Magnetoliposomes: Synthesis and Applications

Magnetoliposomes: synthesis and applications
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1. Magnetoliposomes
In the field of biomedicine, nanotechnology has produced significant advances
in diagnosis, therapy and bioengineering. Liposomes (nanosized vesicles made of
amphiphilic phospholipid molecules in water) have been described as an ideal drug
delivery system [1-3]. Consisting of natural lipids, liposomes are biologically inert and
weakly immunogenic, having low intrinsic toxicity.
Magnetic nanoparticles have been extensively studied for many biomedical
applications [4]. The encapsulation of magnetic nanoparticles into liposomes leads to
the formation of magnetoliposomes (MLs) and the amazing physical properties of these
two systems are brought together. Magnetoliposomes are promising systems that can be
used for both diagnosis and therapy. To both purposes, MLs must cross the endothelium
barrier and accumulate specifically in target cells. Using an external magnetic field, it is
possible to localize these systems in a therapeutic site and minimize cytotoxic effects
for healthy cells. In magnetic resonance imaging (MRI), magnetoliposomes can be
used as carriers of the magnetic nanoparticles are used as T2 contrast agents (negative
contrast enhancement) in MRI [5]. In therapy, these systems have been proposed as a
chemotherapy alternative through controlled drug delivery systems and thermotherapy
[6, 7].
There are two types of magnetoliposomes [8]:
Dry
Ma
are encapsulated in the aqueous phase of the liposomes;
Dry magnetoliposomes (DMLs) a cluster of magneticgne
nanoparticles replaces the inner aqueous phase of the liposomes.
toli
pos
om
es
Aqueous magnetoliposomes (AMLs) - the magnetic nanoparticles
Aqueous
media
Figure 1. Illustration of aqueous magnetoliposomes (on the left) and dry magnetoliposomes (on the right).
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The flow of the MLs in blood results from the competition between magnetic
forces of the magnetic nanoparticles in blood circulation and magnetic forces applied by
the external magnetic field. The magnetic fluid is localized in the site of interest when
the magnetic forces are exerted on linear blood flow of the arteries or capillaries [8]. To
improve cell uptake, cationic amphiphiles (composed of biodegradable moieties and
specifically designed to exert minimal cytotoxic effects) can be inserted into
magnetoliposomes coat [9]. In drug delivery, after the localization of the
magnetoliposomes in the therapeutic site, drug liberation can be induced by different
physico-chemical environment variations, such as pH or temperature changes.
The magnetic force, important for magnetophoresis applications and the spin
spin relaxation time depend on the nanoparticles concentration, and to how close they
are from each other [10]. This way, controlling the number and the time of encapsulated
magnetic nanoparticles in the magnetoliposomes is a key issue.
For in vivo applications, magnetoliposomes must be physically and biologically
stable in order to extend blood time circulation and improve their efficacy. The
encapsulated nanoparticles in MLs allow avoiding earlier dilution and undesirable
interactions with biological structures. There are some chemical adjustments that can be
made in liposomes in order to improve their physical and biological stability; this will
be discussed in the next chapter. The bioavailability of the magnetoliposomes is also
important for in vivo applications. That is, realize if the magnetic fluid leaves the
microcirculation and diffuses into the interstitial space, or if it remains unchanged in the
systemic circulation.
1.1 Liposomes
Liposomes were discovery in 1965 by Bangham, who found that when
phospholipids were dispersed in water, vesicular structures of hydrated bilayers with a
aqueous cavity are form spontaneously [11] (figure 2). This process is known as selfassembling, a bottom-up mechanism of liposome synthesis that occurs due to different
interactions, such as, hydrophilic/hydrophobic electrostatic and van der Waals
interactions.
Liposomes act as encapsulation and transport system that can incorporate different
substances as nutrients, genes and drugs. Due to their amphipathic composition,
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incorporated substances can be either hydrophilic and/or hydrophobic, the first ones will
be incorporated in the aqueous cavity and the second inserted or adsorbed on the
membrane by solubilization in the lipid bilayer or by covalent linkage to active surface
groups in the liposome membrane. Anti-tumor drugs can also be transported by
magnetoliposomes using bifunctional reagents [12], taking advantage of magnetic
targeting.
Figure 2. Structure of liposomes and phospholipid molecule.
Liposomes are structurally flexible in size, composition and fluidity [13]. The
choice of phospholipids determines the rigidity and the charge of the bilayer. Saturated
phospholipids with long acyl chains form a rigid, rather impermeable bilayer structure,
while unsaturated species from natural sources (egg or soy bean phosphatidylcholine)
give much more permeable but less stable vesicles. Phase transition of the liposomes is
also an important characteristics. It is affected by the hydrocarbon length, unsaturation,
charge and headgroup species of the phospholipids. It defines the temperature required
to induce a change in the physical state of the liposomes, from the ordered gel phase,
where the hydrocarbon chains are fully extended and closely packed, to the disordered
liquid crystalline phase, where the hydrocarbon chains are randomly oriented and fluid.
According to size and number of lamellae liposomes are divided into different
subcategories (table 1 and figure 3).
Table 1. Liposome classification based on structural parameters [14].
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Figure 3. Schematic representation

