Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma.
ABSTRACT Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.
- SourceAvailable from: Nataša Poklar Ulrih[Show abstract] [Hide abstract]
ABSTRACT: Iron-oxide nanoparticles of small dimensions that have superparamagnetic properties show immense potential to revolutionize the future of cancer theranostics, the combinatorial diagnosis and therapeutic approach towards cancer. Superparamagnetic iron-oxide nanoparticles (SPIONs) have unique magnetic properties, due to which they show excellent tumor-targeting efficiency, and this paves the way for effective personalized cancer treatment. The aim of this review is to focus on the ability of SPIONs to perform multiple roles in the field of cancer biology, such as in diagnosis, monitoring, targeting and therapy. Also, other topics are discussed, including the synthesis of SPIONs, the challenges and recent advances.Cancer letters 05/2013; · 5.02 Impact Factor
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ABSTRACT: Spurred by recent progress in materials chemistry and drug delivery, stimuli-responsive devices that deliver a drug in spatial-, temporal- and dosage-controlled fashions have become possible. Implementation of such devices requires the use of biocompatible materials that are susceptible to a specific physical incitement or that, in response to a specific stimulus, undergo a protonation, a hydrolytic cleavage or a (supra)molecular conformational change. In this Review, we discuss recent advances in the design of nanoscale stimuli-responsive systems that are able to control drug biodistribution in response to specific stimuli, either exogenous (variations in temperature, magnetic field, ultrasound intensity, light or electric pulses) or endogenous (changes in pH, enzyme concentration or redox gradients).Nature Material 10/2013; 12(11):991-1003. · 35.75 Impact Factor
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ABSTRACT: Gene therapy is a promising new technique for treating many serious incurable diseases, such as cancer and genetic disorders. The main problem limiting the application of this strategy in vivo is the difficulty of transporting large, fragile and negatively charged molecules like DNA into the nucleus of the cell without degradation. The key to success of gene therapy is to create safe and efficient gene delivery vehicles. Ideally, the vehicle must be able to remain in the bloodstream for a long time and avoid uptake by the mononuclear phagocyte system, in order to ensure its arrival at the desired targets. Moreover, this carrier must also be able to transport the DNA efficiently into the cell cytoplasm, avoiding lysosomal degradation. Viral vehicles are the most commonly used carriers for delivering DNA and have long been used for their high efficiency. However, these vehicles can trigger dangerous immunological responses. Scientists need to find safer and cheaper alternatives. Consequently, the non-viral carriers are being prepared and developed until techniques for encapsulating DNA can be found. This review highlights gene therapy as a new promising technique used to treat many incurable diseases and the different strategies used to transfer DNA, taking into account that introducing DNA into the cell nucleus without degradation is essential for the success of this therapeutic technique.International Journal of Pharmaceutics 11/2013; · 3.99 Impact Factor
Surface modified magnetic nanoparticles for immuno-gene therapy of murine
Sara Prijica,1, Lara Prosena,1, Maja Cemazarb,g, Janez Scancarc, Rok Romihd, Jaka Lavrencake,
Vladimir B. Bregarf, Andrej Coerg, Mojca Krzanh, Andrej Znidarsica, Gregor Sersab,*
aKolektor Group, Nanotesla Institute, Ljubljana, Slovenia
bInstitute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia
cJosef Stefan Institute, Department of Environmental Sciences, Ljubljana, Slovenia
dUniversity of Ljubljana, Faculty of Medicine, Institute of Cell Biology, Ljubljana, Slovenia
eInstitute of Oncology Ljubljana, Department of Cytopathology, Ljubljana, Slovenia
fKolektor Group, Kolektor Magma, Ljubljana, Slovenia
gUniversity of Primorska, Faculty of Health Sciences, Izola, Slovenia
hUniversity of Ljubljana, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology, Ljubljana, Slovenia
a r t i c l e i n f o
Received 30 January 2012
Accepted 16 February 2012
Available online 18 March 2012
a b s t r a c t
Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins,
such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against
a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection
involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external
magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles
(SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI)
proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells
with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency
to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid
DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further
refined for cancer immuno-gene therapy.
? 2012 Elsevier Ltd. All rights reserved.
The translation of nanotechnology has already widespread into
biomedicine for diagnostic, therapeutic and theranostic purposes
. Superparamagnetic iron oxide nanoparticles (SPIONs) can be
guided by an external magnetic field, yet due to quantum effects at
the nanometer scale they do not retain residual magnetism in the
absence of an external magnetic field , which makes them
especially suitable for diverse biomedical applications . In the
field of oncology, SPIONs have been exploited for diagnostic
purposes as contrast enhancers for magnetic resonance imaging
and as vehicles for biomarkers detection . For therapeutic
purposes SPIONs have been used for isolation and transfection of
hematopoietic stem cells for gene therapy e magselectofection ,
in magnetic hyperthermia  and as delivery systems for different
Magnetofection is a non-viral transfection method that uses an
external magnetic field to target cells with nucleic acids that are
bound to magnetic nanoparticles . Magnetofection has recently
celebrated its 10th anniversary, and its progress and prospects are
described in the thorough review paper . Many studies regarding
[8,10e18]. The majority were coated and/or functionalized with
positively charged PEI due to its electrostatic interaction with
negatively charged sugar phosphate backbone of nucleic acid and
proton sponge effect, which enables release of SPIONs-PEI-nucleic
acid complexes from endolysosomes into cytoplasm. Although PEI
is a transfection agent per se , it has been shown that when
coupled with SPIONs, magnetofection efficiency increased in
comparison to the transfection efficacy of PEI only [11,20]. Despite
of its extended usage for gene delivery, PEI compromises cell
membrane integrityand induces formation of channels in the outer
mitochondrial membrane , which could lead to cell death.
* Corresponding author.
E-mail address: firstname.lastname@example.org (G. Sersa).
1Authors contributed equally to this work.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ e see front matter ? 2012 Elsevier Ltd. All rights reserved.
Biomaterials 33 (2012) 4379e4391
Similar to PEI, poly(alkylacrylic acid) polymers, including non-
cytotoxic polyacrylic acid (PAA), have been considered as endo-
somolytic polymers . It has been shown that the inclusion of
PAA to PEI-DNA transfection complexes not only increased reporter
gene expression but also reduced toxicity in vivo . However,
there are no reports about SPIONs coated with anionic PAA and
functionalized with cationic PEI for magnetofection of plasmid DNA
encoding either reporter or therapeutic genes. Thus far magneto-
fection of tumors using SPIONs for magnetically-guided immuno-
gene therapy has notresulted in significant antitumoreffect [14,16].
In terms of preparing effective magnetically-guided delivery
system for immuno-gene therapy of tumors, we modified surface of
SPIONs with a double layer of PAA and PEI endosomolytic polymers
(SPIONs-PAA-PEI). After evaluating physicochemical properties and
binding plasmid DNA encoding either reporter gene for enhanced
green fluorescent protein (GFP) (pDNAGFP) or therapeutic gene for
murine interleukin 12 (IL-12) (pDNAIL?12) to SPIONs-PAA-PEI, we
tested cytotoxicity of so prepared SPIONs-PAA-PEI-pDNAGFP. We
implemented magnetofection of cells of different cell lines with
pDNAGFPor pDNAIL?12using SPIONs-PAA-PEI, and we compared its
efficacy to transfection efficacy using commercially available
magnetic nanoparticles and two well-established non-viral trans-
fection methods, electroporation and lipofection. In vivo, we
determined acute toxicity and biodistribution of SPIONs-PAA and
SPIONs-PAA-PEI-pDNAGFP, and tested non-invasive magnetofection
using SPIONs-PAA-PEI. For the proof-of-principle, we determined
antitumor effectiveness after magnetofection of murine mammary
adenocarcinoma tumors with pDNAIL?12using SPIONs-PAA-PEI.
2. Materials and methods
2.1. De novo synthesis of SPIONs-PAA-PEI
SPIONs were synthesized by alkaline co-precipitation of ferrous and ferric
sulfates, (FeSO4? 7H2O, 98% and (Fe2(SO4)3? xH2O)) (Alfa Aesar, Ward Hill, MA), in
an aqueous solution according to the Massart method . Briefly, 250 ml of 0.5 M
aqueous solution containing ferric and ferrous ions in a weight-to-weight (w/w)
ratio of 1.5:1 were precipitated with 150 ml of 25% ammonium hydroxide solution
(NH4OH) (Sigma-Aldrich, St. Louis, MO) under magnetic stirring at 600 rpm for
30 min at room temperature. After SPIONs were obtained, alkaline medium was
removed and replaced with distilled water subsequent to magnetic decantation of
SPIONs (repeated 3 times) in order to obtain a magnetic liquid, i.e. ferrofluid (FF).
