Superparamagnetic Nanoparticle Delivery of DNA Vaccine

Abstract

The efficiency of delivery of DNA vaccines is often relatively low compared to protein vaccines. The use of superparamagnetic iron oxide nanoparticles (SPIONs) to deliver genes via magnetofection shows promise in improving the efficiency of gene delivery both in vitro and in vivo. In particular, the duration for gene transfection especially for in vitro application can be significantly reduced by magnetofection compared to the time required to achieve high gene transfection with standard protocols. SPIONs that have been rendered stable in physiological conditions can be used as both therapeutic and diagnostic agents due to their unique magnetic characteristics. Valuable features of iron oxide nanoparticles in bioapplications include a tight control over their size distribution, magnetic properties of these particles, and the ability to carry particular biomolecules to specific targets. The internalization and half-life of the particles within the body depend upon the method of synthesis. Numerous synthesis methods have been used to produce magnetic nanoparticles for bioapplications with different sizes and surface charges. The most common method for synthesizing nanometer-sized magnetite Fe3O4 particles in solution is by chemical coprecipitation of iron salts. The coprecipitation method is an effective technique for preparing a stable aqueous dispersions of iron oxide nanoparticles. We describe the production of Fe3O4-based SPIONs with high magnetization values (70 emu/g) under 15 kOe of the applied magnetic field at room temperature, with 0.01 emu/g remanence via a coprecipitation method in the presence of trisodium citrate as a stabilizer. Naked SPIONs often lack sufficient stability, hydrophilicity, and the capacity to be functionalized. In order to overcome these limitations, polycationic polymer was anchored on the surface of freshly prepared SPIONs by a direct electrostatic attraction between the negatively charged SPIONs (due to the presence of carboxylic groups) and the positively charged polymer. Polyethylenimine was chosen to modify the surface of SPIONs to assist the delivery of plasmid DNA into mammalian cells due to the polymer's extensive buffering capacity through the "proton sponge" effect.

Full text

181
Monica Rinaldi et al. (eds.), DNA Vaccines: Methods and Protocols, Methods in Molecular Biology, vol. 1143,
DOI 10.1007/978-1-4939-0410-5_12, © Springer Science+Business Media New York 2014
Chapter 12
Superparamagnetic Nanoparticle Delivery of DNA Vaccine
Fatin Nawwab Al-Deen, Cordelia Selomulya, Charles Ma,
and Ross L. Coppel
Abstract
The efficiency of delivery of DNA vaccines is often relatively low compared to protein vaccines. The use of
superparamagnetic iron oxide nanoparticles (SPIONs) to deliver genes via magnetofection shows promise
in improving the efficiency of gene delivery both in vitro and in vivo. In particular, the duration for gene
transfection especially for in vitro application can be significantly reduced by magnetofection compared to
the time required to achieve high gene transfection with standard protocols. SPIONs that have been ren-
dered stable in physiological conditions can be used as both therapeutic and diagnostic agents due to their
unique magnetic characteristics. Valuable features of iron oxide nanoparticles in bioapplications include a
tight control over their size distribution, magnetic properties of these particles, and the ability to carry
particular biomolecules to specific targets. The internalization and half-life of the particles within the body
depend upon the method of synthesis. Numerous synthesis methods have been used to produce magnetic
nanoparticles for bioapplications with different sizes and surface charges. The most common method for
synthesizing nanometer-sized magnetite Fe3O4 particles in solution is by chemical coprecipitation of iron
salts. The coprecipitation method is an effective technique for preparing a stable aqueous dispersions of
iron oxide nanoparticles. We describe the production of Fe3O4-based SPIONs with high magnetization
values (70 emu/g) under 15 kOe of the applied magnetic field at room temperature, with 0.01 emu/g
remanence via a coprecipitation method in the presence of trisodium citrate as a stabilizer. Naked SPIONs
often lack sufficient stability, hydrophilicity, and the capacity to be functionalized. In order to overcome
these limitations, polycationic polymer was anchored on the surface of freshly prepared SPIONs by a direct
electrostatic attraction between the negatively charged SPIONs (due to the presence of carboxylic groups)
and the positively charged polymer. Polyethylenimine was chosen to modify the surface of SPIONs to
assist the delivery of plasmid DNA into mammalian cells due to the polymer’s extensive buffering capacity
through the “proton sponge” effect.
Key words Superparamagnetic iron oxide nanoparticles, SPION, Polyethylenimine, PEI, DNA
vaccine, Magnetofection
1 Introduction
Superparamagnetic iron oxide nanoparticles (SPIONs) have
attracted significant attention in gene delivery applications
because of their relatively low toxicity, low cost of production,

