NOVEL APPROACHES IN DRUG DELIVERY AND TARGETING Students: Coordinating teacher: Alexandra Dulgheru As. Dr. Ing. Alexandru Mihai Grumezescu Monica… [305630]

SPION`S SAFE BY DESIGN:

NOVEL APPROACHES IN DRUG DELIVERY AND TARGETING

Students: Coordinating teacher:

Alexandra Dulgheru As. Dr. Ing. [anonimizat]

2016

[anonimizat], is a [anonimizat]. [anonimizat], nanochemistery is directed towards extensive use in nanomedicine and nanodiagnostic.

[anonimizat][1].

Superparamagnetic iron oxide nanoparticles (SPION`s) are a unique family of magnetic nanoparticles which have more physicochemical properties. Definitely, they show magnetic interactions that don`t persist once the external magnetic field is removed (superparamagnetism) [anonimizat].

Superparamagnetic iron oxide nanoparticles (SPION`s) [anonimizat] a [anonimizat], magnetic resonance imaging (MRI) and hyperthermia; also, SPION`s [anonimizat][2].

[anonimizat]-[anonimizat] a normal limit[3].

Though, because of the variety of SPION`s application, [anonimizat]-loaded SPION to be concentrated at a target region.

So, must to be studied more issues to obtain a good result regarding the SPION`s functionalization.

[anonimizat] a good respond to the further modifications.

Second, the formulation of delivery systems facilitates the loading of drugs as well as SPIONs simultaneously.

Third, [anonimizat][4].

SPION`s [anonimizat] (SPIONs) are one of the most studied biomaterials in this time because they can be used in a wide variety of medical applications[5].

Superparamagnetic iron oxide nanoparticles (SPION) are inorganic nanomaterials of

ferromagnetic substances with sizes between 1 – 100 nm. [anonimizat] (ability to have zero magnetism in the absence of external magnetic field). This enables the particles to have large magnetic susceptibility and single magnetic domain.

Figure 2. 1: Types of magnetism

Superparamagnetism exist when size of a ferromagnetic material is so small that the ambient thermal energy is sufficient to induce free rotation of the whole crystallite.

SPION can be classified into two big types:

SPION with hydrodynamics sizes greater than 50 nm;

SPION with sizes less than 50 nm which are called ultrasmall superparamagnetic iron oxide nanoparticles (USPION)[6].

SPION`s are small synthetic γ-Fe2O3 (maghemite), Fe3O4 (magnetite) or α-Fe2O3 (hermatite) particles with a core ranging from 10 nm to 100 nm in diameter.

However, magnetite and maghemite nanoparticles are the most widely used SPIONs in various biomedical applications[7].

Figure 2.2: SEM images of shaped iron oxides with six different shapes

(a) Nanorod, (b) Nanohusk, (c) Distordted cubes, (d) Nanocubes, (e) Porous spheres and (f) Self-oriented flowers [10]

Size of SPION’s

In time, for in vivo applications, the particles size was discovered as an important factor to better the particles biocompatibility owing to the detecting of particles larger than special size by immune system [11].

The size of nanoparticles largely determines their half-life in the circulation. For example, particles with sizes smaller than 10 nm are mainly removed by renal clearance, whereas particles larger than 200 nm become concentrated in the spleen or are taken up by phagocytic cells of the body, in both cases leading to reduce plasma concentrations.

Anyway, particles with a size range of 10–100 nm are considered to be optimum, with longer circulation times because they can easily escape the reticulo-endothelial system in the body and it was demonstrated that they can also penetrate well in very small capillaries.

Moreover, biomedical applications of SPION`s, like MRI, hyperthermia and magnetic cell separation, depend on the magnetic properties of these particles, which in turn are largely dependent upon size. [7].

.

.

Figure 2.3: Electron micrographs of single and hybrid particles

(a) TEM images of superparamagnetic iron oxide nanoparticles (SPIONs) with core sizes of 5, 10, 20, and 30 nm on 400 mesh formvar carbon grids, imaged at 200000× direct magnification (bars = 100 nm);

(b) SEM images of discoidal porous silicon (pSi) microparticles, unloaded (left) or loaded with 30 nm SPIONs (right with image periphery pseudo-colored black) [12]

Surface properties of SPION

In drug delivery systems, magnetic nanoparticles based drug carriers have more advantages because of their surface properties and their easy modifications [13].

The surface charge of nanoparticles gives an indication of their colloidal stability. Nanoparticles having high positive and negative zeta potential, which exhibit the dispersion stability.

SPION`s having a positive charge are better internalized by human breast cancer cells, than are negatively charged particles. With other words, intake of these nanoparticles depends upon cell type. Thus, particles with a hydrophobic surface, it was demonstrated that are easily adsorbed at the protein surface and are consumed by circulating macrophages. Therefore, they ilustrate a low circulation time. However, particles that are surface-engineered with hydrophilic polymers like PEG (polyethylene glycol) including are able to elude engulfment by the reticulo-endothelial cells or circulating macrophages, thus having better therapeutic efficacy due to increased residence time in the blood. [7]

The organic and inorganic surfactants or capping agents form a protective layer around SPION`s by attaching to the surface atoms of SPION`s via the end functional groups either by electrostatic interactions.

The surfactants or capping agents usually have different end functional groups such as -OH, -COOH, -PO(OH)2, -S(=O)2 -OH, catechol (known as pyrocatechol or 1,2-dihydroxybenzene), which bring stoichiometric modifications on the surface of SPION`s [14].

Furthermore, it was demonstrated the influence on the magnetic properties of SPION`s that differs with the type of end functional groups that are attached to the surface atoms of SPION`s. For example, was showed that the effects of surfactants on the magnetic properties of SPION`s were negligible since no modifications on the surface structure or type of magnetic order in SPION`s were observed [15].

In contrast, the results of canting angle measurements of SPIONs showed that the average surface spin canting of SPION`s had decreased after coating its surface with polyoxyethylene nonylphenylether.

However, this oversight phenomenon was contradicting with another investigation, where the strong affinity of carboxylic end groups of oleic acid towards the surface of SPION`s resulted in increased surface spin canting effects in SPIONs by victimizing the octahedral iron sites.

Conclusive, the usage of surfactants/ capping agents in the synthesis of SPION`s is inevitable to maintain the colloidal stability for extended period of time [16].

