduction efficiency, we tested the
lentiviral complexes of the SO-Mag6-n particles (n≤12%) in
SHSY5Y human neuroblastoma cells at low applied virus
doses per cell (MOI of 0.03–0.5). The results obtained from
the luciferase reporter expression that are shown in Fig. 6
clearly indicate the PEI loading of 9–12w% to be optimal in
achieving high magnetotransduction efficiency.
Storage Stability of Lentiviral Magnetic Complexes
We tested the lentivirus titer in the CMS5 cells for the SO-
Mag6-10 nanoparticle complexes and compared the results
with the titer that was determined using the conventional
procedure of infection in the presence of 8 μg polybrene per
ml medium (polybrene complexes, PB/LV). No magnetic
field was applied during the infection. The dose response
curves were fitted to quantify the dose that resulted in 50%
transduced cells and derived virus titers (Fig. 7a). Freshly
prepared complexes were tested in addition to complexes
that had been prepared and stored at −80°C or 4°C at
different time points during the 5 month storage. The results
presented in Fig. 7b show that even in simple-to-transduce
CMS5 cells without the application of the magnetic field,
the freshly prepared magnetic complexes yielded approxi-
mately 2-fold higher transduction efficiencies. One of the
reasons for this could be the more efficient internalization of
the magnetic complexes even without the application of the
-2024681012
1
10
100
Luciferase expression [ng/mg total protein]
PEI-to-Iron w/w ratio [%]
MOI:0.5 0.25 0.125 0.0625 0.03125
SHSY5Y human neuroblastoma cells
Fig. 6 Magnetotransduction
efficiencies of the lentiviral
magnetic vectors of the silica-iron
oxide MNPs decorated with PEI.
SHSY5Y human neuroblastoma
cells were transduced with mag-
netic SO-Mag6-n/LVSFeGFPLuc
complexes that were constructed
atMNP-to-virusratiosof10fgFe/VP
at varying low doses of infectious
virus particles per cell (MOI) under
a magnetic field. After 72 h of
incubation, luciferase reporter
expression was analyzed in cell
lysates. SO-Mag6-n particles
were obtained by the
attachment of PEI to silica-oxide
SO-Mag5 MNPs at different
PEI-to-iron w/w % ratios n.
Mykhaylyk et al.

Page 15

magnetic field that has previously been established for the
magnetic vectors of the oncolytic adenovirus Ad520 (15,41).
Importantly, the magnetic complexes also showed higher
transduction efficiencies than those of the polybrene-
supported complexes at different time points during the
5-month storage. It is also noteworthy that after 2 days
of storage at +4°C, no deactivation was observed for
either the polybrene or magnetic complexes. This stability of
the complexes over the course of storage could prove to be
favorable in further potential applications.
Cell Association and Cytotoxicity of Silica-Iron Oxide
Nanoparticles Decorated with Polyethylene Imine
Cell association/internalization and cytotoxicity (based on
the MTT-test respiration activity assay) were evaluated in
SHSY5Y human neuroblastoma cells (Fig. 8). The primary
SO-Mag5 particles are not readily internalized, and loading
with 1 w% PEI increased the cell association and inter-
nalization of the particles, whereas with PEI loading
levels of 3–12%, they were very readily internalized
andshowedhighlabelingefficiencies(Fig.8a).Therespiration
rates of the neuroblasts remained higher than 75% of the
reference values for the untreated cells when up to 12 pg of
iron were loaded per cell. For the viral complexes that were
constructed with 10 fg Fe/VP, which was found to be an
optimum MNP-to-virus ratio for numerous transduction
experiments, the 12 pg of applied iron per cell would
correspond to an applied virus dose of 1200 physical
virus particles per cell. This virus dose was found to be
high enough to enable a high magnetotransduction efficiency
using both lentiviral and adenoviral gene delivery vectors
(15,26,41).
The data on the cell respiration rates plotted against
internalized/associated iron (derived from the data shown
in Fig. 8a) show that the effects of the nanoparticles depend
0
00
1
11
2
22
3
33
4
44
Virus titer [10
6 TU/ml]
2 37 72 1492 37 72 149
Storage duration [days]
PB/LV SO-Mag6-10/LV
2 37 72 149
Freshly preparedStored at -80°CStored at +4°C
b
-5-4-3 -2-
2 days2 days 2 days 2 days2 days
LOG10
Freshly prepared Stored at -80°C Stored at +4°C
PB/LV
SO-Mag6-10/LV
72 days72 days72 days 72 days72 days
100
80
60
40
20
0
a
-5 -4-3 -2
eGFP positive CMS5 cells [%]
Fig. 7 Time course of the infectivity of the lentiviral vectors during storage in complexes with silica iron oxide MNPs decorated with PEI.
Complexes of the lentiviral vector LVSFeGFP with either SO-Mag6-10 MNPs that was constructed at 10 fg Fe/VP (SO-Mag6-10/LV) or polybrene
(PB/LV) were (i) freshly prepared and stored at (ii) −80°C or (iii) +4°C. After 2, 37, 72 or 149 days of storage, CMS5 cells were infected with
different doses of the complexes or with freshly prepared complexes, and no magnetic field was applied. After 48 h of cultivation, the percentage
of eGFP-positive cells was determined by FACS analysis and plotted against the applied volume of the virus stock per cell as shown in Figure a for
infections that were performed after 2 days and 72 days of storage. (b) Virus titers were derived from the stock volumes resulting in 50% eGFP-expressing
cells as determined after the fitting of the dose-effect curves with a logistic function. SO-Mag6-10 particles were obtained by the association of PEI to silica-oxide
SO-Mag5 MNPs at a PEI-to-iron w/w % ratio of 10.
Silica-Iron Oxide Nanoparticles for Gene Delivery