182
ability to immobilize biological materials on their surfaces, and
potential for direct targeting using external magnets. Magnetic
particle-assisted gene delivery, also known as magnetic transfec-
tion or magnetofection, has been shown to improve both the
efficiency of gene delivery and the rapidity of uptake in different
tissues in vitro [1]. Magnetofection originated from the concept
of magnetic drug delivery in the late 1970s, with the technique
demonstrating applicability to gene delivery with viral and non-
viral vectors [2]. Magnetic particles appear to be generally use-
able with any gene delivery vector, and the duration of the
transfection process can be significantly reduced down to 10 min,
compared to 4-h incubation usual with standard protocols [2].
Magnetofection is an appropriate tool for rapid and specific gene
transfection needing only low doses in vitro and allowing site-
specific in vivo applications [3, 4].
In biotechnology, the critical characteristics of magnetic
nanoparticles are their nanoscale dimensions, magnetic proper-
ties, and ability to bind particular biomolecules and deliver them
to specific targets. Studies performed over the last decade have
used several types of iron oxides, among them maghemite,
γ-Fe2O3, or magnetite, Fe3O4, which consist of a single domain
of about 5–20 nm in diameter [5, 6]. Magnetite, Fe3O4, is the
most common magnetic iron oxide candidate because its bio-
compatibility in biological systems has already been proved [7].
This form of iron oxide is stable in water or physiological saline
under neutral pH conditions. It has a large surface area that can
be modified to attach biological agents [8]. Nanoparticles of this
composition with suitable surface coating materials can disperse
widely in suitable solvents to produce a homogenous suspension
called ferrofluid that permits further biochemical functionaliza-
tion. Numerous synthesis methods have been used to produce
magnetic nanoparticles for bioapplications including coprecipi-
tation, microemulsions, polyols, sol–gel synthesis, sonochemical
synthesis, hydrothermal, hydrolysis, thermolysis of organic pre-
cursors, flow injection, and electrospray [5, 9]. These methods
have been used to prepare magnetic particles with homogeneous
composition and narrow size distribution. However, the most
common method for synthesizing magnetite particles in solution
within the nanometer range is chemical coprecipitation of iron
salts. The technique is probably the simplest and most efficient
wet chemical route to obtain magnetic particles for biomedical
applications [10].
The stabilization of iron oxide nanoparticles is an important
feature in obtaining ferrofluid colloids that do not aggregate in
both biological media and magnetic field. The hydrophobic sur-
face of magnetic particles means that in the absence of coating
Fatin Nawwab Al-Deen et al.

183
materials, these particles tend to interact with each other to
form large clusters, resulting in the increase of aggregate size
[10]. Coating layers not only provide stability to nanoparticles
in solution but also help to bind various biological ligands to
the particle surface for various biomedical applications. Various
materials have been used as protective coatings for magnetic
nanoparticles.
Polyethyleneimine (PEI) is one of the most efficient cat-
ionic compounds for delivery of plasmid DNA into mammalian
cells due to its extensive buffering capacity through the “proton
sponge” effects [11, 12]. PEI polymer is known to form cat-
ionic complexes with SPIONs that then interact nonspecifically
with negatively charged DNA and enter the cell via endocytosis
[13]. In contrast to other cationic polymers, PEI has high trans-
fection efficiencies even in the absence of endosomolytic agents
such as fusogenic peptides or chloroquine which facilitates cel-
lular uptake [14]. Many types of linkages have been used to
couple magnetic nanoparticles to nucleic acids, and the simplest
one is a physical method based on electrostatic interaction
between positively charged magnetic particles with a cationic
polymer coating layer and negatively charged nucleic acids.
Different factors have been examined for their effects on mag-
netic gene complex preparation such as molecular weight and
different structures (branched and linear structure) of PEI as
well as charge density and charge-to-mass ratio of the polymer
and DNA molecules [13]. For instance, our previous work
showed that SPIONs/branched PEI complexes at pH 4.0
showed a better binding capability for DNA than at a neutral
pH, despite negligible differences in the size and surface charge
of the complexes [15]. This finding might be a result of proton-
ation and mutual charge repulsion between PEI amine groups
in acidic conditions, expanding the polymeric network to
increase the amount of entrapped genetic material and conse-
quently increasing gene expression upon injection. In contrast,
the stiff stable structure of the polymer’s six-membered rings
under neutral conditions would decrease the particle’s ability to
entrap more DNA molecules, subsequently decreasing DNA
dosage (see Fig. 1) [15].
In this chapter, we describe a coprecipitation method to pro-
duce SPIONs. This method is exceptionally useful as it is able to
produce magnetic particles of a specific size within the nanometer
range and with good magnetic properties. The produced particles
can then be coated with PEI polymers as an example of useful cat-
ionic polymers that can form complexes with DNA molecules for
gene delivery.
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