2.2 Synthesis of SPION`s

Among the different nanoparticles employed in biomedical research, functionalized superparamagnetic iron oxide nanoparticles (SPION`s) have been recognized as promising materials due to their high biocompatibility, unique magnetic properties and their capacity for use as a medical innovation [17].

Thus, it was given a increased attention of synthesis methods which can get these SPION`s.

The synthesis method of SPIONs can be classify as:

Physical methods

2.2.2 Chemical methods

2.2.3 Gas Phase

Biological method.

2.2.1 Physical Methods of SPION synthesis

In times, have been reported different physical way of producing SPION such as powder and ball milling and electron beam lithography.

2.2.1.1 Powder and Ball Milling Method

Mechanical powder and ball milling technique is also known as mechanochemistry or mechanical alloying technique. It involves use of impact to reduce micron sized iron precursor to nanometric size.

The particles are produced inside a hollow cylindrical shell rotating about its axis. It is partially filled with micron size balls which serve as grinding media. Samples are grinded in a solvent such as water or mixture of kerosene and oleic acid. The solvent allows effective milling and also serves as capping agent to prevent agglomeration of the particles. Else, the type of solvent and nature of ball (iron, stainless steel and rubber) used influence the formation of either magnetite or maghemite phase.

The SPION produced via this route is often contaminated with non-reduced Fe [7].

2.2.1.2. Electron Beam Lithography.

Electron Beam Lithography (EBL) is another physical method of producing SPION that it involves conversion of iron particles into iron oxides nanoparticles (Fe3O4) below exposure to electron beam (e-beam).

EBL procedure involves two phase: covering surface of a substrate with a thin film of high purity iron material in a patterned style and emission of e-beam over the iron film. The beam causes evaporation and formation of one dimensional nanosized magnetic nanoparticle in on the substrate.

EBL technique is a according method to produce assembled or array of SPION in specific particle geometry [7].

Chemical Method of SPION Synthesis

Wet Chemical Method

Co-precipitation method

The co-precipitation method is considered as the simplest chemical way to synthesize iron oxide nanoparticles. Performed first time by Massart more than 30 years ago, this method gained broad variety of modifications, but it`s main chemistry didn`t change :

Fe2+ + 2Fe3+ 8OH- → Fe3O4 + 4H2O

After:

(Fe3+ (H2O)6)3+ → FeOOH + 3H+ +4H2O

Fe2+ +2OH- → Fe(OH)2

2FeOOH + Fe(OH)2 → Fe3O4 + 2H2O

Fe2+ and Fe3+ ions in molar ratio of 1:2 precipitate into magnetite in a basic solution (pH from 8 to 14). Due to magnetite is sensitive to oxidation, an inert gas purging is needed during the reaction to prevent magnetite transformation to maghemite.

The major advantage of this synthesis method is value of produced nanoparticles per one batch – up to 40 grams. However, the co-precipitation synthesis method possess a weak control of the nanoparticle size distribution due to only kinetic factors influence nucleation and growth of the crystals. Thus, when the concentration of precursors reaches the supersaturation, the nucleation occurs for a short moment of time – “seeds” of synthetising nanoparticles are forming; then slow process of crystal growth via diffusion of precursors on the surface of “seeds” takes place.

The key to produce nanoparticles with narrow size distribution is to separate nucleation from growth. The nucleation time must be very short to form highly monodispersed nanoparticles – final nanoparticle amount is formed at this moment and won`t change during crystal growth.

The LaMer diagram represents the generation of uniform nanoparticles through the nucleation and growth processes.

Figure 2.4: Plot of atomic concentration against time, illustrating the generation of atoms, nucleation, and subsequent growth [18].

Nanoparticle size, morphology, magnetic properties can be controlled by varying of solution pH, ionic strength, temperature, reaction time, type of salts (ferrous and ferric chlorides are most popular) [19].

Hydrothermal

The SPION`s can be also obtain to a method that involves heating various iron salt precursors in aqueous solution at high temperatures (150 °C and higher). The solution blend is sealed in Teflon-line autoclaves to reach high pressure and after the reaction is complete, it is cooled to room temperature and washed with various solvents to clean from the impurities and nonconjugated stabilizing agents.

The SPION`s are generally retrieve as a black powder. The choice of iron salt precursor, the reaction time and temperatures have to be carefully monitored to result SPION`s of different sizes.

Often, it is used methods to produce SPIONs` on the hydrothermal direction make use of a single type of iron salt [20].

Sol-Gel Method

Sol-Gel Method is also known as chemical solution deposition method which involves conversion of precursor solution into an inorganic solid by chemical means.

In materials science, sol-gel process is a well studied and developed procedure to prepare materials and design devices with very specific properties.

Sol-gel synthesis can be class in four steps: hydrolysis and polycondensation of iron precursor in a solvent to form colloidal suspension of the particles (sol), gelation of the sol to form gel, aging and drying of the particles through sintering.

Ironic precursors like alkoxide or non-alkoxide (iron nitrate, iron chloride and metallorganic) compounds have been used to produce SPION.

Thus, based on the solvent used, sol-gel method can be classified into aqueous and nonaqueous process. The former entails the use of water solvent and the organic solvent is used after. The size and magnetic moment of SPION synthesized by this method growth with increased in the annealing temperature [19].

Microemulsion Method

Microemulsion is compound of two immiscible phases (oil and water); different types of microemulsions are known, such as water-in-oil (W/O) and oil-in-water (O/W)[21].

W/O microemulsion method is used to form SPION`s, where the stabilizing agents in continuous oil phase initially protect the droplets formed of iron oxide reactants, which then react to form SPION`s.

The sizes and shapes are controllable by varying the concentrations of iron oxide precursor to base, surfactant and/or solvents.

Nevertheless, the removal of unreacted precursors, base and surfactants is intricate in this microemulsion method [16].

Solvothermal Method

Solvothermal approach involves the use of solvent (reaction medium) other than supercritical water to synthesize iron oxide nanoparticles at moderate to high pressure and temperature of 1-10000 atm and 100-1000 °C.

For this method, it is used some solvents such as methanol, ethylenediamine and hydrazine solution. The complex formation between the surfactant molecules and the iron precursor leads to uniformity of crystallite size and morphology of the particles formed by this route.

Also, in the end of this method was see that the nucleation, growth and size distribution of nanoparticles depend on dispersion of the precursor in suitable solvents, solvothermal temperature and aging time [6].