SPIONs were coated in situ with 45% (w/w) water solution of poly(acrilyc acid,
sodium salt) (PAA) with molecular weight of 8 kDa (Sigma-Aldrich) by mixing
100 ml of FF-SPIONs and 100 ml of PAA water solution of equal mass concentrations
at 10 mg/ml under magnetic stirring at 400 rpm for 5 min at room temperature.
Thereafter FF-SPIONs-PAA was sterilized by filtration using 0.22 mm pore size
syringe filter (Techno Plastic Products e TPP, Trasadingen, Switzerland). For the
evaluation of physicochemical properties and for the experiments stock solution of
the FF-SPIONs-PAA was diluted with distilled water to a working concentration of
1 mg/ml. Functionalization of SPIONs-PAA was performed directly prior to the
experiments with branched cationic polymer polyethylenimine (PEI) with molec-
ular weight of 25 kDa (Sigma-Aldrich). For in vitro experiments, FF-SPIONs-PAA at
1 mg/ml was added into 0.1 mg/ml PEI water solution at mass ratios 0.5:1, 0.6:1,
0.7:1, 0.8:1 and 0.9:1. For in vivo experiments, FF-SPIONs-PAA at 1 mg/ml was added
into 1 mg/ml PEI water solution at the mass ratio 0.6:1.
2.1.1. Binding plasmid DNA to SPIONs-PAA-PEI
Two plasmid DNA (pDNA) were used for binding to SPIONs-PAA-PEI: pDNA
containing reporter gene encoding GFP (pDNAGFP) under the control of the consti-
tutive cytomegalovirus (CMV) promoter(pCMV-EGFP-N1; Clontech, Mountain View,
CA) or pDNA containing therapeutic gene encoding murine IL-12 (pDNAIL?12) under
the control of hybrid promoter EF-1a/HTLV, consisting of elongation factor 1a and 50
untranslated region of the human T-cell leukemia virus, with open reading frame
(ORF) (pORF-mIL-12; InvivoGen, San Diego, CA). Amplification of pDNAGFPand
pDNAIL?12was performed in competent Escherichia coli cells (TOP10; Life Technol-
ogies, Carlsbad, CA), isolation using Qiagen Maxi-Endo-Free Kit (Qiagen, Hilden,
Germany) and subjection to quality control and quantity determination using
agarose gel electrophoresis and spectrophotometer (NanoDrop 2000; Thermo
Scientific, Wilmington, DE). Dilution of pDNAGFPand pDNAIL?12, exhibiting the ratio
between the absorbance at 260 nm and 280 nm wavelengths (A260/A280) more than
1.8, was made with endotoxin-free water to a working concentration of 1 mg/ml.
SPIONs-PAA, PEI and pDNAGFPor pDNAIL?12were prepared at the mass ratio 0.6:1:1
(SPIONs-PAA-PEI-pDNAGFPor SPIONs-PAA-PEI-pDNAIL?12). The ability of SPIONs-
PAA-PEI complexes to bind both pDNA was determined by 45 min electrophoresis
at 100 V on a 1% (w/v) agarose gel stained with 0.5 mg/ml ethidium bromide.
Visualization of the bands was performed under ultraviolet transilluminiscence
(GelDoc-It TS 310; Ultra-Violet Products (UVP), Upland, CA). DNA ladder MassRuler?
DNA Ladders, ready-to-use (Thermo Fisher Scientific, Waltham, MA) was utilized.
2.1.2. Physicochemical properties of SPIONs, SPIONs-PAA, SPIONs-PAA-PEI and
Chemical composition and crystallographic structure of SPIONs and SPIONs-PAA
were determined by X-ray diffractometry (XRD) measuring within the range of
a diffraction angle 2Q from 25?to 80?(AXS, D5005; Bruker, Billerica, MA). The mean
crystallite size was calculated according to the broadening of the (311) characteristic
peak of the XRD pattern using the Scherrer equation . Size and shape of SPIONs
and SPIONs-PAA were evaluated by transmission electron microscopy (TEM) (2000
FX with EDS AN10000; JEOL, Tokyo, Japan). The estimated size from TEM was
obtained by measuring diameters of ten SPIONs and SPIONs-PAA from representa-
tive samples. Also, morphology of SPIONs-PAA-PEI and SPIONs-PAA-PEI-pDNAGFP
was visualized by TEM.
Magnetic characterization of SPIONs and SPIONs-PAA was conducted using
a magnetometer (Quantum Design MPMS XL-5 SQUID, San Diego, CA) equipped
with a 50 kOe magnet, operating in the temperature range 2e400 K. In the first setof
measurements, the magnetization vs. the magnetic field curves, M(H), and the
magnetic susceptibility, c(T), of dry SPIONs and SPIONs-PAA were determined at
T ¼ 300 K. In the second set of measurements, the magnetization vs. the magnetic
field curves, M(H), and the magnetic susceptibility, c(T), of FF-SPIONs and FF-
SPIONs-PAA were determined at T ¼ 300 K.
The zeta (z) potentials of FF-SPIONs at pH ¼ 9.5, FF-SPIONs-PAA at pH ¼ 8.5 and
FF-SPIONs-PAA-PEI at pH ¼ 8 were determined by zetameter measuring electro-
phoretic mobility at 21?C applied to the Henry equation (Zetasizer Nano ZS; Mal-
vern Instruments, Malvern, UK).
2.2. Cell lines
Experiments were performed in two malignant melanoma cell lines, mouse
B16F1 (LGC Standards, Teddington, UK) and human SK-MEL-28 (American Type
Culture Collection (ATCC), Manassas, VA), and two normal cell lines, human meso-
thelial MeT-5A (ATCC) and mouse fibroblasts L929 (LGC Standards). B16F1, SK-MEL-
28 and L929 cells were maintained in advanced minimum essential medium (MEM)
(Life Technologies) whereas MeT-5A cells were maintained in advanced Roswell
Park Memorial Institute (RPMI) 1640 medium (Life Technologies). Both media were
supplemented with 5% fetal bovine serum (FBS; Life Technologies), 10 ml/l L-gluta-
mine (Life Technologies),100 U/ml penicillin (Grünenthal, Aachen, DE) and 50 mg/ml
gentamicin (Krka, Novo mesto, Slovenia). Cells were grown in Petri dishes of 15 cm
diameter (TPP) and incubated in a humidified atmosphere of 5% CO2at 37?C until
they reached at least 90% confluence. Then the medium was removed, cells were
washed with phosphate-buffered saline (PBS) and detached with 0.25% trypsin/
EDTA in Hank’s buffer (Life Technologies). An equal volume of medium with FBS for
trypsin inactivation was then added, cells were collected, centrifuged, counted and
used for subsequent experiments.
2.2.1. Cytotoxicity of SPIONs-PAA-PEI-pDNAGFP
Cytotoxicity of SPIONs-PAA-PEI bound to pDNAGFP(SPIONs-PAA-PEI-pDNAGFP)
was tested on cells derived from B16F1, SK-MEL-28, L929 and MeT-5A cell lines by
cell viability alamarBlue? (Life Technologies) assay measuring metabolic activity of
cells. For the experiments 2.5 ? 104B16F1, 5 ? 104SK-MEL-28, 2.5 ? 104L929 and
7.5 ?104MeT-5A cells were plated in 1 ml of cell culture medium on clear-bottomed
24-well plates (TPP). Immediately thereafter SPIONs-PAA, PEI and pDNAGFPalone
were added to cells in concentrations of 1.2 mg/ml, 2 mg/ml and 2 mg/ml, respectively.
SPIONs-PAA-PEI complexes, PEI-pDNAGFPand SPIONs-PAA-PEI-pDNAGFPtrans-
fection complexes were prepared at mass ratios 0.6:1, 1:1 and 0.6:1:1, respectively,
and added to cells at same concentrations as described above. Cells were either
directly incubated or exposed to Nd-Fe-B magnets with surface magnetic flux
density B ¼ 403 mT and magnetic gradient G ¼ 38 T/m (i.e. an external magnetic
field) for 15 min. After 72 h incubation, 500 ml of cell culture medium was removed
and 50 ml of alamarBlue? reagent (Life Technologies) was added, followed by 2-h
incubation. The fluorescence of formed resorufin product was quantified using
a fluorescence microplate reader (Infinite F200, Tecan, Männedorf, Switzerland) at
560 nm excitation wavelength and 590 nm emission wavelength. Cell survivals of
exposed cells are presented as the percentages of the fluorescence obtained from
untreated control cells.