of different liposomes.
Liposomes are very important for biomedical applications as they can overcome
many of the problems associated with other systems used for therapy, such as those
involving solubility, pharmacokinetics, in vivo stability and toxicity [15, 16].
Liposomes stability is affected by different processes that can be chemical,
physical or biological. Chemical stability is related with liposome composition while
physical stability is associated with the aggregation and fusion of the vesicles. The
electrostatic repulsion reduces vesicle fusion and aggregation; this way, including
electrical charge in the liposomes will improve their physical stability. The addition of
cholesterol to the liposomes composition will prevent substance release as it will
decrease liposome permeability and improve their chemical stability. In fact, Egg-PC
(egg yolk phosphatidylcholine) liposomes with cholesterol, in (7:3) proportion, are
commonly used as models of cell membranes [17, 18]. On the other hand, biological
stability is concerned with interaction of the liposomes and so it is related with
administration pathway [19].
For in vivo applications liposomes must be very small (100 nm) in order to
reduce the recognition and phagocytosis, thereby increasing the probability of
penetration into the tissues of interest [20]. Recent studies have revealed that the
functionalization of the liposomes surface improves their efficacy. Functionalize the
lipid bilayer with FAB portions (a part of the antibody molecule that binds antigen),
make liposomes to be uptake only by cells that have specific antigens. In order to make
the system less recognizable by the immune system, a PEG (polyethylene glycol) crown
can be added. This will also provide stealth ability (long-circulating) and substance
accumulation in the site of interest [6]. It has also been reported that cationic liposomes
preferably target vasculature [21], while anionic ones are captured by monocytes/blood
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neutrophils [3]. This last behavior has been used to target the brain through the blood
brain barrier (BBB) [22].
1.2 Magnetic nanoparticles
Magnetic nanoparticles are a class of nanoparticles that have at least one
dimension less than 100 nm and one metallic component on its composition. This way,
its location can be manipulated with an external magnetic field. Due to their size,
nanoparticles have a large surface/volume ratio and thus the properties of mass and heat
transfer are better than in common materials [19].
Magnetic effects in the nanoparticles are caused by movements of particles as
electrons and protons that have both mass and electric charges; the spinning electriccharged particle creates a magnetic dipole, so-called magneton. Magnetic behavior of
ferromagnetic materials like iron, nickel and cobalt arises from magnetic domain
structures. Domains result from the action of several interactions that exist in magnetic
materials, such as, exchange, anisotropy and dipolar interactions. Domains are regions
in a sample that has uniform magnetization where the individual magnetic moments are
aligned and point in the same direction [23]. When the size of a ferromagnetic material
decreases, it becomes single domain, where the magnetization is equal to the saturation
magnetization.
Since it is now accepted that magnetic fields are not especially contraindicated
for humans, the potential of magnetism has been recognized in many biological
applications [4]. Magnetic materials have attracted great interest in recent years because
of novel effects that arise due to size reduction, as is the case of superparamagnetism.
This type of magnetism is present in magnetic nanoparticles due to their size and it only
occurs below critical sizes in which nanoparticles are single domain. Critical sizes (dc)
of superparamagnetic nanoparticles depend on the material; iron nanoparticles have
superparamagnetic behavior below 20nm, while in nickel nanoparticles it occurs below
30nm [24, 25].
Superparamagnetic particles are characterized by an enormous magnetic
moment under an external magnetic field. However, when the external magnetic field is
removed, there is no remanescent magnetic moment and the net moment of the particles
is randomized to zero [26].
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Superparmagnetic behavior is easily observed by the hysteresis loop of the