Sonochemical Method

The sonochemical process is a good route of synthesising nanostructures that involves decomposition of organometallic or inorganic iron precursor in deoxygenated water via ultrasonic irradiation [6].

Sound energy such as ultrasound can be used for synthesizing SPION`s, where the cavitation bubbles produced by such ultrasound transform the reactants into desired products at ambient temperatures.

The size and shapes of SPIONs can be varied by controlling the refluxing time, irradiation time and power [22].

Electrochemical Method

Electrochemical method of producing SPION is based on reduction and oxidation reaction of iron-based electrode in an electrolyte. The anode can be oxidized to metal ion species in the electrolyte and the metal ion is further reduced to metal at the cathode with the help of stabilizers.

The as-synthesized SPION is normally deposited on the electrode in the form of a coating or a small film.

However, amine or other surfactant can be used as supporting electrolytes to produce functionalized and stabilized SPION [6].

Thermal Decomposition Method

The thermal decomposition (pyrolysis) method is a well known technique for synthesis of highly crystalline SPION.

Highly crystalline and monodisperse SPION`s with diverse sizes and shapes can be synthesized via thermal decomposition method in the presence of surfactants (for example, oleic acid and oleylamine) and organic solvents with high boiling points.

Solvent free thermal decomposition of iron precursors can also be utilized for preparing magnetic nanoparticles. However, the resulting hydrophobic SPIONs tend to show good dispersibility only in organic solvents (for example, tetrahydrofuran) because of their hydrophobic interactions between surfactants and solvents.

Many synthesis parameters such as concentration of surfactants, reaction temperatures, reaction timings, ratio of precursors to surfactants, solvents and heating rate during reflux govern the physicochemical characteristics and magnetic properties of SPION`s [22].

2.2.3. Gas Phase Method

Gas phase synthesis of SPION involves evaporating large particle size of coarse grained iron precursor such as FeO/Fe2O3 and Fe2O in a reactor to nano-size. In this class of synthesis, nanoparticle formation starts abruptly when a sufficient degree of supersaturation of condensable products is reached in the vapour phase. The process of nanoparticles formation via this route involves 3 stages: homogeneous nucleation in the gas phase, condensation and coagulation.

The gas phase process is often conducted inside a reactor to decompose iron precursor into nanoparticles. Based on sources of energy used in this technique, the various type of reactors which can be found in the literature include flame, laser and plasma reactors.

2.2.4. Biological Method

Biological method is a green chemistry process that connects nanotechnology with microbial biotechnology for the synthesis of nanoparticles. Several micro-organisms such as fungi, bacteria, virus and protein have been reportedly used to synthesis SPION.

The microbes can be used to synthesize SPION through either reduction or precipitation of soluble toxic inorganic ions to insoluble non-toxic metal nanoclusters. Majorly, the SPION synthesised via biological means are magnetite. The detoxification and synthesis of the particles can be done via intra or extracellular production. However, in the intracellular production, additional process such as ultrasound treatment or reaction with suitable detergents is required to release the nanoparticles. The SPION synthesized via this route is strongly coated with biomolecules.

Table 2. 1: Summarizing the various methods used [6]

3.Drug deleivery and targeting system-systematic design of SPION’s surface

3.1 General aspects

Magnetic nanoparticle is an attractive material in magnetic recording but also it has applications in biomedical, optical, electronics, chemical and mechanical field . For these uses, parameters such as size, size distribution of magnetic nanoparticles, and its physical properties (e.g., magnetic, optical, and electronic) are critical [23].

The principle behind the development of superparamagnetic iron oxide nanoparticles (SPION’s) as novel drug delivery vehicles is guiding magnetic iron oxide nanoparticles with the help of an external magnetic field to its target[24].Due to superparamagnetic these nanoparticles have large magnetic susceptibility, single magnetic domain and controllable magnetic behavior [25].

SPION’s are promising tool in the biomedical applications . There have been considerable efforts on research focusing on the development of magnetically guided drug delivery systems because an externally applied magnetic can make the drug-loaded SPION to be concentrated on a specific region [26].

Magnetic nanoparticles can localize heat on particular organs which makes them effective in hyperhermia and also can kill cancer cells by utilizing alternating magnetic fields. Due to their ease of preparation,non-toxicity and their ability to tailor their properties for precise applications, especially in tumor treatment, superparamagnetic iron oxide nanoparticles (SPION’s) have received wide recognition in recent decades [27].

Figure 3.1: Large MNPs (>200 nm) will be easily detected by the immune system and removed from the blood and delivered to the liver and the spleen.320 Very small MNPs (o5.5 nm) can be excreted through the kidneys.321 The optimal MNP size for drug delivery treatments ranges between 10 to 100 nm, as these will have the longest blood circulation time. Different magnetic biocomposites can be transported to reach the tumor area inside the body thanks to the applied magnetic field [35]

3.2 Essential characteristics for drug delivery

Shape

The reaction conditions and chemicals involved affect the morphology of Fe2O3 nanoparticles. In the presence of surfactants with bulky hydrocarbon chain structures, like oleylamine and adamantane amine, the steric hindrance exerted by surfactants has been shown to affect the shape of growing crystals of iron oxide during synthesis.

Size

The size of nanoparticles determines their half-life in the circulation.15 Particles with sizes smaller than 10 nm are removed by renal clearance,but particles larger than 200 nm are concentrated in the spleen or they are taken up by phagocytic cells.

Surface properties

The surface charge of nanoparticles gives an indication of their colloidal stability. Nanoparticles having high positive and negative zeta potential show dispersion stability and as a result do not agglomerate on storage. SPIONs having a positive charge are better internalized by human breast cancer cells than are negatively charged particles

Figure 3.2: Physicochemical characteristics of SPION`s

Coating

After SPION’s cores are fabricated,the next step is represented by their coating. This can be obtained with suitable polymers which endows essential characteristics to these nanoparticles that are important for their use as drug delivery vehicles. This step is essential because can reduce the aggregation tendency of the uncoated particles improving their dispersibility and colloidal stability; protects the surface from oxidation; provides a surface for conjugation of drug molecules and targeting ligands; increases the blood circulation time by avoiding clearance by the reticuloendothelial system; makes the particles biocompatible and minimizes nonspecific interactions, thus reducing toxicity; and increases their internalization efficiency by target cells[22].