2.2.2. Internalization of SPIONs-PAA-PEI-pDNAGFPinto cells
Internalization of SPIONs-PAA-PEI
pDNAGFP) intocells was evaluatedqualitativelyby TEM (CM100;Philips, Amsterdam,
the Netherlands) and quantitatively by inductively coupled plasma mass spectrom-
eter (ICP-MS) (Agilent 7700, Tokyo, Japan) determining iron (Fe) concentrations in
bound to pDNAGFP
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
the samples of digested cells at m/z 56 and 57. Internalization of SPIONs-PAA-PEI-
the amount of Fe per cell normalized to control cells. For the experiments 5 ? 104
of cell culture medium on clear-bottomed, 24-well plates (TPP). After 24 h or 48 h of
incubation, complexes of SPIONs-PAA, PEI and pDNAGFPwere prepared at the mass
ratio of 0.6:1:1, respectively, and given to cells at pDNAGFPconcentration of 2 mg/ml.
Cells were exposed to an external magnetic field for 15 min. After 4 h or 24 h of
SPIONs-PAA-PEI-pDNAGFPqualitatively, cells were fixed in a mixture of 4% (w/v)
paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4,
for 1 h at 4?C. Post-fixation was carried out in 1% osmium tetroxide in 0.1 M caco-
dylate buffer for 2 h, followed by dehydration in graded ethanol and embedding in
Epon 812 resin. Ultrathin sections (60 nm) were cut, counterstained with uranyl
acetate and lead citrate and examined with TEM. To determine the internalization of
4.5 ml cryo tubes (BD Biosciences, Two Oak Park, Bedford, MA) and stored at ?18?C.
Prior to analysis, samples were equilibrated to room temperature and digested with
90?C forat least24h toobtain clearsolutions. Samples werethen dilutedwith water
to 10 mL for the analysis with ICP-MS.
2.2.3. Magnetofection of cells with pDNAGFPusing SPIONs-PAA-PEI
To optimize the mass ratio between SPIONs-PAA, PEI and pDNAGFPfor magne-
tofection of cells using SPIONs-PAA-PEI, 5 ? 104B16F1, 1 ?105SK-MEL-28, 5 ? 104
L929 and 1.5 ?105MeT-5A cells were plated in 1 ml of cell culture medium on clear-
to cells at pDNAGFPconcentration of 2 mg/ml. Cells were exposed to an external
magnetic field for 15 min. After 24-h incubation, transfection efficacy was evaluated
qualitatively with fluorescent microscope by recording images with digital camera
(Olympus DP50) attached to fluorescent microscope (Olympus IX70, Hamburg,
Germany) at 488 nm excitation wavelength and 507 nm emission wavelength. The
differences in transfection of cells with pDNAGFPbetween the absence and presence
of an external magnetic field are presented as potentiating factors. Thereafter cells
were trypsinized, collected in 15 ml conical falcon tubes (TPP) and centrifuged. The
supernatant was removed and cells were resuspended to 5 ml polystyrene round-
bottom tubes (BD Biosciences) in 1 ml of PBS for quantitative determination of
transfection efficacy with flow cytometer (BD FACSCanto II; Becton Dickinson, San
Jose, CA), identifying the percentage of GFP-positive (fluorescent) cells and
measuring the median fluorescence intensity of the GFP. Statistics between the
magnetofection efficacies using SPIONs-PAA-PEI, prepared as SPIONs-PAA-PEI-
pDNAGFP, at different mass ratios were analyzed by one way ANOVA. Statistics
between the transfection efficacies using SPIONs-PAA-PEI in the absence and pres-
ence of an external magnetic field at the particular mass ratio were analyzed by
Furthermore, comparison of the transfection efficacy of B16F1 cells with
pDNAGFPwas made between: SPIONs-PAA-PEI prepared as SPIONs-PAA-PEI-
pDNAGFPat the mass ratio 0.6:1:1, PEI prepared as PEI-pDNAGFPat the mass ratio 1:1,
3 different commercially available magnetic nanoparticles for magnetofection
(CombiMAG and PolyMag purchased at chemicell GmbH, Berlin, Germany and
MATra bought from IBA GmbH, Göttingen, Germany), electroporation, lipofection
using Lipofectamine? 2000 (Life Technologies) and SPIONs-PAA-PEI in the absence
of an external magnetic field. Cells for transfection with PEI and SPIONs-PAA-PEI in
the absence and presence of an external magnetic field as well as for electroporation
were maintained in the medium as described above whereas cells for magneto-
fection with commercially available magnetic nanoparticles and lipofection were
maintained in MEM, supplemented with 10% FBS and 10 ml/l L-glutamin. For
transfection with PEI and SPIONs-PAA-PEI in the absence and presence of an
external magnetic field, 5 ? 104cells were plated in 1 ml of cell culture medium on
clear-bottomed, 24-well plates (TPP). After 24-h incubation, 2 mg/ml of pDNAGFPwas
prepared as PEI-pDNAGFPand SPIONs-PAA-PEI-pDNAGFPat mass ratio of 1:1 and
0.6:1:1, respectively, and added to cells. Cells transfected with SPIONs-PAA-PEI-
pDNAGFPwere either exposed to Nd-Fe-B magnets for 15 min or not. After 24 h of
incubation, cells were prepared for analysis by flow cytometer as described above.
For electroporation a dense cell suspension with a concentration of 1 ?106cells and
10 mg of pDNAGFPin 50 ml of electroporation buffer  was placed between two flat
parallel stainless steel electrodes with a 2 mm gap connected to the GT-1 electro-
porator (University of Ljubljana, Faculty of Electrical Engineering, Ljubljana,
Slovenia) and subjected to 8 square-wave electric pulses with an amplitude per
distance ratio 600 V/cm, 5 ms duration time, and 1 Hz repetition frequency. After
electroporation, cells were incubated for 5 min at the room temperature and then
plated to 6 cm diameter Petri dishes (TPP). After 24-h incubation, cells were
prepared for analysis by flow cytometer as described above. For lipofection and
magnetofectionwith commerciallyavailable magnetic nanoparticles either 2.5 ?104
or 5 ? 104cells were plated in 1 ml of cell culture medium on clear-bottomed, 24-
well plates (TPP). After 24-h incubation, 1.7 mg/ml of pDNAGFPwas used for lip-
ofection in accordance to manufacturer’s instructions whereas for magnetofection
with commercially available magnetic nanoparticles 4 mg/ml of pDNAGFPwas
coupled to nanoparticles according to the instructions of the manufacturer and
given to cells with subsequent 15-min exposure to Nd-Fe-B magnets. After 24-h
incubation, cells were prepared for analysis by flow cytometer as described above.
2.2.4. Magnetofection of cells with pDNAIL?12using SPIONs-PAA-PEI
The comparison of the transfection efficacy of B16F1 cells with pDNAIL?12was
made between: magnetofection using SPIONs-PAA-PEI prepared as SPIONs-PAA-
PEI-pDNAIL?12at the mass ratio 0.6:1:1, PEI prepared as PEI-pDNAIL?12at the mass
ratio 1:1, electroporation, lipofection and SPIONs-PAA-PEI in the absence of
magnetic field. The transfections of cells with pDNAIL?12was performed likewise the
transfection of pDNAGFP-based experiments. After either 24-h incubation, the
medium was transferred into 1.5 ml cryo tubes (Corning Incorporated, Corning, NY)
and centrifuged in order to eliminate cell debris. Thereafter the supernatant was
analyzed for the amount of IL-12 p70 by enzyme-linked immunosorbent assay
(ELISA) kits (R&D Systems, Minneapolis, MN) in accordance to the manufacturer’s
instructions. Transfection efficacy is presented in picograms of secreted biologically
active heterodimer p70 of IL-12 per cell.
In vivo experiments were performed on female BALB/c and C57Bl/6 mice
obtained fromthe Instituteof Pathology, Facultyof Medicine, Universityof Ljubljana,
Slovenia. At the beginning of the experiments mice were 10e12 weeks old and were
housed in specific pathogen free colony at constant room temperature 20e24?C,
relative humidity 55 ? 10% and 12 h light/dark cycle. Food and water were
period of 7e10 days before the experiments were carried out. All procedures were
performed in compliance with the official guidelines of the Ministry of Agriculture,
Forestry and Food of the Republic of Slovenia (permission no. 34401-11/2009/6).