material. Magnetic hysteresis is a function of the applied field at a given temperature
(M(H)). It is obtained on cycling the external field to values beyond the magnetic fields
where the magnetization reaches its saturation. The curves cross zero external field at
the remnant magnetization and the magnetization becomes zero at the coercive field.
Figure 4. Cobalt hysteresis for different size samples; the larger particles shows hysteresis while the smaller are
superparamagnetic and do not have hysteresis [24].
Superparamagnetic nanoparticles do not present remnant magnetization and are

characterized by the absence of hysteresis and coercivity (red line in figure 4) [27]. As a
balance between magnetic energy and thermal energy, superparamagnetism occurs in a
limited range. The underlying physics of this magnetic behavior is founded on an
activation law for the relaxation time, , of the new magnetization of the particle. It
depends on KV/kbT, where K is the particle anisotropy constant, V is particle volume, kb
is Boltzmanns constant and T is the absolute temperature. When the size (volume) of
the particles is sufficiently small, KV becomes comparable to the thermal energy, kbT,
and so magnetization of the particle fluctuates rapidly from one direction to another due
to thermal agitation, leaving no net magnetic moment. In this condition, particles
present superparamagnetic behavior [26, 28]. Below a certain temperature, the blocking
temperature, the thermal energy is not enough to allow the spins to easily rearrange,
coercivity appears and particles have a ferromagnetic behavior. Blocking temperature
has a time component and is often defined as the point at which a particle dipole is able
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to reorient under the influence of a specified magnetic field in 100 s (a typical timescale
for a measurement). This temperature is important because it represents the maximum
in susceptibility and the lower limit of superparamagnetic behavior [24]. Blocking
temperature is an important characteristic for many magnetic applications that depend
on particle size. A wide particle size distribution will result in a wide range of blocking
temperatures, and so, in a non-ideal magnetic behavior for the magnetic fluid.
Iron and nickel are considered metals of biological interest as they present
magnetic properties at room temperature [29]. However, particles of these metals have
some issues as its toxicity, high reactivity and also the fact that they are easily degraded
due to high surface/volume ratio. In order to overcome these problems and make them
compatible for biological applications, core-shell structures are used. Core-shell
structure consists of a metal or metallic oxide core, encapsulated in an inorganic or a
polymeric coating; silica is commonly used (figure 5). Silica coating will separate the
particles, thereby preventing a cooperative switching. It will also adjust the magnetic
properties of nanoparticles, as the extent of dipolar coupling is related to the distance
between particles and this, in turn, depends on the thickness of the inert silica shell.
Figure 5. Representation of core-shell nanoparticles structure.
With this structure, the undesirable effects are avoided and the properties of the
magnetic nanoparticles, such as thermal and chemical stability and solubility, are
improved. Core-shell structures also allow the conjugation of other molecules [30]. For
in vivo applications, nanoparticles must have high magnetization so that their movement
in blood circulation can be easily controlled. Nanoparticles with superparamagnetic
behavior are preferred because the risk of forming agglomerates is negligible at room
temperature [31, 32]. They can be guided to a therapeutic site and are not subject to
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strong magnetic interactions in the dispersion. Also, they are readily stabilized in
physiological conditions [28].
Colloidal stability of the magnetic fluid is important and it can be controlled by
size, charge and surface chemistry of the nanoparticles. Magnetic nanoparticles must be
small in order to prevent precipitation from gravitation forces and must be stable in
water at pH 7 and in physiological environment. The size of nanoparticles depends on
the preparation methods and can be controlled using a surfactant [23]. Citric acid avoids
nanoparticles aggregation and can preserve monodipersity [33]. Surface charges, which
give rise to both steric and coulombic repulsions [34], can also be controlled.
1.3 Biomedical applications
The high biocompatibility and versatile nature of liposomes have made these
systems keystone components in many biomedical research areas. Liposomes can be
combined with a large variety of nanomaterials, such as magnetic nanoparticles which
have greatest applications in biomedicine (figure 6).
Because the unique features of both the magnetic colloid and the versatile lipid
bilayer can be joined, the resulting so-called magnetoliposomes can be exploited in a
great array of biomedical applications. In therapy, the most promising applications of
magnetoliposomes are magnetic controlled drug delivery and hyperthermia [6, 7].
Otherwise, in diagnosis, magnetic nanoparticles have been used as contrast agents in
MRI (magnetic resonance imaging) [5].
Figure 6. Biomedical applications of magnetic nanoparticles [36].
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Cancer is a disease that causes more than six million deaths per year worldwide
[19]. In pharmaceutical industry new cytotoxic agents for cancer treatment have been
produced. However, the administration of these drugs continues to be a problem
because of toxicity to healthy cells. To overcome this problem, magnetoliposomes have
been proposed as carrier systems that protect and transport the drug to the target site by
means of an external magnetic field.
Drug delivery based on magnetoliposomes is one of the most promising
applications with largest impact on the scientific community. The efficacy of