There are significant points that should be to considered in achieving the successful development of magnetically guided drug delivery systems:

the surface of SPIONs has to be coated with materials which can provide the water dispersity to the nanoparticles and functional groups for the further modifications .

the formulation of delivery systems facilitates the loading of drugs as well as SPIONs simultaneously .

-the efficient magnetic guided targeting, modifications and formulations of SPIONs should not

interfere with the superparamagnetic properties of the SPIONs.[24]

3.3 Surface modification of SPION’s

Surface modification of SPION’s with biocompatible materials is used as a strategy to achieve biomedical applications. Materials such as glucose, chitosan, , carboxylic and amine group, surfactant, polymer and inorganic materials (silica or gold) are used to modify the surface of SPION’s. Agglomeration of SPION’s through either electrostatic repulsion or steric stabilization is prevent bu using these materials[25] .Oleic acid, alkane sulphonic acids, lauric acid, and alkane phosphonic acids, have been used significantly for the stabiliza- tion of small-sized SPIONs [27].

Figure 3.3 : Scheme showing representative groups that can be used to stabilize the SPIONs[18]

Silica materials, biological molecules, small organic molecules ,natural and synthetic polymers are the main SPION’s coating agents . The strategy used to modify the surface is the formation of a silica shell which requires alkoxysilane molecules or tetraethyl orthosilicate use [24]. Silica coating is advantageous because the silane groups can be covalently bound onto the NPs’ surface [29].

The formation of silica layer around the particles is induced by subsequent crosslinking[8].

Figure 3.4: Showing the various methods of producing SPION and its surface modification with

silica nanoparticles [24]

SPION’s modified with proper polymers can carry drugs and can be accumulated in tumor near a magnet.This property and nanoparticle size are characteristics that influence the magnetic targeting effects[31].

Surface modification can be achieved using different hydrophilic coatings as chitosan.This natural polysaccharide has the ability to degrade in biological environments.Chitosan has hydroxyl groups and active amine, which produce chemical modifications and introduce desired functional groups[26].

To increase the biomimmetic nature of the particles for various biomedical applications,coating with biocompatible phases is used. Surface modifications of SPION’s strongly influence their biodistribution and cellular interactions in vivo[32].

The applications of superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers and contrast agents of magnetic resonance imaging (MRI) to develop biomedical platforms for therapy, simultaneous imaging and diagnosis have been actively explored

SPION’s synthesized by thermal decomposition method are preferred because their higher saturation magnetization. Thesenanoparticles exhibit high r2 relaxivity and are efficient MRI contrast agents . MRI can noninvasively obtain cross section images of tissues or organs with high resolution , and MRI-guided targeting chemotherapy is promising for the theranostics of cancers.

Folic acid (FA) is used to target various human carcinomas that overexpress FA receptors. FAmodified SPIONs loaded with therapeutic agents can enter into cancer cells via the receptor mediated endocytosis, this significantly enhances the therapeutic efficacy .

In vitro studies on FA conjugated magnetic nanoparticles revealed that the uptake of nanoparticles by cancer cells (HeLa cells) was more than that by normal cells .

Figure3.5: (a) The synthesis and surface coating of SPIONs and FA-SPIONs. (b) The DOX loaded FA-SPIONs for FA-mediated andmagnetically targeted drug delivery to the tumor and the MR imaging [9]

3 .4 SPION’s applications

SPION’s have demonstrated their efficiency as nonviral gene vectors that facilitate the introduction of plasmids into the nucleus at rates multifold those of routinely available standard technologies. SPION-induced hyperthermia has also been utilized for localized killing of cancerous cells[24].

Most drug delivery studies have been directed to the treatment of cancer. Treatment of solid and malignant tumors is a challenge that is complicated by low drug internalization due to a hard-to-penetrate

Figure 3.6: Superparamagnetic iron oxide nanoparticles can be guided to their site of action using an externally applied magnetic field [32]

SPION’s synthesis and coating material determine their applications.Silica nanoparticle is one of the preferred inert inorganic materials used for surface modification because this material has several advantages like chemical stability, optical transparency, size-selective, porous structure.

There are strategies for modifying the surface silica nanoparticles.All the reported processes fall under chemical method which can be categorized into wet phase and gas phase method[24].

In combination with a target-directed magnetic gradient field SPION’s are sensitive devices for magnetic drug targeting after intravenous nanoparticles administration,. This technique has been applied in an attempt to target dry nanoparticle aerosols. Recently, inert SPIONs added to the nebuliser solution make aerosol to be guided to the affected region of the lung by means of a strong external magnetic field.This concept is used to administrate various type of therapeutic agents. [28].

SPION’s provide an additional targeting capability; the SPION-drug complex can be transported by blood circulation and by applying a magnetic field on the specific site they accumulate in the tumor region.

MRI can be used to validate the localization of magnetic DDSs.The loaded drugs are released by diffusion, endocytosis, vehicle rupture or dissolution. It is generally coupled to NPs’ surface by covalent or ionic bonds and the link between the magnetic core and the drug must be cleaved to release the drug.

Using different methods for the preparation, adjustable physicochemical properties (e.g., size, surface charge, morphology,shell thickness, etc.), a wide variety of raw materials for preparation, and functional versatility makes the nanoparticles very important for biomedical applications. relevant Parameters such as colloidal stability, release kinetics,encapsulation efficiency, and interactions at the nano–bio interface (e.g., with proteins) should be studied. Nanoencapsulation protect the encapsulated drugs against degradation by pH and light, minimize tissue irritation and provide controlled release by external features such as temperature,light radiation reduction and pH changes [33].

When SPIONs are involved in in vivo cancer treatments,there are problems about their toxicity. SPIONs have superparamagnetic behavior at size below 30 nm at room temperature. Due to the increased surface area-to-volume ratio, the surfaces of SPION’s at reduced sizes are reactive.

To prevent agglomeration in colloidal solution, aggregation to form bulk structures and settle down in colloidal solutions and to maintain the size and shape,the surfaces of SPION’s are usually coated with surfactants/capping agents/polymers .

The SPION’s are conjoined with other contrast agents, quantum dots, fluorescence tags/dyes, etc. to impruve cancer cells’s images through fluorescence imaging, ,single photon emission computed tomography (SPECT), computed tomography (CT), positron emission tomography (PET), near infra-red (NIR) imagingultrasound imaging,etc.