2.3.1. In vivo acute toxicity and biodistribution of SPIONs-PAA and SPIONs-PAA-PEI-
Acute toxicity and biodistribution of SPIONs-PAA and SPIONs-PAA-PEI bound to
pDNAGFP(SPIONs-PAA-PEI-pDNAGFP) were assessed in BALB/c mice. Acute toxicity of
SPIONs-PAA was evaluated using Up-and-Down Procedure (UDP) protocol by the
Organization for Economic Co-operation and Development (OECD). Biodistribution
of SPIONs-PAA and SPIONs-PAA-PEI-pDNAGFPwas determined quantitatively by
measuring iron concentration in samples of digested internal organs, i.e. lungs,
heart, spleen, liver and kidneys, using ICP-MS, and qualitatively by recording images
of histological specimens of liver with digital camera (Nikon DXM1200F; Nikon,
Tokyo, Japan) attached to light microscope (Nikon Eclipse 80i). According to the
OECD guidelines, food was restrained 3 h before and 1 h after the experiment
whereas water was provided ad libitum. For the acute toxicity determination
assessing the median lethal dose (LD50) mice were injected intraperitoneally (i.p.)
with 1 ml of high doses of SPIONs-PAA (175 mg/kg and 550 mg/kg). The highest dose
suggested by the guidelines (2000 mg/kg) was not possible to inject due to the
restricted i.p. injection volume and limited highest concentration of SPIONs-PAA
stock solution. Additionally, body weight was monitored by weighing mice at the
commencement and throughout the experiment every 4 days after the initial
administration of SPIONs-PAA. Changes in body weight are presented as the ratios of
the intermediate and final body weights to the initial body weight. Based on the
results of our in vitro experiments and regarding pDNAIL?12dosage optimization
after i.t. administration followed by electroporation of tumors , three mice per
group were also injected i.p. with low doses of SPIONs-PAA (1.2 mg/kg) and SPIONs-
PAA-PEI-pDNAGFP(1.2-2-2 mg/kg, respectively). Control group of animals was
injected i.p. with distilled water in the same volume as SPIONs-PAA and SPIONs-
PAA-PEI-pDNAGFPwere given (1 ml). Animals were sacrificed 14 days after the
commencement of the experiment. Organs were removed, weighted and stored in
4 ml cryo vials (BD Biosciences) at ?18?C for ICP-MS analysis. Before analysis,
microwave digestion of organs was applied, and then the measurement was per-
formed likewise the quantitative internalization of SPIONs-PAA and SPIONs-PAA-
PEI-pDNAGFPinto cells. Additionally, qualitative evaluation of biodistribution of
SPIONs-PAA was made in liver, which was immediately after the excision fixed in
10% (w/v) buffered formalin phosphate and embedded in paraffin. From paraffin
10 mm thick sections were cut and stained with Perl’s Prussian blue histochemical
method. The Perls histochemical reaction is adequate for the detection of iron as
ferritin and hemosiderin in higher vertebrates .
2.3.2. Magnetofection of tumors with pDNAGFPusing SPIONs-PAA-PEI
Magnetofection of B16F1 melanoma tumors, syngeneic to C57Bl/6 mice, and TS/
A mammary adenocarcinoma tumors, syngeneic to BALB/c mice, with pDNAGFPwas
evaluated in frozen tumor sections, which were examined for the GFP level and
spatial distribution by recording images with digital camera (Olympus DP72)
attached to fluorescent microscope (Olympus BX51). Tumors were induced by
inoculating 1.0 ? 106B16F1 cells or 2.0 ? 106TS/A cells in 0.1 ml of 0.9% NaCl
subcutaneously into the right flanks of mice. Cells of both cell lines were prepared
from in vitro cultures growing in advanced MEM. After 7e9 days, when the tumors
reached approx. 40 mm3, mice were randomly divided into 6 groups consisting of 3
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
animals and subjected to different experimental protocols for 3 days consecutively.
First group of animals was injected intratumorally (i.t.) with 40 ml of distilled water.
Second group of animals was injected i.t.15 mg of pDNAGFPin 40 ml of endotoxin-free
water. Third group of animals was injected i.t. with 15 mg of pDNAGFPprepared in
40 ml of PEI-pDNAGFPcomplexes at the mass ratio 1:1. Fourth and fifth group of
animals were injected i.t. with 15 mg of pDNAGFPprepared in 40 ml of SPIONs-PAA-
PEI-pDNAGFPcomplexes at the mass ratio of 0.6:1:1. Thereafter, Nd-Fe-B magnets
(same magnets as used for in vitro experiments) were placed above the tumors of
the fourth group and fixed with the tape for 30 min. Sixth group of animals was
injected i.t. with 15 mg of pDNAGFPin 40 ml of endotoxin-free water. After 10 min,
electroporation of tumors was performed by applying eight square-wave electric
pulses, delivered in 2 sets of 4 pulses in perpendicular directions, at a frequency of
1 Hz, amplitude overdistance ratio of 600 V/cm and 5 ms duration through2 parallel
stainless steel electrodes with a 7 mm gap connected to GT-1 electroporator. All
injections were performed using 0.3 ml insulin syringes with 28-gaugee needles
(Terumo, Tokyo, Japan) on anesthetized animals. Also, fourth group of animals was
subjected to anesthesia during the fixation of Nd-Fe-B magnets above the tumors.
General anesthesia was induced and maintained by placing mice in a clear plastic
chamber while administering 1.5e3% vapor set of isoflurane with oxygen flow at 1 l/
min (Draeger, Lübeck, Germany). Tumors were excised 24 h after the last injection,
embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA) and stored
at ?18?C. Frozen tumor sections of 20 mm were cut every 150 mm of the tumor with
cryostat (Leica CM1850; Leica Mycrosystems, Wetzlar, Germany) for the GFP level
and spatial distribution examination.
2.3.3. Magnetofection of tumors with pDNAIL?12using SPIONs-PAA-PEI
For the proof-of-principle, transfection of TS/A tumors with pDNAIL?12was
performed in BALB/c mice. Transfection efficacy of tumors was determined by
calculating tumor volumesafter measuring 3 orthogonal tumordiameters (e1, e2and
e3) with Vernier caliper, using the formula V ¼p ? e1? e2? e3
induced likewise the tumors for the pDNAGFP-based experiments. After 7e9 days,
when the tumors reached approx. 40 mm3, mice were randomly divided into 9
groupsconsisting of 4 or5 animals and subjected todifferentexperimental protocols
for 3 days consecutively. The protocol for the first six groups was the same as for the
Eighth group of animals was injected i.t.15 mg of PEI in 40 ml of endotoxin-free water.
Ninthgroupof animals was injectedi.t. 9mgof SPIONs-PAA in 40ml of endotoxin-free
water with subsequent exposure to Nd-Fe-B magnets for 30 min that were placed
above the tumors with the tape. Tumor growth and regression was monitored until
the tumors reached between 300 and 350 mm3; then the animals were euthanized.
Tumor growth delay was calculated when the mean value of the tumor volume of all
experimental groups reached 200 mm3because the tumors of SPIONs-PAA-PEI-
pDNAIL?12-treated mice and after pDNAIL?12electrotransfer started to delay in
growth after they had already reached doubling or tripling volume.
. TS/A tumors were
2.4. Statistical analysis
All quantitative data were tested for normality of distribution by Shapiro-Wilk
test and are presented as means ? standard errors (s.e.m.). Student’s t-test was
used to evaluate the differences in the transfection efficacy between the absence and
presence of magnetic field regarding optimization of magnetofection of cells with
pDNAGFPusing SPIONs-PAA-PEI. Allotherquantitative datawere analyzed byone way
ANOVA, and tested for significance by Holm-Sidak test. Alpha level was set to 0.05.
When P values were 0.05 or less, differences were considered statistically significant.
All statistical analyses were performed by SigmaPlot 11 (Systat Software, San Jose, CA).
3.1. De novo synthesis of SPIONs-PAA-PEI
We synthesized SPIONs for magnetofection by alkaline co-
precipitation of ferrous and ferric ions in aqueous solution
according to the Massart method . To increase magnetofection
efficacy and reduce PEI-related cytotoxicity, we coated SPIONs in
situ with pH-responsive anionic polymer PAA and functionalized
obtained SPIONs-PAA with cationic polymer PEI through electro-
static interaction between highly negative surface of SPIONs-PAA
SPIONs-PAA with PEI was performed directly prior to the pDNAs
binding due to incipient stability of the ferrofluid containing
SPIONs-PAA-PEI. All results regarding physicochemical properties
of SPIONs, SPIONs-PAA and SPIONs-PAA-PEI stand for a represen-
3.1.1. Physicochemical properties of SPIONs, SPIONs-PAA, SPIONs-
PAA-PEI and SPIONs-PAA-PEI-pDNAGFP
From the TEM images (Fig.1a), diameter of spherical SPIONs was
estimated to be 8 ? 1 nm whereas estimated diameter of SPIONs-
PAA was 10 ? 1 nm. After functionalization of SPIONs-PAA with PEI
crystalline SPIONs-PAA was observed. After binding pDNAGFPto
PAA-PEI was noted. Crystallographic structure of SPIONs and
SPIONs-PAA was iron oxide maghemite (g-Fe2O3) as observed from
electrophoresis (Fig. 1c). Contrariwise, highly negative surface of
SPIONs-PAA without PEI functionalization remained unattached to
electronegative oxygens of phosphate groups of pDNA.