magnetically-controlled drug targeting (MDT) depends on physiological parameters
[35].
In order to specifically target cancer cells, it is possible to attach antibodies [37],
creating a subclass which is known as immuno-MLs, use PEG-folate conjugate to target
folate receptors (overexpressed in cancer cells) [38], and incorporate small peptides that
target specific membrane proteins [22]. Recently, it has been shown that the use of MLs
tagged with folate results in superior doxorubicin cell uptake [39]. The inclusion of the
anionic lipid cholesteryl hemisuccinate (CHEMS) in the magnetoliposomes allows them
to be pH-sensitive, which is important as the tumor cells have a lower pH than normal
cells. These liposomes fuse after the pH is lowered below a critical value between 4.0
and 6.7 [40]. It has also been reported that the incorporation of the tripeptide arginineglycine-aspartic acid (RGD) into liposomes resulted in specific and efficient binding of
the liposomes to integrin-expressing endothelial and melanoma cells [41].
The concomitant use of magnetic targeting greatly increased drug loading
efficiency [38], by use of a simultaneous biological and physical targeting. MDT has
been widely studied, due to the large potential and advantages, such as [42]:
the ability to target specific locations in the body;
the reduction of the quantity of drug needed to attain a particular
concentration in the vicinity of the target;
the decrease of the concentration of the drug at non-targeted sites, that
minimize severe side effects.
Magnetoliposomes have also been used in thermotherapy, as a chemotherapy
alternative [7]. Tumor cells are more sensitive to high temperatures than healthy ones
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[42, 43] and hyperthermia makes use of magnetic nanoparticles to deliver toxic amount
of thermal energy to targeted tumors. Therefore, magnetic nanoparticles are used as
hyperthermia agents. For a better temperature control, nanoparticles should be uniform
in size and shape. Also, they should be subdomain in order to absorb much more power
at tolerable AC (alternating current) magnetic fields [44, 45].
In hyperthermia, magnetic nanoparticles are placed in alternating current and so
magnetic fields randomly flip the magnetization direction between the parallel and
antiparallel spin orientation. This random orientation allows the transfer of magnetic
energy, in the form of heat, to the particles. Fixing the magnetic nanoparticles in a
tumor, this property can be used to increase the temperature of the tumor tissues and so
destroy the pathological cells by hyperthermia [23]. The greatest advantage associated
with thermotherapy is that it allows the heating to be restricted to the tumor area and so
healthy cells are preserved.
In diagnosis, magnetoliposomes are a valuable system for magnetic resonance
imaging (MRI). MRI is one of the most powerful non-invasive imaging modalities used
in clinical medicine today. Magnetoliposomes, with the benefits of the magnetic
nanoparticles, could improve image quality, as they are used as contrast agents to
enhance nuclear magnetic resonance. Magnetoliposomes based on magnetite have been
reported as the less toxic formulation, with a maximum load of 67pg Fe/cell, that
corresponds to a minimum of 50 cells/microliter detection limit by MRI [46], settling
the high efficient induced contrast already reported [47].
Reticuloendothelia system or mononuclear phagocyte system is a network of
cells, part of the immune system, responsible for remove foreign substances from
bloodstream located in reticular connective tissue. Tumor cells do not have the effective
reticuloendothelial system as healthy cells, and so their relaxation times are not altered
by contrast agents [5]. This theory has been used for diagnosis of malignant lymph
nodes [48], liver tumors [49], and brain tumors [50].
Body tissues contains lots of water, so, when a magnetic field is applied, the
average magnetic moment of the many water protons become aligned with the direction
of the field. When a radio frequency current is briefly turned on, an electromagnetic
field with resonance frequency is absorbed, flipping the spin of the protons in the
magnetic field. When the field is turned off, the spins of the protons return to the
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thermodynamic equilibrium and the bulk magnetization is reorganized with the static
magnetic field. Protons in different tissues return to their equilibrium state at different
relaxation rates. The image is obtained from different variables, as spin density and T1
and T2 relaxation times (T1 is the spin-lattice or longitudinal relaxation time, and T2 is
the spin-spin or transverse relaxation time) that are used to create a MRI contrast which
relies on the differential uptake of different tissues.
2. Preparation techniques
Because of the widespread applications of magnetoliposomes in biomedicine and
engineering, much attention has been paid to the synthesis of different kinds of
magnetic nanoparticles [51-53] and liposomes [54].
Each potential application requires different properties. Synthesis methods of
magnetoliposomes and their constituents will determine their final shape, size
distribution, surface chemistry and magnetic properties [55-57].
2.1 Synthesis of magnetic nanoparticles by soft chemical methods
Magnetic nanoparticles with spherical shape can be synthesized by plasma
atomization, wet chemistry, from gas phases and aerosols. Depending on the
mechanism of formation, spherical nanoparticles obtained in solution can be crystalline
or amorphous, if they result from a disordered or ordered aggregation, respectively.
Synthesis method also determines a great extent of the structural defects or impurities in
the particle, as well as the distribution of such defects within the particles, therefore
determining their magnetic behavior [58, 59].
Techniques for magnetic nanoparticle synthesis have been developed to yield