The SPION’s are also combined with nucleic acids (deoxyribonucleic acids and ribonucleic acids), chemotherapeutic drugs (such as anthracyclines, antimetabolites, platinum-based-drugs, taxanes, vinca alkaloids and so on), targeting agents (peptides, proteins, and small biological), unconjugated monoclonal antibodies (for instance, rituximab, trastuzumabphotodynamic, photothermal and sonodynamic agents/nanoparticles to form combinatorial nanopackages for effective cancer treatment. Unfortunately, the magnetic properties of SPIONs are deteriorated when they are combined with these drugs/antibodies/other nanoparticles [34].

The size, surface chemistry of the magnetic nanoparticles and charge are very important and strongly affect the blood circulation time and the bioavailability of the particles within the body, which dictates the biomedical applications [32].

SPIONs for tumor targeting applications by loading anticancer agents to them is now a field with with a lot of perspectives

Curcumin is a natural diphenol, traditionally used for the prevention of various clinical disorders like Alzheimer’s disease, diabetes, HIV replication, arthritis, wound healing, etc

Figure 3.7: (a) Chemical structure of curcumin. (b) SPIONs attracted by a magnet. (c) Dispersion of OA-SPIONs in n-hexane. (d) Aqueous CUR-OA-SPIONs. (e) Experi-mental setup for in vitro localization study of nanoparticles [27]

In vitro localization study of CUR- OA-SPION’s shows that nanoparticles are localized or aggregated at the side wall of the glass capillary with the use of a permanent magnet located close to the glass capillary.For optimize the shape and size of the aggregation micro/nano-magnets should be developed because CUR-OA-SPION’s is a promising therapeutic agent and its anti-cancer is valuable for targeted drug delivery systems[27].

However, drug delivery using the surface of SPION’s conjugated with drugs suffers has some disadvantages like:

low entrapment efficiency of drug ;

highly stable linkages as a result of covalent bonding between drug molecules and the surface of SPIONs, leading to failure to release the drug molecule at the target site ;

vivo toxicity if it is not purified properly;

difficult to control the orientation of binding ligands when attaching them to the surface of magnetic nanoparticles[23].

More recently, a field called “molecular imaging” has appeared, this technique allows the in vivo visualization of molecular events occurring at the cellular level and needs theevolution of high affinity ligands and their grafting to SPIONs [41]. SPIONs are also good substrates for bioconjugation, they are used as reporters for many physiologic processes and have a lot of clinical applications such as liver and spleen imaging, inflammation, apoptosis, and cardiovascular disease [42,46]

Figure 1: Superparamagnetic oxide nanoparticles (SPIONs) as a tool to study and promote axon growth.(A) Functionalized SPIONs specifically bind cell receptor proteins present in the axon membrane. SPIONs can be magnetized by close apposition of electromagnet to induce clustering of receptors and a resulting intracellular signaling cascade. (B) Functionalized SPIONs attach to growth cone membrane and then accumulate at the end of filopodia when an electromagnet is turned on. The magnetic force exerted by these SPIONs induces rapid filopodia elongation (Pita-Thomas et al., 2015). (C) A hollow electromagnet made by tightly wrapping copper wire around a hollow metallic cylinder will generate a magnetic force (green arrow) inside the cylinder that is perpendicular to the electric current flowing through the copper wire (orange arrows). SPIONs located at the axon cell membrane may generate magnetic forces and induce axon elongation when axons are located inside the hollow electromagnet.

Targeting towards other tissues/organs was the second important challenge in the development of nanoparticles for pharmaceutical applications. It is interesting to note that the concept to decorate nanoparticles with specific antibodies to target cancer cells had already been proposed in the early eighties [32] and the proof of concept done in cell culture in vitro, but in vivo applications remained completely unsuccessful until the above mentioned opsonization process was sufficiently understood.

However, once the surface-derivatized NPs are inside the cells, the coating is likely digested leaving behind the bare particles exposed to other cellular compo- nents and organelles thereby potentially influencing the overall integrity of the cells [51,52]. It is hypothesized that rigid coatings such as crosslinked PEGF could postpone this shortcoming [51].

The major limitation of SPIONs for drug delivery applications is the deficient magnetic gradient, in order to control the residency time of NPs at the targeted site.

SPIONs-assisted drug delivery systems have been designed to deliver peptides, DNA molecules, and chemotherapeutic, radioactive and hyperthermic drugs. The most recent delivery systems are centred on anti-infective, blood clot dissolving, anti-inflammatory, anti-arthritic, photodynamic therapy, and paralysis-inducing drugs as well as on stem cell differentiating/tracking [55,56].

By targeting the proteins are able to recognize a specific biological target became possible, allowing promotion of the delivery to a specific cell population and/or control over the intracellular trafficking of the nanoparticles. Modern bioconjugation approaches, offer a great versatility for nanoparticle functionalization.

The surface engineered SPIONs used together with the aid of an external magnetic field is recognized as a modern technology to introduce particles to the desired site where the drug is released locally. Such a system has the potential to increase the minimum side effects and the required dosage of the drugs [48,49,50].

Figure 3.8: Targeting surface [50]

4.Development and progress in drug delivery using SPIONs

At present, nanoparticles are used for various biomedical applications where they promote laboratory diagnostics and therapeutics. More specifically for drug delivery purposes, the use of nanoparticles is attracting increasing attention due to their unique capabilities and their negligible effects not only in cancer therapy but also in the treatment of other ailments. Among all types of nanoparticles, biocompatible superparamagnetic iron oxide nanoparticles (SPIONs) with proper surface architecture and conjugated targeting proteins have attracted a great deal of attention for drug delivery applications.[31]

Today, the development of nanoparticle suspensions which contain medicines has made it possible to increase the therapeutic index of many components (improvement of the activity) by selectively directing them towards the dis-eased tissues and cells (‘drug targeting’). The shift in size from tens of micrometers to hundreds of nanometers has thus been a significant technological and medical breakthrough.

Notably, it was a purely physico-chemical concept (the so-called “steric repulsion”[33]) which allowed the prevention of opsonization and subse-quent liver capture, thanks to the decoration of nanoparticles with poly(ethylene glycol), a hydrophilic and flexible polymer[34]. These so-called “long circulating” or “PEGylated” nanoparticles were found

to be capable of specific extravasation in diseased tissues not located in the reticuloendothelial area, as a result of the enhanced permeability and retention effect (EPR).