The ferrimagnetic shape of M(H) curves and saturated magne-
tization values of dry SPIONs and SPIONs-PAA (amounting M0¼ 69
emu/g and M0¼ 62 emu/g, respectively) were typical for g-Fe2O3-
composed nanoparticles (Fig. 1d). The zero-field-cooled (zfc) and
field-cooled (fc) magnetic susceptibilities, c ¼ M/H, of dry SPIONs
and SPIONs-PAA, measured in a magnetic field H ¼ 100 Oe, showed
czfc- cfcsplitting at about 280 K, whereas a broad maximum in czfc
was observed at the temperature TB¼ 185 K. This could be asso-
ciated with the superparamagnetic blocking temperature below
which SPIONs’ spin reorientation is frozen on the experimental
time scale. The TB ¼ 185 K value was consistent with TEM-
estimated diameters of SPIONs and SPIONs-PAA, as for larger
SPIONs TBshifts above the room temperature. Similarly, czfc- cfc
splitting and TBz 300 K were observed for SPIONs-PAA, where
slightly higher values, as compared to SPIONs, verylikelyoriginated
from their slightly larger size. Magnetic characterization of dry
SPIONs and SPIONs-PAA thus confirmed their superparamagnetic
nature and small size with diameters in the range 8e10 nm. The
M(H) curves and the magnetic susceptibility c ¼ M/H of ferrofluids
(FFs), denoting water-based suspension of SPIONs (FF-SPIONs) and
SPIONs-PAA (FF-SPIONs-PAA), were also determined. The M(H)
shapes remained those of the dry samples (the diamagnetic
susceptibility of water is negligible as compared to the ferrimag-
netic susceptibility of g-Fe2O3-composed SPIONs). The difference in
magnetization values between FF-SPIONs and FF-SPIONs-PAA
originated from different concentration of nanoparticles in the
suspensions (FF-SPIONs > FF-SPIONs-PAA). The temperature-
dependent zfc and fc susceptibilities of FFs displayed discontinu-
ities in the vicinity of T ¼ 0?C ¼ 273 K (marked by vertical dashed
line), which was a consequence of FFs freezing upon cooling and
melting upon heating. By measuring the fc susceptibility in a cool-
ing run, starting at 300 K, FFs freezed at 260 K. In the zfc suscep-
tibility measurements performed in a heating run, starting at 2 K,
melting of FFs started at 273 K.
Zeta potentials of FF-SPIONs at pH 9.5 and FF-SPIONs-PAA at pH
8.5 were z ¼ ?24 ? 2 mV and z ¼ ?47 ? 2 mV, respectively, indi-
cating negative surface charge of SPIONs as well as SPIONs-PAA and
good stability of FF-SPIONs-PAA due to strong mutual electrostatic
repulsion. Zeta potential of the FF-SPIONs-PAA-PEI at pH 8 was
z ¼ 20 ? 1 mV, indicating positive surface charge of SPIONs-PAA-
PEI due to the functionalization of SPIONs-PAA’s surface with PEI.
3.2. In vitro
3.2.1. Cytotoxicity of SPIONs-PAA-PEI and SPIONs-PAA-PEI-pDNAGFP
on cells of different cell lines
We tested cytotoxicity of SPIONs-PAA-PEI and SPIONs-PAA-PEI
bound to pDNAGFP(SPIONs-PAA-PEI-pDNAGFP) on mouse mela-
noma B16F1 cells, human melanoma SK-MEL-28 cells, mouse
fibroblasts L929 and human mesothelial MeT-5A cells, and
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
compared it to cytotoxicity of PEI-pDNAGFP. No additional cyto-
toxicity of an external magnetic field generated by Nd-Fe-B
magnets on cells of all four cell lines in comparison to cells not
exposed to an external magnetic field was observed (Fig. 2a).
Therefore, results regarding cytotoxicity of a certain substance as
well as comparisons between the substances will be interpreted
referring to the presence of an external magnetic field. No
substance decreased cell survival below 50% in all cell lines. In
general, cells of all cell lines exhibited similar toxicity-related
patterns to added substances. Only for MeT-5A cells, survival was
significantly decreased after exposure to pDNAGFP
comparison to SPIONs-PAA, PEI and SPIONs-PAA-PEI. Pronounced
sensibility of MeT-5A cells to pDNAGFPmight be due to their
spontaneous transfection which we observed during the experi-
ments (data not shown). Survivals of cells of all cell lines, treated
with SPIONs-PAA-PEI, alternated between 82% and 86%. Binding of
pDNAGFPto SPIONs-PAA-PEI (SPIONs-PAA-PEI-pDNAGFP) signifi-
cantly decreased cell survival, probably due to the compromised
cell membrane integrity after internalization of SPIONs-PAA-PEI-
pDNAGFP, which was followed by magnetofection (See further
results). Similar was observed after treatments of cells with PEI-
pDNAGFP. SPIONs-PAA-PEI-pDNAGFPdisplayed tendency of being
less cytotoxic than PEI-pDNAGFPin all cell lines, however, only in
L929 cells significant difference in cell survival after the exposureto
Fig. 1. Physicochemical properties of SPIONs, SPIONs-PAA, SPIONs-PAA-PEI and SPIONs-PAA-PEI-pDNAGFP: (a) Transmission electron micrographs of spherical SPIONs, SPIONs-PAA,
SPIONs-PAA-PEI and SPIONs-PAA-PEI-pDNAGFP.12 nm-sized SPION-PAA-PEI has an amorphous 1e2 nm edge (double arrows). SPIONs-PAA-PEI are entrapped inside organic bubble-
like-formation (pDNA) forming SPIONs-PAA-PEI-pDNAGFP(arrows). (b) X-ray diffractograms of SPIONs and SPIONs-PAA exhibit characteristic peaks for iron oxide maghemite (g-
Fe2O3). (c) Retardation of pDNAIL?12and pDNAGFPbound to SPIONs-PAA-PEI (lanes 1 and 5, respectively), and to PEI (lanes 2 and 6, respectively) (arrows). Separation of pDNAIL?12
and pDNAGFP(lanes 3 and 7, respectively) as well as pDNAIL?12and pDNAGFPthat did not bound to SPIONs-PAA (lanes 4 and 8, respectively). Molecular weight markers (M). (d) M(H)
curves, the saturated magnetization and temperature dependent zero-field-cooled (zfc) and field-cooled (fc) magnetic susceptibilities, c ¼ M/H, of dry SPIONs and SPIONs-PAA and
ferrofluids containing SPIONs (FF-SPIONs) and SPIONs-PAA (FF-SPIONs-PAA). All data are for a representative sample.
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
Fig. 2. Cytotoxicity and internalization of SPIONs-PAA-PEI-pDNAGFP: (a) Cytotoxicity of SPIONs-PAA-PEI and SPIONs-PAA-PEI-pDNAGFPon B16F1, SK-MEL-28, L929 and MeT-5A cells
in comparison to PEI-pDNAGFPin the absence and presence of an external magnetic field. *P < 0.05, **P < 0.01, between the compared groups (n ¼ 12). (b,c) Internalization of
SPIONs-PAA-PEI-pDNAGFPinto cells of different cell lines after 4-h incubation with SPIONs-PAA-PEI-pDNAGFP. (b) Quantitative determination of internalized SPIONs-PAA-PEI-
pDNAGFPcorresponds to the Fe concentration per cell. (c) Transmission electron micrographs of B16F1, SK-MEL-28, L929 and MeT-5A cells with internalized SPIONs-PAA-PEI-
pDNAGFPcomplexes after 15-min exposure to an external magnetic field. SPIONs-PAA-PEI-pDNAGFPinside endocytotic compartments with intact membranes (arrowheads).
Pseudopodia-like formations (arrows) coinciding with the invagination of the cell membrane. Scale bar, 500 nm first and third column; 200 nm second and fourth column. (d)
Internalization of SPIONs-PAA-PEI-pDNAGFPinto B16F1 cells after 24-h incubation with SPIONs-PAA-PEI-pDNAGFPsubsequent to 15-min exposure to an external magnetic field.
Transmission electron micrographs (clockwise): The internalized SPIONs-PAA-PEI-pDNAGFPinside the endocytotic compartment (arrow) beneath the cell surface. SPIONs-PAA-PEI-
pDNAGFPin the endocytotic compartment with intact membrane, and organic compound separation (arrow). SPIONs-PAA-PEI-pDNAGFPin the endocytotic compartment with
disrupted membrane (arrow). Vacuoles without SPIONs-PAA-PEI-pDNAGFP(arrowheads). Scale bar, 200 nm. (e) Schematic showing the process of internalization of SPIONs-PAA-
PEI-pDNAGFPfollowed by subsequent magnetofection of the cell after exposure to an external magnetic field in a 24 h time scale.
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
SPIONs-PAA-PEI-pDNAGFP(67%) and PEI-pDNAGFP(50%) in disfavor
to the latest was observed.