monodispersed colloids, consisting of uniform nanoparticles both in size and shape. In
these systems, the entire uniform physicochemical properties directly reflect the
properties of single particles [60, 61].
As mentioned in the previous chapter, for biomedical applications, nanoparticles
with core-shell structure should be used in order to reduce adverse effects. After
purification, synthesized metal nanoparticles can be covered with a silica shell that can
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be obtained by hydrolysis of TEOS (tetraethyl orthosilicate, a compound that is easily

converted into silicon dioxide). Different shell sizes are obtained by the addition of
different TEOS concentrations, either in a dispersed nanoparticle solution in ethanol, or
in AOT/cyclohexane. MDA (mercaptododecanoic acid) is usually used to promote the
binding between TEOS and the metal nanoparticles.
2.1.1 Nickel nanoparticles
Simple soft chemical methods have been widely used as they do not demand
extreme pressure or temperature control, are easy to handle, and do not require special
or expensive equipment. Nickel nanoparticles can be obtained with different soft
chemical methods, namely by the reduction of nickel chloride with hydrazine (N2H4):
Surfactants can act as size control agents in the synthesis of nanoparticles.

Superparamagnetic nickel nanoparticles with mean diameter between 10 and 36 nm
were obtained by the reduction nickel chloride with hydrazine in an aqueous solution of
the cationic surfactant CTAB (cetyltrimethylammonium bromide) [5] (figure 7).
However, size monodispersity is not tightly controlled. Nanoparticles size is influenced
by nickel chloride and hydrazine concentration.
Figure 7. Nanoparticles synthesis in aqueous solution of CTAB.
Size monodispersity can be improved using water-in-oil microemulsions in

metallic nanoparticles synthesis [62]. Microemulsion method is one among the various
low-temperature routes to tailor nanoparticles. It is a soft technique that can be used
with almost all chemical reactions which have been studied to obtain nanoparticles in
homogeneous solutions.
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A microemulsion is a stable dispersion of reverse micelles of water in an organic

solvent, that is, two immiscible liquids in the presence of a surfactant that form
nanometer-size water droplets which are dispersed in a continuous oil medium and
stabilized by surfactant molecules accumulated in the oil/water interface. These are
categorized as water-in-oil (w/o) microemulsions.
In this process, reagents are separated in two different microemulsions that are
then mixed under agitation. After mixing, the reverse micelles collide among
themselves to exchange the reactants solubilized in the nanoreactors (individual reverse
micelles) and then again break apart (figure 8). This way, the reagents undergo
homogeneous mixing and the polydispersity of the particles decrease. Decoalescence
ensures the presence of the protective coating for the controlled nucleation and growth
and it also prevents aggregation [63].
Figure 8. Mechanism showing the intermicellar exchange for the formation of nanoparticles.
The main problem associated with the synthesis of small magnetic nanoparticles
are the magnetic attractions of the metallic nanoparticles. This contributes to
agglomeration/coalescence into larger particles and their subsequent settling out of the
reaction environment. A novel route for the synthesis of smaller magnetic nanoparticles
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has been proposed. This method is based on the reduction of metal chloride ionic
clusters
in
the
confined
space
of
the
anionic
surfactant
AOT
(bis(2-
ethylhexyl)sulfosuccinate) reverse micelles [64].