The design of multifunctional nanoparticles is certainly another exciting future challenge in the field. For instance, the combination of many drugs acting on complementary biological targets (for ex-ample, an antiangiogenic compound and a DNA intercalating agent) is an appealing approach which could lead to an enhanced therapeutic ac-tivity, much higher than the single addition of the individual pharmaco-logical activity of each compound[34]. Association of a drug and an imaging agent in the same nanoparticle to get simultaneous personal-ized patient treatment and diagnosis (“Nanotheragnostics”) is another example of multifunctional nanoparticle construction[36].

5.Benefits and risks of SPIONs

The potential benefits of SPION are considerable, there is a distinct need to identify any potential cellular damage associated with these nanoparticles. Besides focussing on cytotoxicity, the most commonly used determinant of toxicity as a result of exposure to SPION[52].

In fact, exposure to SPION has been related with significant toxic effects such as inflammation, the formation of apoptotic bodies, impaired mitochondrial function (MTT), membrane leakage of lactate dehydrogenase (LDH assay) generation of reactive oxygen species (ROS), increase in micronucleic (indicators of gross chromosomal damage; a measure of genotoxicity), and chromosome condensation[10-15]

Figure 5.1: Cellular toxicity induced by SPION. Exposure to SPION could potentially lead to toxic side effects such as membrane leakage of lactate dehydrogenase, impaired mitochondrial function, inflammation, formation of apoptotic bodies, chromosome condensation, generation of reactive oxygen species (ROS) and DNA damage.

SPION have attracted much attention not only because of their superparamagnetic properties but also because they have been shown to be associated with low toxicity in the human body [42, 43, 36,52].

Another mechanism which SPION can induce (geno)toxicity is via the generation of ROS. SPION are presumably degraded on iron ions within the lysosomes by hydrolysing enzymes effective at low pH [56, 52, 53].

Several studies have examined the cytotoxic potential of several different specimen of SPION with a range of surface coatings and have broadly found low or no cytotoxicity associated with these NPs until high exposure levels (>100 g/ml). The toxicity was also found to be dependent factors such as type of surface-coating or its crack-up products, chemical composition of cell-medium, oxidation state of iron in SPION and protein–SPION interaction [57, 48, 42, 53].

Common to all NPs, SPION are associated with unique physico-chemical features, such as nanometric sizes and a large surface area to mass degree that also facilitate novel applications. On the other hand, the alike nanoscale properties can potentially induce cytotoxicity that can manifest itself by impairing the functions of the major components of the cell, mitochondria, nucleus and DNA [53].

For biological applications, nanoparticles must be highly stable in aqueous ionic solutions at physiological pH. Vectors grafted on their surface have to be capable to recognize the target cells or tissues. Particles must be non-toxic and stay in the circulation for a sufficient amount of time to accord targets to be reached. Intensive research are currently undertaken to acquirespecific contrast agents in targeted cancer imaging [49, 50].

6.Conclusions

In summary, SPIONs may be used as an agent in the context of cancer, in other words it can serve as both a diagnostic and therapeutic agent[49].

Multifunctional SPIONs play an important role in the elaboration of diagnosis and treatment of cancerous tumors in vivo.

Further understanding on the protein corona compositions at the surface of nanoparticles are constitutivel for development of nanoparticle specific uptake by desired cells in vivo. In addition, control of the protein corona which is formed at the surface of magnetic particles, which are decorated by cancer-specific bond agents, would make targeted delivery and magnetic fluid hyperthermia treatment much more selective than traditional chemotherapy and even accepted hyperthermia. Furthermore, multifunctional magnetic particles can be magnetically targeted and focused in the target tissue, and drug release together with heating are then only induced to significant burst effects and temperatures where the magnetic nanoparticles have been deposited. In annexation, tissue-deposited magnetic particles will generally stay where they were initially deposited, thus allowing for repeated and concentrated drug absolve and hyperthermia treatments in the same area[54].

At the moment the aggregate of particles delivered to the cancerous tissues or cells by means of antibody targeting is too low for a sufficient drug release and temperature increase.

The main challenges in this field will be the design of covertness nanoparticles able to circulate in the blood compartment for a long time and the surface grafting of ligands able to help their specific internalization in cancerous cells. Even if the results obtained from the first clinical trials of magnetic nanoparticles are very promising, it would be premature to claim that these molecules contribute therapeutic advantages because survival and disease progression benefits were not defined endpoints of the feasibility studies[55].

Multiple studies on SPION including those on commercially available and clinically approved MRI contrast agents have reported that these NPs are biocompatible and absence of cytotoxicity. In this moment, the measure of biocompatibility largely focuses on the extent of cytotoxicity observe[52].

So,the criteria to define toxicity of NPs needs to be clearly defined , particularly as ascension studies have begun to highlight cellular responses including DNA damage, oxidative stress, mitochondrial membrane dysfunction and adjust in gene expression as a result of SPIONs exposure, all in the absence of cytotoxicity.

References

1. Scialabba, C., et al., Inulin-based polymer coated SPIONs as potential drug delivery systems for targeted cancer therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2014. 88(3): p. 695-705.

2. Darroudi, M., et al., Superparamagnetic iron oxide nanoparticles (SPIONs): Green preparation, characterization and their cytotoxicity effects. Ceramics International, 2014. 40(9, Part B): p. 14641-14645.

3. Sabareeswaran, A., et al., Effect of surface-modified superparamagnetic iron oxide nanoparticles (SPIONS) on mast cell infiltration: An acute in vivo study. Nanomedicine: Nanotechnology, Biology and Medicine, 2016. 12(6): p. 1523-1533.

4. Jeon, H., et al., Poly-paclitaxel/cyclodextrin-SPION nano-assembly for magnetically guided drug delivery system. Journal of Controlled Release, 2016. 231: p. 68-76.

5. Tong, H.-I., et al., Physiological function and inflamed-brain migration of mouse monocyte-derived macrophages following cellular uptake of superparamagnetic iron oxide nanoparticles—Implication of macrophage-based drug delivery into the central nervous system. International Journal of Pharmaceutics, 2016. 505(1–2): p. 271-282.

6. Sodipo, B.K. and A.A. Aziz, Recent advances in synthesis and surface modification of superparamagnetic iron oxide nanoparticles with silica. Journal of Magnetism and Magnetic Materials, 2016. 416: p. 275-291.