3.2.2. Internalization of SPIONs-PAA-PEI-pDNAGFP into cells
Internalization of SPIONs-PAA-PEI bound to pDNAGFP(SPIONs-
PAA-PEI-pDNAGFP) was evaluated quantitatively and qualitatively.
Quantitative analysis demonstrated internalization of SPIONs-PAA-
PEI-pDNAGFPinto cells of all cell lines at 4 h after the incubation
magnetic field for different time intervals (Fig. 2b). Internalization
of SPIONs-PAA-PEI-pDNAGFPcorresponds to the cellular iron (Fe)
concentration between the cell lines were noticed. In all cell lines
directly proportional tendency between the time interval exposure
to an external magnetic field and Fe concentration was found.
However, no statistically significant differences in Fe concentration
between 0 and 30 min exposures toan externalmagnetic field were
determined. Additionally, in B16F1 and L929 cells plateau level at
15-min exposure to an external magnetic field was observed.
According to these results, intermediate time interval exposure
(15 min) to an external magnetic field was chosen for the imple-
mentation into further experiments for magnetofection of cells
with pDNAGFPor pDNAIL?12.
In agreement with the results of quantitative analysis, at 4 h
after incubation with SPIONs-PAA-PEI-pDNAGFPthat included 15-
min exposure of cells to an external magnetic field, internaliza-
tion of complexes was observed in cells of all cell lines (Fig. 2c). Due
to the size of the complexes (approx. 200e400 nm), the cellular
uptake was determined to be phagocytosis. Moreover, distinctive
pseudopodia-like formations were observed in cells of all cell lines.
The complexes were observed in the endocytotic compartments
with intact membranes. No SPIONs-PAA-PEI-pDNAGFPcomplexes
outside the endocytotic compartments neither in the cytoplasm
nor in the nucleus were observed. At 24 h after incubation with
SPIONs-PAA-PEI-pDNAGFP, the complexes were observed in the
endocytotic compartments of B16F1 cells with intact as well as
disrupted membranes (Fig. 2d). No SPIONs-PAA-PEI-pDNAGFPwere
observed in the cytoplasm, however, separation of organic
compound (pDNAGFP) from the complexes within the endocytotic
compartments and vacuoles without SPIONs-PAA-PEI-pDNAGFP
were detected. Schematic showing the processes of internalization
of SPIONs-PAA-PEI-pDNAGFPfollowed by subsequent magneto-
fection after exposure of a cell to an external magnetic field in
a 24 h time scale (Fig. 2e).
and exposure to an external
3.2.3. Magnetofection of cells with pDNAGFPusing SPIONs-PAA-PEI
Magnetofection with pDNAGFPusing SPIONs-PAA-PEI was per-
formed in four cell lines: B16F1, SK-MEL-28, L929 and MeT-5A, and
compared to the transfection using PEI only (Fig. 3a). Optimization
of magnetofection was conducted by preparing SPIONs-PAA, PEI
and pDNAGFPat different mass ratios. Among the cell lines B16F1
cells exhibited the highest percentage of fluorescent cells (47%) at
the mass ratio of 0.6:1:1, however, MeT-5A cells displayed the
highest fluorescence intensity (11,943 a.u.) at the mass ratio 0.5:1:1.
The lowest percentage of fluorescent cells (2%) as well as the lowest
fluorescence intensity (520 a.u.) was observed in L929 cells. The
percentage of fluorescent SK-MEL-28 cells (13%) was similar to that
of MeT-5A cells (12%), however, their fluorescence intensity (4,169
a.u.) was only higher than that of L929. In all cell lines, exposure of
cells to an external magnetic field contributed to the increase in the
percentage of fluorescent cells only at the mass ratio 0.9:1:1,
however the highest percentage of fluorescent B16F1 cells was
observed at the mass ratio 0.6:1:1. Interestingly, an external
magnetic field did not contribute to the increase in the percentage
of fluorescent B16F1 cells only at that mass ratio. In comparison to
the transfection with pDNAGFPusing PEI prepared as PEI-pDNAGFP
at the mass ratio 1:1, significant increase in the percentage of
fluorescent B16F1 cells at the mass ratio 0.6:1:1 was observed.
According to all these results, mass ratio 0.6:1:1 was chosen to be
implemented in further experiments on B16F1 cells (Fig. 3b).
Photomicrographs taken under fluorescence epi-illumination
supportthe quantitative results
observed in all cell lines in the absence and presence of an external
magnetic field (Fig. 3c). The GFP levels observed from the photo-
micrographs indicated differences in transfection efficacy at the
level of the cell line as well as some alterations in the absence and
presence of an external magnetic field were noticed. In agreement
with the results obtained from quantitative analysis, B16F1 cells
showed the highest GFP level whereas L929 cells resulted in the
Magnetofection of B16F1 cells with pDNAGFPusing SPIONs-PAA-
PEI was compared to transfection using PEI, 3 commercially avail-
able magnetic nanoparticles, electroporation, lipofection and
SPIONs-PAA-PEI in the absence of an external magnetic field
(Fig. 3d). Magnetofection using SPIONs-PAA-PEI resulted in excel-
lent transfection efficacy, which was comparable to electro-
poration, lipofection and SPIONs-PAA-PEI in the absence of an
external magnetic field. However, SPIONs-PAA-PEI outperformed
PEI and all commercially available magnetic nanoparticles for
magnetofection either in the percentage of fluorescent cells or in
the fluorescence intensity.
3.2.4. Magnetofection of cells with pDNAIL?12using SPIONs-PAA-PEI
Magnetofection of B16F1 cells with pDNAIL?12using SPIONs-
PAA-PEI was compared to transfection using PEI, electroporation,
lipofection and SPIONs-PAA-PEI in the absence of an external
magnetic field (Fig. 3e). The secretion of IL-12 from B16F1 cells into
medium was observed after transfection with all the methods
tested.Magnetofection resulted in 12.6-fold and 7.2-fold increase in
transfection efficiency in comparison to PEI and lipofection,
respectively. No statistically significant differences in transfection
efficacy using SPIONs-PAA-PEI in the absence and presence of
magnetic were observed, however, IL-12 secretion was increased
for 1.2-fold in the presence over the absence of an external
3.3. In vivo
3.3.1. Acute toxicity and biodistribution of SPIONs-PAA and SPIONs-
The acute toxicity was determined according to the OECD
guidelines. The i.p. administration of the highest dose injected,
550 mg/kg of SPIONs-PAA, did not result in any death in the time
period of 14 days after injection. Therefore, LD50determinationwas
not possible. The highest dose suggested to be used by OECD
guidelines (2000 mg/kg) was not possible to inject due to the
restricted i.p. injectionvolume and limited highest concentration of
SPIONs-PAA in stock solution. A slight but non-significant decrease
in body weight was observed on day 4 after the first administration
of SPIONs-PAA as well as distilled water, but after day 4 the body
weight started to rise (Fig. 4a).
After the i.p. administration of 175 mg/kg and 550 mg/kg of
SPIONs-PAA, Fe concentrations were elevated in spleen and liver
(Fig. 4b). In spleen, Fe concentrations were increased for 1.9-fold
and 2.5-fold after administration of 175 mg/kg and 550 mg/kg of
SPIONs-PAA, respectively, in comparison to the Fe concentration
measured in the spleen of control mice. Similarly in liver, Fe
concentrations were increased after administration of 175 mg/kg
and 550 mg/kg of SPIONs-PAA for 1.5-fold and 3.4-fold, respec-
tively, in comparison to the Fe concentration measured in the liver
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
Fig. 3. Magnetofection of cells using SPIONs-PAA-PEI: (a) Determination of the GFP level, measured as the percentage of fluorescent (FL) cells and median fluorescence (FL)
intensity, in B16F1, SK-MEL-28, L929 and MeT-5A cells after transfection with pDNAGFPusing SPIONs-PAA-PEI prepared as SPIONs-PAA-PEI-pDNAGFPat mass ratios from 0.5:1:1 to
0.9:1:1 in the absence and presence of magnetic field. Mass ratio 1:1 denotes PEI-pDNAGFP. *P < 0.01, **P < 0.001, comparison between mass ratios.yP < 0.05,yyP < 0.01,yyyP < 0.001,
compared to the absence of an external magnetic field (n ¼ 4 or 11). (b) The GFP level and increase after magnetofection of cells using SPIONs-PAA-PEI prepared as SPIONs-PAA-PEI-
pDNAGFPat the mass ratio 0.6:1:1. (c) Micrographs of B16F1, SK-MEL-28, L929 and MeT-5A cells after transfection with pDNAGFPusing SPIONs-PAA-PEI prepared as SPIONs-PAA-PEI-
pDNAGFPat the mass ratio 0.6:1:1 in the absence and presence of magnetic field under phase contrast (PC) and fluorescence epi-illumination (FL) (?60 magnification). (d,e)
Magnetofection of B16F1 cells with pDNAGFPor pDNAIL?12using SPIONs-PAA-PEI, prepared as SPIONs-PAA-PEI-pDNAGFPor SPIONs-PAA-PEI-pDNAIL?12at the mass ratio 0.6:1:1, in
comparison to other methods. (d) The GFP level in B16F1 cells. *P < 0.01, compared to SPIONs-PAA-PEI magnet (FL cells).yP < 0.05,yyP < 0.01, compared to SPIONs-PAA-PEI magnet
(FL intensity) (n ¼ 7e13). (e) The secretion of IL-12 from B16F1 cells.*P < 0.01, compared to SPIONs-PAA-PEI magnet (n ¼ 3).