184
2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water dd H2O to attain a sensitivity of 18 MΩ cm at
25 °C) and analytical grade reagents. Prepare and store all reagents
at room temperature (unless indicated otherwise).
1. Fe (III) chloride (FeCl3.6H2O) and Fe (II) chloride
(FeCl2.7H2O) (from Ajax Finechem and Ajax Chemicals,
respectively).
2.1 Iron Oxide
Nanoparticle
Preparation from Iron
Salts
+
+
+
+
+
+
+
+
+
+
+
NH2
NH2
NH NH
N
H
NH
NH
NH2
NH
NH
NH
N
H
NH
NH2
NH
NH2
N
H
NH2
NH
NH2
+
+
+
+
+
+
+
+
+
+
+
NH2
CH3
+
+
+
NH2
NH
NH2
NH
NH NH
N
H
NH
NH
NH2
NH
NH
NH
N
H
NH
NH
NH
NH2
N
H
NH2
NH
NH2
NH2
NH2
+
+
+
+
+
+
+
+
+
+
NH2
NH
NH2
NH
NH2
NH
NH
NH NH
NH
NH
NH2
NH
NH
NH NH
NH
NH
NH2
NH
NH2
NH
NH2
NH2
NH2
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
NH2
CH3
+
+
+
N
H
2
NH
NH2
NH
NH NH
N
H
NH
NH
NH2
NH
NH
NH
N
H
NH
NH
NH
NH2
N
H
NH2
NH
NH2
N
H
2
NH2
Fe3O4
Fe3O4
OH-
H+
H
H
H
H
NH
NH2
H
N
H
N
H
NH
N
H
NH
N
N
H
NH
NH
N
H
N
H
N
N
NH
NH
NH
N
NH
NH
NH
N
H
NH2
NH 2
CH3
+
++
+
+
+
+
+
+
++
+
+
H
H
H
H
H
H
H
H
H
H
H
H
NH
NH2
H
N
H
N
H
NH
N
H
NH
N
N
H
NH
NH
N
H
N
H
N
N
NH
NH
NH
N
NH
NH
NH
N
H
NH2
NH2
+
++
+
+
+
+
+
+
++
+
+
Hydrogen bonds
Acidic pH condition Neutral pH condition
NH
NH
NH2
NH2
NH2
N
N
N
2
NH
NH2
NH
NH2
NH
N
N
NH
NH2
NH
NH2
NH
NH2
NH2
+
+
+
+
+
+
+
+
+
+
+
NH
NH
NH2
NH2
NH2
N
N
NH2
NH
NH2
NH
NH2
NH
N
N
NH
NH2
NH
NH2
NH
NH2
NH2
+
+
+
+
+
+
+
+
+
+
+
H
H
H
H
H
H
H
H
DNA
Fe3O4
Fe3O4
Fig. 1 A schematic demonstrating PEI structures under acidic and neutral pH conditions, showing a relatively
branched structure due to mutual charge repulsion between amine groups under acidic condition and a stiff
structure under neutral pH condition, with DNA likely to be entrapped within the respective structures. Adapted
from Al-Deen et al. [15], with permission from American Chemical Society (ACS) publications of Langmuir
Fatin Nawwab Al-Deen et al.

