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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.
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).
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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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Figure 4. Cobalt hysteresis for different size samples; the larger particles shows hysteresis while the smaller are
superparamagnetic and do not have hysteresis [24].
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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.
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].
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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].
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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-
Figure 9. SEM images of Ni nanoparticles synthesized in the confined space of AOT reversed micelles.
strategies,
including
co-precipitation,
microemulsion
approaches,
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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
Figure 10. Fe3O4 nanoparticles synthesized by co-precipitation method, the scaler bar is 30 nm [65].
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Figure 11. Fe3O4 nanoparticles prepared by thermal decomposition of iron oleate Fe(OA)3 [65].
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Passive loading
techniques
Mechanical dispersion
methods
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
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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].
phospholipid
molecules,
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