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
of control mice. Based on the results of our in vitro experiments and
regarding pDNAIL?12dosage optimization after i.t. administration
followed by electroporation of tumors , mice were also injected
i.p. with low doses of SPIONs-PAA (1.2 mg/kg) and SPIONs-PAA-PEI-
pDNAGFP(1.2-2-2 mg/kg) to evaluate the biodistribution. No
significant differences in Fe concentration within the organs
between the control group and low doses-treated groups of mice
with SPIONs-PAA and SPIONs-PAA-PEI-pDNAGFPwere detected
In agreement with the results of quantitative analysis, Perl’s
Prussian blue staining of the control liver indicated SPIONs-PAA
free organ whereas after the administration of high doses of
SPIONs-PAA blue precipitates with Fe3þ, indicating the accumula-
tion of SPIONs-PAA, in the phagocytes of the reticulo-endothelial
system were detected (Fig. 4d).
3.3.2. Magnetofection of tumors with pDNAGFPusing SPIONs-PAA-
Magnetofection of tumors with pDNAGFPusing SPIONs-PAA-PEI
was tested in two tumor models: B16F1 melanoma syngeneic to
C57Bl/6 mice due to the highest magnetofection efficacy of B16F1
cells in vitro, and TS/A mammary adenocarcinoma syngeneic to
BALB/c mice, in which acute toxicity and biodistribution were
determined. Also, we were interested in how weakly immunogenic
TS/A mammary adenocarcinoma  would respond to immuno-
gene therapy with pDNAIL?12.
As demonstrated from the frozen tumor sections, magneto-
fection of B16F1 melanoma and TS/A mammary adenocarcinoma
tumors with pDNAGFPusing SPIONs-PAA-PEI was effective (Fig. 5a).
The GFP level in B16F1 tumors was observed either after 30-min
exposure to an external magnetic field or in the absence of an
external magnetic field. However, without an external magnetic
field exposure, the GFP level in TS/A tumors after transfection with
SPIONs-PAA-PEI was barely visible. Importantly, no transfection of
both tumors with pDNAGFPusing PEI was observed (data not
shown). Hence, by combining PEI with SPIONs-PAA, increased
transfection efficiency in vitro was achieved and magnetofection of
two different murine tumors was proved in vivo. Gene electro-
transfer of tumors was used as a positive control since it is known
and well established non-viral transfection method of tumors
[30,31]. Gene electrotransfer resulted in homogenous GFP spatial
distribution whereas after magnetofection scattered GFP spatial
distribution was observed (Fig. 5a). The effect of an external
magnetic field on magnetofection of melanoma B16F1 and
mammary adenocarcinoma TS/A tumors with pDNAGFPusing
SPIONs-PAA-PEI was more pronounced in TS/A than in B16F1
3.3.3. Magnetofection of tumors with pDNAIL?12using SPIONs-PAA-
Magnetofection of TS/A mammary adenocarcinoma tumors
with pDNAIL?12using SPIONs-PAA-PEI resulted as effective treat-
ment in BALB/c mice (Fig. 5b). Three consecutive treatments lead to
statistically significant reduction in the tumor volume compared to
non-treated tumors. The effect of the treatment was noticed 2 days
after the completion of the treatment. The significant antitumor
effect was seen only when the tumors were exposed to an external
magnetic field whereas in the absence there was no antitumor
effect. Gene electrotransfer of pDNAIL?12has in other studies
already resulted in tumor growth regression [27,32]. Thus, we used
Fig. 4. Change in body weight after the i.p. administration of high doses of SPIONs-PAA and biodistribution of SPIONs-PAA and SPIONs-PAA-PEI-pDNAGFPafter i.p. administration of
low and high doses: (a) Change in body weight. Symbols represent the ratios of intermediate and final body weights to initial body weight. The experiment was performed in one
mouse per dosage due to the OECD guidelines. (b) Biodistribution of high doses of SPIONs-PAA. The experiment was performed in one mouse per dosage due to the OECD guidelines.
Bars represent absolute values. (c) Biodistribution of low doses of SPIONs-PAA and SPIONs-PAA-PEI-pDNAGFP. (d) Micrographs of mice liver stained with Perl’s Prussian blue after
administration of distilled water (control) and two different high doses of SPIONs-PAA.
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
Fig. 5. Magnetofection of tumors with pDNAGFPor pDNAIL?12using SPIONs-PAA-PEI: (a) Micrographs of frozen murine melanoma B16F1 and mammary adenocarcinoma TS/A
tumor sections under phase contrast (PC) and epi-fluorescence illumination (FL) after transfection with pDNAGFPusing (A) magnetofection, (B) SPIONs-PAA-PEI in the absence of
magnetic field, (C) electrotransfer. Scale bar, 50 mm. (b) Antitumor effect of i.t. administration of pDNAIL?12on TS/A mammary adenocarcinoma tumors after magnetofection with
SPIONs-PAA-PEI and electrotransfer. Blue arrows represent three consecutive treatments with pDNAIL?12. Tumor growth delay was calculated on the 10th day. (c) The effect of
cancer immuno-gene therapy with pDNAIL?12on TS/A mammary adenocarcinoma tumors. *P < 0.05, **P < 0.01, compared to control (n ¼ 5).
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
gene electrotransfer as a positive control, and similarly to magne-
tofection it resulted in statistically significant antitumor effect in
comparison to untreated control tumors (Fig. 5c). Tumor growth
delay was calculated at the tumor volume 200 mm3because
tumors of SPIONs-PAA-PEI-pDNAIL?12-treated mice and after
pDNAIL?12electrotransfer started to delay in growth after they had
already reached doubling or tripling volume (Fig. 5b). The effect of
magnetofection and gene electrotransfer was comparable: gene
therapy of tumors with pDNAIL?12resulted in 7.8 ? 1.3 and 6.6 ?1.1
days tumor growth delays, respectively. In all the other control
groups no antitumor effect was observed (Fig. 5c).
Biocompatibility and biodistribution of every nanoparticulate
delivery system should be determined since the physicochemical
properties, such as size, shape and surface characteristics, might
govern their brand new behavior and safety in vitro and invivo .
In vitro, survival of cells of all cell lines was after treatment with
SPIONs-PAA-PEI subsequent to 15-min exposure to an external
magnetic field still around 80%, which is similar to the cytotoxicity
of silica-coated SPIONs . PEI-related cytotoxicity of PEI-
pDNAGFPreduced cell survival up to 50%, and was statistically
significantly diminished byassociating PEI-pDNAGFPto SPIONs-PAA
only in fibroblasts L929. In general, SPIONs-PAA-PEI-pDNAGFP
decreased survival of cells to approximately 70%, which indicated
that they were not vastly cytotoxic, and could be further used in
animal studies. In vivo, i.p. administration of 175 mg/kg and
550 mg/kg of SPIONs-PAA did not reach the LD50. To date, there are
no other reports about the in vivo toxicity of g-Fe2O3-composed,
PAA-coated and PEI-functionalized SPIONs. Different research
groups selected different coatings, functionalization and cargos for
SPIONs as delivery systems, thus the comparison between the
studies evaluating LD50 of so diversified SPIONs is virtually
impossible. Nonetheless, acute toxicity-based study in mice
showed that LD50for magnetite (Fe3O4)-composed and dextran-
coated magnetic nanoparticles after i.p. administration is above
2000 mg/kg . Other studies in mice revealed that after i.p.