185
2. Trisodium citrate dihydrate (C6H5Na3O7. 2H2O) (Sigma
Aldrich).
3. Sodium hydroxide ACS reagent, ≥97.0 % in pellets (Sigma
Aldrich).
4. Cooking oil for oil bath.
5. Zetasizer Nano ZS (Malvern Instruments Ltd., UK).
6. 1140 PW diffractometer with nickel-filtered Cu Kα radiation
(λ = 1.5405 Å) (Philips).
7. Vibrating sample magnetometer (VSM) (Riken Denshi).
8. Transmission electron microscope (TEM) CM20 (Philips).
3 SPION/PEI Complexes
1. PEI solution: 10 % PEI in water (w/v), pH 7.9. Weigh 10 g
of PEI (molecular weight of 25 kDa branched, Sigma Aldrich)
and dissolve in 75 ml of H2O. Adjust pH to 7.9 with concen-
trated HCL and add water to a volume of 100 ml. Filter the
PEI solution through a 0.22 μm nitrocellulose filter. Store the
solution at 4 °C (see Note 1).
2. 0.5 M HCl.
3. 0.5 M NaOH.
4. Zetasizer Nano ZS (Malvern Instruments Ltd., UK).
5. Dialysis tubing Spectra/Por® membranes (MWCO = 12,000–
14,000) (Spectrum Medical Industries, Inc., Los Angeles,
CA).
1. Endotoxin-free plasmid DNA: 10 μg/ml.
2. 1× PBS, pH 7.4.
1. Agarose.
2. 6× Sample loading buffer: Weigh ~0.05 mg bromophenol
blue and transfer to a 2 ml tube with 1 ml sterile H2O and
1 ml glycerol. Add enough bromophenol blue to make the
buffer deep blue. For long-term storage, keep the sample
loading buffer frozen.
3. Ethidium bromide (EtBr) stock solution (10 mg/ml): 0.02 g
in 1 ml sterile H2O.
4. DNA ladder standard.
5. 50× tris–acetate–EDTA (TAE) buffer: 242 g tris base, 100 ml
of 0.5 M EDTA solution, 57.1 ml glacial acetic acid, pH 8.5.
Add 800 ml water to a 1 l graduated cylinder. Weigh 242 g tris
base and transfer to the cylinder. Add 100 ml of 0.5 M EDTA
and 57.1 ml glacial acetic acid, mix, and adjust the pH to 8.5
3.1 SPION/PEI/DNA
Polyplexes
3.2 Agarose Gel
Electrophoresis
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

186
using KOH. Add up to 1 l with H2O. Store the buffer at room
temperature (see Note 2).
6. Electrophoresis chamber.
7. Power supply.
8. Gel casting tray and combs.
1. 100 ml three-necked round-bottom flask.
2. Dropping funnel.
3. Air evacuation vacuum pump.
4. Ultrasonic bath (Power Sonic 405, 40 kHz and 350 W).
5. Probe sonicator (Sonics vibra cell, 40 kHz and130 W).
6. N2 gas cylinder.
7. Water-cooled condenser.
8. Temperature controller.
9. Heating magnetic stirrer.
10. Stir bar.
4 Methods
This method involves coprecipitation of ferrous and ferric salts in
an alkaline solution by the addition of a base such as concentrated
ammonium hydroxide (NH4OH) or sodium hydroxide (NaOH)
in a non-oxidizing environment (N2 gas atmosphere) with the fol-
lowing chemical reaction [16, 17]:
Fe Fe OH Fe
23
34 2
28 4
++ -
++ ®+
OH
O.
Control over size and shape of nanoparticles depends on the
Fe2+ and Fe3+ ratio, the type of salts (e.g., sulphate, nitrate, chlo-
ride), and the pH of the reaction media [10].
1. Weigh 1.35 g (0.005 mol) of Fe (III) chloride (FeCl3.6H20)
and 0.70 g (0.0025 mol) of Fe (II) chloride (FeCl2.7H2O)
dihydrate (1:2 M ratios), and dissolve them in 20 ml of H2O
in the first beaker [1].
2. Weigh 1.2 g (1.5 mol) of NaOH and 1.47 g (0.005 mol) of
trisodium citrate dihydrate, and dissolve them in 20 ml of
Milli-Q H2O in the second beaker [2] (see Note 3).
3. Sonicate these beakers in an ultrasonic bath with the homog-
enization shaking rate (3,600–9,000 rpm) for 10–15 min.
4. Transfer the solution in the first beaker [1] into 100 ml three-
necked flask, and place a small magnetic bar inside the flask.
Place the flask in the oil bath which has already been placed on
the magnetic stirrer, and set the stirring rate to 1,000–
1,500 rpm (see Note 4). Heat the oil bath to 80 °C (see Fig. 2).
3.3 Reflux System
4.1 Synthesis
and Characterization
of SPIONs
Fatin Nawwab Al-Deen et al.