7. Wahajuddin and S. Arora, Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. International Journal of Nanomedicine, 2012. 7: p. 3445-3471.

8. Butoescu, N., et al., Magnetically retainable microparticles for drug delivery to the joint: efficacy studies in an antigen-induced arthritis model in mice. Arthritis Research & Therapy, 2009. 11(3): p. R72-R72.

9. Kopanja, L., et al., Core–shell superparamagnetic iron oxide nanoparticle (SPION) clusters: TEM micrograph analysis, particle design and shape analysis. Ceramics International, 2016. 42(9): p. 10976-10984.

10. Sayed, F.N. and V. Polshettiwar, Facile and Sustainable Synthesis of Shaped Iron Oxide Nanoparticles: Effect of Iron Precursor Salts on the Shapes of Iron Oxides. Scientific Reports, 2015. 5: p. 9733.

11. Ghazanfari, M.R., M. Kashefi, and M.R. Jaafari, Modeling and optimization of effective parameters on the size of synthesized Fe3O4 superparamagnetic nanoparticles by coprecipitation technique using response surface methodology. Journal of Magnetism and Magnetic Materials, 2016. 405: p. 88-96.

12. Lundquist, C.M., et al., Characterization of Free and Porous Silicon-Encapsulated Superparamagnetic Iron Oxide Nanoparticles as Platforms for the Development of Theranostic Vaccines. Medical sciences : open access journal, 2014. 2(1): p. 51-69.

13. Theumer, A., et al., Superparamagnetic iron oxide nanoparticles exert different cytotoxic effects on cells grown in monolayer cell culture versus as multicellular spheroids. Journal of Magnetism and Magnetic Materials, 2015. 380: p. 27-33.

14. Yuen, A.K.L., et al., The interplay of catechol ligands with nanoparticulate iron oxides. Dalton Transactions, 2012. 41(9): p. 2545-2559.

15. Masih, D., et al., Nanoscale size effect on surface spin canting in iron oxide nanoparticles synthesized by the microemulsion method. Journal of Physics D: Applied Physics, 2012. 45(19): p. 195001.

16. Tefft, B.J., et al., Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles. 2015(104): p. e53099.

17. Mirsadeghi, S., et al., Effect of PEGylated superparamagnetic iron oxide nanoparticles (SPIONs) under magnetic field on amyloid beta fibrillation process. Materials Science and Engineering: C, 2016. 59: p. 390-397.

18. Chang, J. and E.R. Waclawik, Colloidal semiconductor nanocrystals: controlled synthesis and surface chemistry in organic media. RSC Advances, 2014. 4(45): p. 23505-23527.

19. Marinin, A., Synthesis and characterization of superparamagnetic iron oxide nanoparticles coated with silica. 2012. p. 50.

20. Lam, T., et al., Superparamagnetic iron oxide based nanoprobes for imaging and theranostics. Advances in Colloid and Interface Science, 2013. 199–200: p. 95-113.

21. Malik, M.A., M.Y. Wani, and M.A. Hashim, Microemulsion method: A novel route to synthesize organic and inorganic nanomaterials: 1st Nano Update. Arabian Journal of Chemistry, 2012. 5(4): p. 397-417.

22. Kandasamy, G. and D. Maity, Recent advances in superparamagnetic iron oxide nanoparticles (SPIONs) for in vitro and in vivo cancer nanotheranostics. International Journal of Pharmaceutics, 2015. 496(2): p. 191-218.

23. Darroudi, M., et al., Superparamagnetic iron oxide nanoparticles (SPIONs): Green preparation, characterization and their cytotoxicity effects. Ceramics International, 2014. 40(9, Part B): p. 14641-14645.

24. Wahajuddin,Sumit Arora,Superparamagnetic iron oxide nanoparticles:magnetic nanopplatforms as drug carries,International Journal of Nanomedicine,5 July 2012

25. Sodipo, B.K. and A.A. Aziz, Recent advances in synthesis and surface modification of superparamagnetic iron oxide nanoparticles with silica. Journal of Magnetism and Magnetic Materials, 2016. 416: p. 275-291.

26. Jeon, H., et al., Poly-paclitaxel/cyclodextrin-SPION nano-assembly for magnetically guided drug delivery system. Journal of Controlled Release, 2016. 231: p. 68-76.

27. Mansouri, M., et al., Magnetic responsive of paclitaxel delivery system based on SPION and palmitoyl chitosan. Journal of Magnetism and Magnetic Materials, 2017. 421: p. 316-325.

28. Anwar, M., et al., Synthesis and in vitro localization study of curcumin-loaded SPIONs in a micro capillary for simulating a targeted drug delivery system. International Journal of Pharmaceutics, 2014. 468(1–2): p. 158-164.

29. Tewes, F., C. Ehrhardt, and A.M. Healy, Superparamagnetic iron oxide nanoparticles (SPIONs)-loaded Trojan microparticles for targeted aerosol delivery to the lung. European Journal of Pharmaceutics and Biopharmaceutics, 2014. 86(1): p. 98-104.

30. Janko, C., et al., Strategies to optimize the biocompatibility of iron oxide nanoparticles – “SPIONs safe by design”. Journal of Magnetism and Magnetic Materials.

31. Huang, Y., et al., Superparamagnetic iron oxide nanoparticles conjugated with folic acid for dual target-specific drug delivery and MRI in cancer theranostics. Materials Science and Engineering: C, 2017. 70, Part 1: p. 763-771.