administration LD50for Fe3O4-composed and daunorubicin-loaded
magnetic nanoparticles is 1010 mg/kg , and for Fe3O4-
composed and Au-coated magnetic nanoparticles LD50is 8390 mg/
kg . In our study, the i.p. administration of high doses of
SPIONs-PAA resulted in theiraccumulation inside the phagocytes of
the reticulo-endothelial system of spleen and liver. Histologically,
no damages in the liver of low and high dose SPIONs-PAA-treated
mice were detected because iron-induced hepatocellular injury
does not occur when SPIONs are taken up by reticuloendothelial
cells . The i.p. administration of low doses of SPIONs-PAA as
well as SPIONS-PAA-PEI-pDNAGFPdid notresultin anyalterations in
the histological specimen (data not shown) as well as in the level of
iron measured in the internal organs of mice. On the other hand,
Trubetskoy et al. observed extensive parenchymal destruction of
the mice liver after i.v. administration of complexes composed of
PAA, PEI and pDNA, but without SPIONs, at slightly higher dose
than ours . This suggests that in vivo usage of our SPIONs-PAA-
PEI-pDNAGFPis safe when their mass ratio and dosage is carefully
Cellular uptake of SPIONs for magnetofection is the doorway for
efficient nucleic acid delivery to the cell nucleus. It has been shown
in many studies that malignant cells internalize more SPIONs than
normal cells [34,39,40]. After 4-h incubation, we detected inter-
nalized SPIONs-PAA-PEI-pDNAGFPat the mass ratio 0.6:1:1 in cells
of all four cell lines, however, at that time no statistically significant
differences in the internalization between normal and malignant
cells were measured. On the other hand, after 24-h incubation,
diverse GFP levels among the cell lines were determined: malig-
nant mouse B16F1 and human SK-MEL-28 melanoma cells out-
performed normal human mesothelial MeT-5A cells and mouse
fibroblasts L929. After 24-h incubation, no SPIONs-PAA-PEI-
pDNAGFPinside the nuclei of B16F1 cells were detected, but we
observed internalized SPIONs-PAA-PEI-pDNAGFPin the endocytotic
compartments with disrupted membranes, which indicates the
infamous PEI-related proton sponge effect mechanism . The
direct evidence that PAA as an endosomolytic polymeric anion also
contributed to the membrane disruption could not be observed,
however, increased levels of GFP and IL-12 obtained after magne-
tofection of B16F1 cells with SPIONs-PAA-PEI in comparison to PEI
is an indirect indication.
To further evaluate SPIONs-PAA-PEI as delivery systems for
pDNA in vitro and in vivo, we first compared in vitro efficacy of
magnetofection of mouse melanoma B16F1 cells with pDNAGFPand
pDNAIL?12using SPIONs-PAA-PEI to the efficacy of transfection
using PEI, 3 commercially available magnetic nanoparticles for
magnetofection, electroporation, lipofection and SPIONs-PAA-PEI
in the absence of an external magnetic field. PEI is well known
transfection agent with pronounced endosomolytic properties ,
however, when coupled with SPIONs, synergistic effect in trans-
fection efficacy has been observed [11,20,21]. Coating and func-
tionalization of SPIONs with PAA and PEI, respectively, significantly
increased magnetofection efficacy of mouse melanoma B16F1 cells
with pDNAGFPand pDNAIL?12in comparison to the transfection
using PEI only. Moreover, SPIONs-PAA-PEI outperformed all
commercially available magnetic nanoparticles that we tested
either at the percentage of GFP-positive cells or at the fluorescence
intensity of GFP. Magnetofection efficacy of B16F1 cells with
pDNAGFPusing SPIONs-PAA-PEI was about the same as that
obtained by lipofection and electroporation. In another study
transfection of B16F1 cells with pDNAGFPusing electroporation
resulted in approx. 1.5-fold more GFP-positive cells that we
observed after electroporation . However, for more accurate
evaluation of transfection efficacy also the fluorescence intensity of
GFP, indicating the amount of the protein synthesized, should be
taken into account. The amount of secreted IL-12 from B16F1 cells
was statisticallysignificantlyhigheraftermagnetofection than after
transfection with PEI and lipofection. Cationic lipids efficiently
deliver pDNA by endocytosis , and destabilize fluid lipid bila-
yers of cell membranes by promoting the formation of non-bilayer
lipid structures . Significantly lower amount of secreted IL-12
from B16F1 cells after lipofection in comparison to magneto-
fection and electroporation might indicate liposome-induced
alterations in cell membrane resulting in diminished exocytosis-
mediated protein secretion.
Magnetofection of tumors with pDNAGFPusing SPIONs-PAA-PEI
was the first step in evaluating the efficacy of our gene delivery
system also in vivo. To date, only two research groups report about
efficient non-virally-associated magnetofection of tumors with
pDNA encoding reporter gene with PEI-PEG-chitosan copolymer-
coated magnetic nanoparticles or magnetic crystal-lipid nano-
structure [45e47]. In our study, the effect of an external magnetic
field on magnetofection of melanoma B16F1 and mammary
adenocarcinoma TS/A tumors with pDNAGFPusing SPIONs-PAA-PEI
was more pronounced in TS/A tumors than in B16F1 tumors. In the
second step we evaluated the antitumor effectiveness of pDNAIL?12
after magnetofection with our SPIONs-PAA-PEI in weakly immu-
nogenic TS/A mammary adenocarcinoma . The therapeutic
effect after magnetofection of TS/A tumors with pDNAIL?12using
SPIONs-PAA-PEI was significantly better than administration of
SPIONs-PAA-PEI-pDNAIL?12in the absence of an external magnetic
field. In fact, there was no antitumor effect without the exposure of
tumors to an external magnetic field. Interestingly, the contribution
S. Prijic et al. / Biomaterials 33 (2012) 4379e4391
of an external magnetic to the magnetofection efficacy was not
seen in vitro but only in vivo. In vitro, internalization of SPIONS is
usually limited by the lack of contact between SPIONs and cellular
surface, and can be enhanced by increasing either gravitational or
magnetic force. The gravitational force is a function of particle
density and radius, and in standard in vitro conditions causes the
sedimentation of SPIONs onto the cellular surface . The gravi-
tational force might have enabled 250 nm-sized SPIONs-PAA-PEI-
pDNAGFPand SPIONs-PAA-PEI-pDNAIL?12to sediment onto the
cellular surface during 4-h incubation, thus the exposure of cells to
an external magnetic field could not additionally contribute to the
sedimentation onto the cellular surface, subsequent internalization
and nevertheless magnetofection of cells. In vivo, i.t. administration
of SPIONs-PAA-PEI-pDNAIL?12with subsequent exposure to an
external magnetic field resulted in significant antitumor effect. The
exposure of tumors to an external magnetic field after adminis-
tration of SPIONs prolonged their retention at the targeted site
[8,11,49], which might have contributed to the increased magne-
tofection efficacy of the magnet-exposed tumors. However, further
studies are needed in order to elucidate the importance of an
external magnetic field for the treatment of different tumors using
To the best of our knowledge, pDNA encoding therapeutic genes
was delivered into tumors by magnetofection in only two studies
by the same research group, dealing with dose-escalation neo-
adjuvant gene therapy of feline fibrosarcomas before surgery
[14,16]. Magnetofection of fibrosarcomas turned out to be safe and
efficient with the use of intermediate pDNA dose, which was
explained by the bell-shaped dose dependence of IL-2 . In our
study, magnetofection of TS/A tumors with pDNAIL?12resulted in
significant antitumor effect as the only treatment modality.
Furthermore, the effect of IL-12-based treatments of TS/A tumors
after magnetofection was equal to the one of well-established and
efficacious non-viral gene delivery methods in vivo e gene elec-
trotransfer [27,51]. Three repetitive treatments with the same
dosage of pDNAIL?12resulted in the same significant antitumor
effect. Magnetofection of tumors with pDNAIL?12using SPIONs-
PAA-PEI can be further refined for cancer immuno-gene therapy,
which can be very efficient in combination with other treatment
modalities, e.g. irradiation , particularly for the patients whose
tumors cannot be removed by surgery .
The combination of coating and functionalization of SPIONs
using PAA and PEI pH-responsive endosomolytic polymers as
membrane disruptive agents proved to be nontoxic and effective
for in vitro magnetofection of different cells with pDNA encoding
either GFP or IL-12, and even superior in transfection efficacy than
some other non-viral transfection approaches. In vivo, we demon-
strated that magnetofection of mammary adenocarcinoma TS/A
tumors with pDNA encoding IL-12 using SPIONs-PAA-PEI resulted
in significant antitumor effect and could be further developed for
cytokine-based tumor gene therapy.
We dedicate this work to A. Znidarsic, PhD, who passed away
unexpectedly during the preparation of this manuscript. We
sincerely acknowledge prof. J. Dolinsek, PhD, Josef Stefan Institute,
Ljubljana, Slovenia, and prof. Z. Jaglicic, Institute of Mathematics,
Physics and Mechanics, for facilitating us to do the measurements
regarding the magnetic properties of our magnetic nanoparticles.
We appreciate all the help by M. Lavric, B. Markelc, A. Sedlar and N.
Rajnar that eased our work in achievement of our objectives. This
work was financially supported by Slovenian Research Agency
(program P3-0003, projects J3-2069, J3-4211) and conducted in the
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