187
Water in
Water out
Cooling system
Gas
N2
Oil
A
B
C
D
E
1
2
3
Fig. 2 A reflux system for synthesis of superparamagnetic iron oxide nanoparticles (SPIONs). (A) Heating mag-
netic stirrer, (B) temperature controller, (C) water-cooled condenser, (D) water-cooled condenser, (E) air evacu-
ation vacuum pump
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

188
5. Transfer the solution in the second beaker [2] into a dropping
funnel, and connect the funnel with the three-necked
round- bottomed flask via neck [2]; make sure that its stopcock
is closed (see Note 5) (see Fig. 2).
6. Introduce a separate hose of the nitrogen stream to the three-
necked round-bottomed flask via neck 3 (see Fig. 2). Separate
hose connected to the condenser which has been connected to
N2 gas cylinder via a gas-trap arrangement connected to the
top of the condenser.
7. Connect the water-cooled condenser to the three-necked
round-bottomed flask via neck 1 (see Fig. 2) (see Note 6).
Start water circulation through turning on a water tap. Be sure
that cold water is flowing through the condenser in moderate
water flow rates.
8. Wrap the connection of equipment in the reflux system with
each other by a thin strip of paraffin film to avoid any outside
air entering the system.
9. Remove the air out of the system by using a vacuum pump for
4–5 min. Open the cylinder tap cautiously to allow N2 gas to
enter the system for 4–5 min at a steady but controlled rate
until 18.2 g to provide a nitrogen blanket to the reaction.
Flowing N2 gas through the reaction medium during the syn-
thesis reaction can afford protection to the produced iron
oxide particles from oxidation. The three-necked flask is kept
under a positive nitrogen pressure by means of a gas-trap
arrangement connected to the top of the condenser.
10. Once the system temperature reaches 80 °C, turn the funnel
stopcock partially to start the aqueous coprecipitation of the
iron salt solution with NaOH and trisodium citrate dihydrate
solution.
11. After 1 h of reaction, collect the resulting black precipitates
and remove them from solution by applying an external mag-
net. Then wash the precipitates four times, firstly with ddH2O,
then twice with ethanol, and finally with deionized (DI) water
to remove excess ions and salts from the suspension. The con-
centration of the solution will be about 8 mg/ml.
12. Disperse the washed precipitate in 20 ml DI water. Zetasizer
Nano ZS is used to determine the hydrodynamic diameter and
zeta potential of these particles in suspension, while TEM
CM20 is used to confirm the size and morphology of dry par-
ticles. X-ray powder diffraction (XRD), by means of diffrac-
tometer with nickel-filtered Cu Kα radiation (λ = 1.5405 Å), is
used to determine the crystallinity and phase of iron oxide
particles. Magnetic saturation is measured using a VSM under
a magnetic field of up to 15 kOe at room temperature.
Fatin Nawwab Al-Deen et al.

189
An example of size and morphology of prepared SPIONs
under TEM and magnetic saturation using a VSM is shown in
Fig. 3.
In magnetofection, magnetic nanoparticles need appropriate sur-
face coatings to form gene complexes, which also increase their
stability in solution. The stability of magnetic nanoparticles in bio-
logical fluid can be improved by modifying their surface using
materials including inorganic and polymeric materials to increase
repulsive forces between particles, thus balancing magnetic and
van der Waals attractive forces [18] (see Note 7).
1. Mix the prepared iron oxide suspension (0.1 mg/ml) with 10 %
(w/v) PEI solution (25 kDa branched PEI), at PEI/Fe mass
ratios of (R) = 10, while sonicating using a probe sonicator using
a Sonics vibra cell 130 W apparatus at 40 kHz for 5 min.
2. Dialyze the produced SPION/PEI complexes using Spectra/
Por membranes (MWCO = 12,000–14,000) against deionized
water for 3 days to remove any unbound/excess PEI.
3. Acidify the mixture of SPION/PEI complex to pH 2.0 using
0.5 M HCl, and retain at this pH for 10 min to stabilize the
complexes.
4. Divide each sample into two aliquots: increase the pH of the
first part to 4.0 (referred to as SPION/PEI-A), while the
other part is neutralized to pH 7.0 (referred to as SPION/
PEI-N) using 0.5 M NaOH.
4.2 Coating SPIONs
with PEI Polymer
20 nm -80
-60
-40
-20
0
20
40
60
80
-20000 -10000 0 10000 20000
M(emu/g)
Applied field /kOe
ab
Fig. 3 (a) A TEM image of as-synthesized SPIONs, (b) VSM data for SPIONs, with X- and Y-axes in the graph
indicating the applied field (kOe) and magnetization (emu/g), respectively
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