From this method, smaller nanoparticles, with diameters lower than 100 nm, were
obtained (figure 9). The resulting particles are covered with an AOT layer that prevents
particles agglomeration, due to electrostatic repulsions of the reverse micelles.
Figure 9. SEM images of Ni nanoparticles synthesized in the confined space of AOT reversed micelles.
2.1.2 Iron oxide magnetic nanoparticles

Iron oxide-based magnetic nanoparticles (Fe3O4, Fe2O3, ) are prepared through
bottom-up
strategies,
including
co-precipitation,
microemulsion
approaches,
hydrothermal processing and thermal decomposition techniques.
Table 2. Summary comparison of different nanoparticles synthesis methods [66].
Iron-based nanoparticles can be obtained by the precipitation of suitable mixtures

of Fe(II) and Fe(III) chlorides (figure 10). Co-precipitation is a simple and suitable
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method to synthesize iron oxides from aqueous Fe2+ and Fe3+ salt solutions by the
addition of a base under inert atmosphere at room temperature or at elevated
temperature:
+
Fe+
2 + Fe3 + 8HO Fe3 O4 + 4H2 O
Magnetite nanoparticles characteristics, like size and shape, depend on the

Fe2+/Fe3+ ratio, the reaction temperature, pH value and ionic strength of the media.
Figure 10. Fe3O4 nanoparticles synthesized by co-precipitation method, the scaler bar is 30 nm [65].
Maghemite nanoparticles can be easily obtained from the deliberate oxidation of

the resulting magnetite nanoparticles by the dispersion of magnetite nanoparticles in
acidic medium, followed by addition of iron (III) nitrate. The maghemite particles
obtained are then chemically stable in alkaline and acidic medium [66].
The experimental challenge in the preparation of iron oxide nanoparticles by coprecipitation lies in control of particles size and polydispersity. This method suffers
from a narrow particle size distribution and poor crystallinity. Small molecules and
amphiphilic polymeric molecules can be introduced to enhance the ionic strength of the
medium, protect the synthesized nanoparticles from further growth, and stabilize the
magnetic fluid [67]. Recent studies have revealed that a short burst of nucleation and
subsequent slow controlled growth is crucial to the synthesis of monodisperse
nanoparticles. Organic additives that act as stabilization and/or reducing agents can be
used to prepare monodisperse magnetite nanoparticles of different sizes [66].
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A modification of the co-precipitation method is the microemulsion method, in

which the Fe(II) and Fe(III) salts are precipitated with bases in reverse micelle droplets
stabilized by surfactant. The final size and shape of the nanoparticles prepared by this
technique can be tuned by the adjustment of surfactant or reactants concentration [68].
Small magnetic iron oxide nanoparticles with sizes lower than 10nm were
synthesized by this method [69]. Also, MnFe2O4 nanoparticles with sizes between 4nm
and 15 nm were synthesized in microemulsions of water-in-toluene reverse micelles
with surfactant sodium dodecylbenzenesulfonate (NaDBS). The synthesis of MnFe2O4
nanoparticles in microemulsions of water-in-toluene starts with a first clear aqueous
solution consisting of Mn(NO3)2 and Fe(NO3)3. A NaDBS aqueous solution is mixed
with the metal salt solution, and subsequent addition of a large volume of toluene forms
reverse micelles [70].
The disadvantages of this method are the low yield and poor crystallinity of the
nanoparticles, which limit its practical use. Also, the large amount of organic solvents
used would lead to not only higher costs, but also a non-friendly impact in the
environment.
In order to improve monodispersity and crystallinity, magnetic nanoparticles can
be prepared by thermal decomposition method (figure 11). In this method, the
organometallic complexes are decomposed in refluxing organic solvent in the presence
of surfactant [71]. Metallic iron nanoparticles with size between 2 and 10nm and good
polydispersity were obtained by thermal decomposition of Fe(CO)5 and in the presence
of polyisobutene in decalin [72].
Figure 11. Fe3O4 nanoparticles prepared by thermal decomposition of iron oleate Fe(OA)3 [65].
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In the thermal decomposition technique, nanoparticles size and morphology can

be controlled by temperature, reaction time, surfactant concentration and type of
solvent. The result nanoparticles are usually hydrophobic. Recently, it has been
demonstrated that using strong polar solvents (e.g. dimethylformamide or 2pyrrolidone) resulted in hydrophilic iron oxide nanoparticles, which are preferred for
biomedical applications [73].
2.2 Liposomes preparation