32. A. Sabareeswaran, E.B. Ansar, P.R. Harikrishna Varma, P.V. Mohanan,T.V. Kumary,Effect of Surface-Modified Superparamagnetic Iron Oxide Nanoparticles(SPIONS) on Mast Cell Infiltration: An Acute In vivo Study,Nanomedicine: Nanotechnology,Biology, and Medicine (2016), doi: 10.1016/j.nano.2016.02.018

33. Sophie Laurent†, Amir Ata Saei, Shahed Behzadi, Arash Panahifar &Morteza Mahmoudi,Superparamagnetic iron oxide nanoparticles for delivery oftherapeutic agents: opportunities and challenges,Expert Opinion on Drug Delivery , May 2014 DOI: 10.1517/17425247.2014.924501

34. Kandasamy, G., Maity, D.,RECENT ADVANCES IN SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONs) FOR IN VITRO AND IN VIVO CANCER NANOTHERANOSTICS, International Journal of Pharmaceutics(2015),http://dx.doi.org/10.1016/j.ijpharm.2015.10.058

35. Miriam Colombo,wa Susana Carregal-Romero,wb Maria F. Casula,c Lucı´a Gutie´rrez, Marı´a P. Morales,d Ingrid B. Bo¨hm,f Johannes T. Heverhagen,f Davide Prosperi*a and Wolfgang. J. Parak*b,Biological applications of magnetic nanoparticles,: Chem. Soc. Rev., 2012, 41, 4306–4334

35. Anbarasu, M., Anandan, M., Chinnasamy, E., Gopinath, V., Balamurugan, K., 2015.Synthesis and characterization of polyethylene glycol (PEG) coated Fe3O4 nanoparticles by chemical co-precipitation method for biomedical applications. Spectrochim. Acta A Mol. Biomol. Spectrosc. 135, 536-539.

37. Juliano R: Nanomedicine: is the wave cresting? Nat Rev Drug Discov 2013, 12:171-172.

38.Duncan R, Gaspar R: Nanomedicine(s) under the microscope.Mol Pharm 2011, 8:2101-2141.

39. Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron oxide nanoparticles (SPIONs): Development, surface modification and applications in chemotherapy. Advanced Drug Delivery Reviews. 2011;63:24–46

40. LaVan DA, McGuire T, Langer R. Small-scale systems for in vivo drug delivery. Nature Biotechnology. 2003;21:1184–91

41. Vinogradov SV, Bronich TK, Kabanov AV. Nanosized cationic hydrogels for drug delivery: preparation, properties and interactions with cells. Advanced Drug Delivery Reviews. 2002;54:135–47.

42.Mahmoudi M, Milani AS, Stroeve P, Arbab SA. New York: Nova Science Publisher; 2011. Superparamagnetic Iron Oxide Nanoparticles: Synthesis, Surface Engineering, Cytotoxicity and Biomedical Applications. ISBN: 978-1-61668-964-3.

43. Mahmoudi M, Simchi A, Imani M, Milani AS, Stroeve P. Optimal design and characterization of superparamagnetic iron oxide nanoparticles coated with polyvinyl alcohol for targeted delivery and imaging. Journal of Physical Chemistry B. 2008;112:14470–81.

44. Mahmoudi M, Sahraian MA, Shokrgozar MA, Laurent S. Superparamagnetic iron oxide nanoparticles: Promises for diagnosis and treatment of multiple sclerosis. ACS Chemical Neuroscience. 2011;2:118–40.

45. Mahmoudi M, Amiri H, Shokrgozar MA, Sasanpour P, Rashidian B, Laurent S, Casula MF, Lascialfari A. Raman active jagged-shaped gold-coated magnetic particles as a novel multimodal nanoprobe. Chemical Communications. 2011;47:10404–6.

46. Mahmoudi M, Simchi A, Imani M. Recent advances in surface engineering of superparamagnetic iron oxide nanoparticles for biomedical applications. Journal of the Iranian Chemical Society. 2010;7:S1–S27.

47. Laurent S, Forge D, Port M, Roch A, Robic C, Vander Elst L, Muller RN. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chemical Reviews. 2008;108:2064–110

.

48. Febvay S, Marini DM, Belcher AM, Clapham DE. Targeted Cytosolic Delivery of Cell-Impermeable Compounds by Nanoparticle-Mediated, Light-Triggered Endosome Disruption. Nano Letters. 2010;10:2211–9

49. Mahmoudi M, Amiri H, Shokrgozar MA, Sasanpour P, Rashidian B, Laurent S, Casula MF, Lascialfari A. Raman active jagged-shaped gold-coated magnetic particles as a novel multimodal nanoprobe. Chemical Communications. 2011;47:10404–6.

50. Choi, S.Y., Kwak, B.K., Shim, H.J., Lee, J., Hong, S.U., Kim, K.A., 2014. MRI traceability of superparamagnetic iron oxide nanoparticle-embedded chitosan microspheres as an embolic material in rabbit uterus. Diagn. Interv. Radiol. 21, 47-53.

51. Stampfl, U., Sommer, C.M., Bellemann, N., Holzschuh, M., Kueller A., Bluemmel, J., Gehrig, T., Shevchenko, M., Kenngott, H., Kauczor, H.U., Radeleff, B., 2012. Multimodal visibility of a modified polyzene-F-coated spherical embolic agent for liver embolization: feasibility study in a porcine model. J. Vasc. Interv. Radiol. 23, 1225-1231.

52. Peng, M.L., Li, H.L., Luo, Z.Y., Kong, J., Wan, Y.S., Zheng, L.M., Zhang, Q.L., Niu, H.X., Vermorken, A., Van de Ven, W., Chen, C., Zhang, X.K., Li, F.Q., Guo, L.L., Cui, Y.L., 2015. Dextran-coated superparamagnetic nanoparticles as potential cancer drug carriers in vivo. Nanascale 7, 11155-11162

53. 161. Shi J, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in Drug Delivery and Tissue Engineering: From Discovery to Applications. Nano Letters. 2010;10:3223–30.

54. Hergt R, Andra W, D'Ambly CG, Hilger I, Kaiser WA, Richter U, Schmidt HG.

Physical limits of hyperthermia using magnetite fine particles. IEEE Transactions on Magnetics. 1998;34:3745–3754

55. Dudeck O, Bogusiewicz K, Pinkernelle J, Gaffke G, Pech M, Wieners G, Bruhn H, Jordan A, Ricke J. Local arterial infusion of superparamagnetic iron oxide particles in hepatocellular carcinoma: A feasibility and 3.0 T MRI study. Investigative Radiology. 2006;41:527–35.

56. Johannsen M, Gneveckow U, Eckelt L, Feussner A, Waldfner N, Scholz R, Deger S, Wust P, Loening SA, Jordan A. Clinical hyperthermia of prostate cancer using magnetic nanoparticles: Presentation of a new interstitial technique. International Journal of Hyperthermia. 2015;21:637–47.

57. Johannsen M, Gneveckow U, Taymoorian K, Cho CH, Thiesen B, Scholz R, Waldöfner N, Loening SA, Wust P, Jordan A. Thermal therapy of prostate cancer using magnetic nanoparticles. Actas Urol Esp. 2007;31:660–7