190
Zetasizer Nano ZS (Malvern Instruments Ltd., UK) is used to
determine the hydrodynamic diameter and zeta potential of
SPIONs/PEI in suspension, while TEM CM20 is used to confirm
the size and morphology of dry particles. An example of the small
aggregate size of prepared SPIONs/PEI at pH 4.0 compared with
bare SPIONs under TEM is shown in Fig. 4.
1. Mix plasmid DNA at a concentration of 10 μg/ml in PBS (pH
7.4) with SPION/PEI complexes at R of 10 at different N/P
ratios (i.e., the molar ratio of PEI nitrogen to DNA phos-
phate) (see Note 8 for details of calculating different N/P
ratios).
The DNA binding capabilities of SPION/PEI/DNA polyplexes
are determined using 1 % agarose gel electrophoresis. SPION/PEI
complexes with plasmid DNA were formed at N/P ratios of
0.5–30.
1. Measure out 1 g of agarose.
2. Pour agarose powder into flask along with 100 ml of 1×TAE.
3. Boil with swirling the agarose solution on a heater (until all of
the small translucent agarose particles are dissolved, the solu-
tion becomes clear, and there is a nice rolling boil) (approxi-
mately 10 min).
4. Allow the agarose solution cool to about 50–55 °C, swirling
the flask occasionally to cool regularly.
4.3 Preparation of
SPION/PEI/DNA
Polyplexes
4.4 DNA
Retardation Assay
4.5 Agarose Gel
Electrophoresis of
SPION/PEI/DNA
Polyplexes
Fig. 4 TEM images of (a) as-synthesized SPIONs and (b) SPIONs/PEI (ratio = 10) at pH 4 displaying better dis-
persion. Adapted from Al-Deen et al. [15], with permission from American Chemical Society (ACS) publications
of Langmuir
Fatin Nawwab Al-Deen et al.

191
5. Add EtBr to a final concentration of approximately 0.5 μg/ml
(usually about 2–3 μl of stock solution per 100 ml gel)
(see Note 9).
6. Seal the ends of the casting tray with two layers of tape.
7. Place the combs in the gel-casting tray.
8. Pour the melted agarose solution into the casting tray and let
cool until it is solid.
9. Carefully pull out the combs, and remove the tape. Place the
gel in the electrophoresis chamber.
10. Add enough TAE buffer so that there is about 2–3 mm of buf-
fer over the gel (see Note 10).
1. For each N/P ratio, mix the appropriate amount of SPIONs/
PEI with 0.5 μg plasmid DNA in the 25 μl PBS.
2. Incubate all SPION/PEI/DNA polyplex solutions at 37 °C
for 30 min.
3. Add 5–6 μl of 6× sample loading buffer to each 25 μl SPION/
PEI/DNA polyplexes.
4. Carefully pipette 20 μl of each sample/loading buffer mixture
into separate wells in the gel.
5. Pipette 10 μl of the DNA ladder standard into at least one well
of each row on the gel.
Carry out the electrophoresis at 60 V for 90 min, and then visual-
ize the DNA bands using a UV illuminator.
An example of agarose gel of gel electrophoresis of SPION/
PEI/DNA polyplexes at different N/P ratios is shown in Fig. 5.
4.6 Loading the Gel
4.7 Running the Gel
Fig. 5 Agarose gel electrophoresis of SPION/PEI/DNA polyplexes. Lane N: Plasmid DNA (naked). Lanes 0.5–30
correspond to SPION/PEI/DNA polyplexes at different N/P ratios
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