Liposomes can be obtained by means of several different methods. The properties
of the liposomes depend on the composition and concentration of the lipids and also on
the preparation method (figure 12). Liposomes incorporating different types of
substances could be manufactured by passive or active loading. The methods involving
the loading of the entrapped agents before or during the manufacturing procedure are
called passive loading. Otherwise, in active loading, certain type of compounds with
ionizable groups, and those which display both lipid and water solubility, are introduced
into the liposomes after the formation of intact vesicles.
Methods of Liposomes preparation
Passive loading
techniques
Mechanical dispersion
methods
Lipid film hydration by

hand shaking, non-hand
shaking or freeze drying.
Micro-emulsification
Sonication
French pressure cell
Membrane extrusion
Dried reconstituted
vesicles
Freeze-thawed liposomes
Active loading
techniques
Solvente dispersion
methods
Ethanol injection
Ether injection
Double emulsion
Reverse phase
evaporation vesicles
Stable pluri lamellas
vesicles
Detergent removal
methods
Detergent (Chocolate,
alhylglycoside, triton x100) removal from
mixed micelles by:
- Dialysis
- Column
chromatography
- Dilution
- Reconstituted sendai
virus enveloped
Figure 12. Scheme of the methods for liposome preparation.
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The adequate choice of liposome preparation depends on the following parameters

[74, 75]:
1. the physicochemical characteristics of the material to be entrapped and
those of the lipids;
2. the nature of the medium in which the lipid vesicles are dispersed;
3. the effective concentration of the entrapped substance and its potential
toxicity;
4. additional processes involved during application/delivery of the vesicles;
5. optimum size, polydispersity and shelf-life of the vesicles for the intended
application;
6. reproducibility and possibility of large-scale production of safe and
efficient liposomal products.
2.2.1 Synthesis of aqueous magnetoliposomes by ethanolic injection

One of the simplest methods used to obtain magnetoliposomes for in vivo
application is the so-called ethanolic injection. This is a simple technique, which
provides a reasonably homogeneous population vesicle, although rather dilute.
Figure 13. Representation of ethanolic injection.
In the ethanolic injection method (figure 13), a lipid solution in ethanol is rapidly
injected in a buffer solution under vortex. The buffer solution must be at a temperature
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above the transition temperature of the lipid [76-78]. Small liposomes in a range of 30110 nm are obtained. The advantages of ethanolic injection include its potential for
scale up, the simplicity of the procedure, low cost and low expenditure of time [79]. On
the other hand, the drawbacks of this method are that the population is poorly
homogeneous, liposomes are dilute and it is also difficult to remove all the ethanol [54].
Magnetoliposomes result from the encapsulation of magnetic nanoparticles into
liposomes, by the injection of the ethanolic lipid solution in an aqueous solution of
nanoparticles. The magnetoliposomes obtained have an aqueous pool inside and are
known as aqueous magnetoliposomes [47].
2.3. Synthesis of dry magnetoliposomes

From the alkaline co-precipitation of iron salts in the presence of the lipid DOPG
(1,2-Dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)])
phospholipid
molecules,
magnetite nanoparticles covered by a monolayer of DOPG molecules were synthesized

[8]. Dry magnetoliposomes are based on the growth of a second lipid layer around these
nanoparticles. Adding slowly another volume of lipid solution, equal to that used in the
synthesis of those nanoparticles, a second phospholipid layer is formed around the iron
oxide core [8]. This way, a lipid bilayer is created around the nanoparticles and dry
magnetoliposomes are obtained. Dry magnetoliposomes can also be obtained by mixing
SUV liposomes (obtained by strong sonication with tip) with MNPs, followed by
dialysis [6].
In order to decrease toxic effects, magnetoliposomes are washed by magnetic
decantation, taking advantage of the magnetic properties. Also, centrifugation or gel
permeation chromatography can be performed by magnetophoresis. This way, the nonencapsulated nanoparticles are removed and the toxicity of the magnetic fluid is
reduced.
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