192
5 Notes
1. The PEI 10 % (w/v) solution is the working PEI reagent solu-
tion that should be stable for a long period of time at 4 °C. If
a 6 % PEI solution is needed, add 6 ml of 10 % of PEI to 4 ml
of H2O.
2. For convenience, a concentrated stock of TAE buffer (either
10× or 50×) is often made ahead of time and diluted with
water to 1× concentration prior to use.
3. The presence of trisodium citrate (C6H5Na3O7.2H2O) on the
magnetic nanoparticles works as an electrostatic stabilizer
[15]. Trisodium citrate has three carboxyl groups which pro-
mote their adsorption onto the iron oxide particles, whilst ion-
ization of carboxyl groups supplies the coated magnetic
particles with a negative charge to give a stable dispersion in
water due to a strong mutual electrostatic repulsion. Moreover,
these negatively charged particles are able to adsorb cationic
polyelectrolytes such as PEI.
4. High stirring rate (1,500 rpm) might play effective roles dur-
ing particle nucleation in preventing the nanocrystals from
growing further into large single crystals. Furthermore, the
size of nanoparticles becomes smaller when the stirring rate
increases due to the increasing amount of energy that is trans-
ferred to the suspension. SPIONs with this narrow size range
could be used for the delivery of gene vector because the small
size of nanoparticles has been shown to influence the rate of
their uptake as well as their cytotoxicity [19].
5. Ensure that the dropping funnel stopcock is completely closed
to prevent the coprecipitation reaction from starting while
contents are being added to the three-necked flask at this stage.
6. A condenser is attached to the heated three-necked flask, and
cooling water is circulated to condense the vapor, returning it
back to the flask as a liquid.
7. A highly positively charged coating agent for magnetic
nanoparticles such as PEI cationic polymer has advantages
over other polycations in that it not only increases repulsive
forces between the particles but also readily associate with
negatively charged plasmid DNA accompanied by intrinsic
endosomolytic activity [20].
8. The calculation of the N/P ratio for the SPION/PEI/DNA
complexes is defined as the molar relation of primary amine
groups in the PEI cationic molecule (secondary and tertiary
amines are neglected in this calculation due to their lower pKa
values), which represent the positive charges, to phosphate
groups in the DNA, which represent the negative charges. The
Fatin Nawwab Al-Deen et al.

193
calculation of the N/P ratio was based on the assumption that
one repeating unit of PEI containing one nitrogen (N) corre-
sponds to 43.1 g/mol and one repeating unit of DNA containing
one phosphate group (P) corresponds to 330 g/mol [21].
For example, if we need to prepare SPION/PEI/DNA poly-
plexes containing 10 μg DNA at N/P ratio of 2:
For DNA, 330 g/mol corresponds to one phosphate atom.
1 μg × 330 g/1 μg = 1 mol phosphate.
1 μg DNA = 1 mol phosphate × 10 − 6/330.
1 μg DNA = 3.03 × 10 − 9 mol phosphate.
10 μg DNA = 10 × 3.03 × 10 − 9 mol phosphate.
10 μg DNA = 30.3 × 10 − 9 mol phosphate.
For PEI, the number of N atom in 25 KDa of PEI = 25,000/
43.1 = 580.0464 ≈ 580.05.
If we need to prepare SPION/PEI/DNA polyplexes with an
N/P ratio of 2:
N/P = 2.
N = 2 × P.
N = 2 × 30.3 × 10 − 9 = 60.6 × 10 − 9.
(1 mol of PEI = 580.05 mol of N.)
How many moles of PEI are in 60.6 × 10 − 9 mol of N?
60.6 × 10 − 9/580.05 = 0.10 × 10 − 9 mol of PEI.
PEI mass = 0.10 × 10 − 9 mol × 25,000 g/mol = 2.61 × 10 −
6 g = 2.61 μg.
Alternatively, we can use the ratio to calculate the amounts of
PEI and DNA required.
The g/mol ratio for one N (PEI) to one P (DNA) is
N:P = 43.1:330. For N/P = 2, the g/mol ratio is 86.2:330.
Thus, mass of PEI:mass of DNA = 86.2:330.
For 10 μg DNA, the ratio becomes
Mass of PEI:10 μg = 86.2:330 or, in the form of division,
Mass of PEI/10 μg = 86.2/330.
Rearranging, mass of PEI = 86.2/330 × 10 μg = 2.61 μg.
Since we previously coated SPION with PEI at PEI/Fe mass
ratios of (R) = 10 (see Subheading 3.2), the mass of SPION
could be calculated depending on the mass of PEI, assum-
ing that all SPIONs are coated completely with PEI
polymer:
R (10) = PEI/SPIONs.
Mass of SPIONs = 2.61 μg/10 = 0.261 μg.
Superparamagnetic Nanoparticle Delivery of DNA Vaccine

194
9. EtBr is an intercalating agent commonly used as a fluorescent
tag (nucleic acid stain) that binds to the DNA and allows the
visualization of DNA under ultraviolet (UV) light.
10. Gels can be made several days prior to use and sealed in a plas-
tic wrap (without combs).
References
1. Krotz F, Sohn HY, Gloe T et al (2003)
Magnetofection potentiates gene delivery to
cultured endothelial cells. J Vasc Res 40:
425–434
2. Scherer F, Anton M, Schillinger U et al (2002)
Magnetofection: enhancing and targeting gene
delivery by magnetic force in vitro and in vivo.
Gene Ther 9:102–109
3. Plank C, Scherer F, Schillinger U et al (2003)
Magnetofection: enhancing and targeting gene
delivery with superparamagnetic nanoparticles
and magnetic fields. J Liposome Res 13:29–32
4. Dobso J (2006) Gene therapy progress and
prospects: magnetic nanoparticle-based gene
delivery. Gene Ther 13:283–287
5. Tartaj P, Morales MD, Veintemillas-Verdaguer
S et al (2003) The preparation of magnetic
nanoparticles for applications in biomedicine.
J Phys D Appl Phys 36:R182–R197
6. Roca AG, Costo R, Rebolledo AF et al (2009)
Progress in the preparation of magnetic
nanoparticles for applications in biomedicine.
J Phys D Appl Phys. doi:10.1088/
0022-3727/42/22/224002
7. Schwertmann U, Cornell RM (1991) Iron
oxides in the laboratory: preparation and char-
acterization. Wiley-VCH, Weinheim, NY
8. Ankamwar B, Lai TC, Huang JH et al (2010)
Biocompatibility of Fe3O4 nanoparticles evalu-
ated by in vitro cytotoxicity assays using
normal, glia and breast cancer cells. Nano-
technology. doi:10.1088/0957-4484/ 21/7/
075102
9. Laurent S, Forge D, Port M et al (2008)
Magnetic iron oxide nanoparticles: synthesis,
stabilization, vectorization, physicochemical
characterizations, and biological applications.
Chem Rev 108:2064–2110
10. Gupta AK, Gupta M (2005) Synthesis and
surface engineering of iron oxide nanoparticles
for biomedical applications. Biomaterials 26:
3995–4021
11. Boussif O, Lezoualch F, Zanta MA et al (1995)
A versatile vector for gene and oligonucleotide
transfer into cells and in-vivo- polyethylenimine.
Proc Natl Acad Sci U S A 92:7297–7301
12. Behr JP (1997) The proton sponge: a trick to
enter cells the viruses did not exploit. Chimia
51:34–36
13. Godbey WT, Wu KK, Mikos AG (1999)
Poly(ethylenimine) and its role in gene delivery.
J Control Release 60:149–160
14. Kichler A, Leborgne C, Coeytaux E et al
(2001) Polyethylenimine-mediated gene deliv-
ery: a mechanistic study. J Gene Med 3:
135–144
15. Al-Deen FN, Ho J, Selomulya C et al (2011)
Superparamagnetic nanoparticles for effective
delivery of malaria DNA vaccine. Langmuir
27:3703–3712
16. Massart R (1981) Preparation of aqueous mag-
netic liquids in alkaline and acidic media. IEEE
Trans Magn 17:1247–1248
17. Massart R, Dubois E, Cabuil V et al (1995)
Preparation and properties of monodisperse
magnetic fluids. J Magn Magn Mater 149:1–5
18. Wu W, He QG, Jiang CZ (2008) Magnetic
iron oxide nanoparticles: synthesis and surface
functionalization strategies. Nanoscale Res
Lett 3:397–415
19. Steitz B, Hofmann H, Kamau SW et al (2007)
Characterization of PEI-coated superparamag-
netic iron oxide nanoparticles for transfection:
Size distribution, colloidal properties and
DNA interaction. J Magn Magn Mater 311:
300–305
20. Lungwitz U, Breunig M, Blunk T et al (2005)
Polyethylenimine-based non-viral gene deliv-
ery systems. Eur J Pharm Biopharm 60:
247–266
21. Geisse S, Jordan M, Wurm FM (2005) Large-
scale transient expression of therapeutic pro-
teins in mammalian cells. Methods Mol Biol
308:87–98
Fatin Nawwab Al-Deen et al.