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2013 77 Ana Arizaga Páez Synthesis of core-shell magnetic nanoparticles for biomedical applications Departamento Director/es Física de la Materia Condensada Millán Escolano, Ángel Palacio Parada, Fernando Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Ana Arizaga Páez SYNTHESIS OF CORE-SHELL MAGNETIC NANOPARTICLES FOR BIOMEDICAL APPLICATIONS Director/es Física de la Materia Condensada Millán Escolano, Ángel Palacio Parada, Fernando Tesis Doctoral Autor 2013 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Synthesis of Core-Shell Magnetic Nanoparticles for Biomedical Applications Synthesis of Core-Shell Magnetic Nanoparticles for Biomedical Applications Ana Arizaga Páez A Jose Luís y Ainhoa Index Preface ..................................................................................................... 1 1 Introduction and goals........................................................................... 3 1.1 Advantages of MNPs in biomedical uses .......................................... 3 1.1.1 Magnetic resonance imaging (MRI) ........................................ 6 1.1.2 Magnetic hyperthermia ............................................................ 7 1.1.3 Biosensors ................................................................................ 8 1.1.4 Drug delivery ........................................................................... 8 1.1.5 Targeting .................................................................................. 9 1.2 Magnetic properties of nanoparticles............................................... 10 1.2.1 Superparamagnetism.............................................................. 12 1.2.2 Magnetic relaxation................................................................ 14 1.2.3 Specific absorption rate (SAR) .............................................. 15 1.2.4 Surface effects........................................................................ 16 1.3 Core-Shell structures of MNPs used in medicine ............................ 16 1.3.1 Materials for the core ............................................................. 18 1.3.1.1 Iron oxides: An introduction ........................................ 19 1.3.1.2 Synthesis of iron oxide MNPs...................................... 21 1.3.1.3 Strategies for the control of size in nanoparticles ........ 25 1.3.2 Materials for the shell............................................................. 26 1.3.2.1 Organic coatings........................................................... 27 1.3.2.2 Inorganic coatings ........................................................ 32 viii Index 1.4 Purpose of the thesis and goals ........................................................ 35 1.4.1 Ferrofluids for biomedical applications ................................. 35 1.4.2 Superparamagnetic beads for a biosensor.............................. 36 1.4.3 MNPs encapsulated in a polymer for drug delivery............... 37 1.5 Bibliography .................................................................................... 38 2 Experimental and methods.................................................................. 49 2.1 Introduction...................................................................................... 49 2.2 Experimental.................................................................................... 49 2.2.1 A survey of methods for the production of ferrofluids in organic media..................................................................... 50 2.2.2 Synthesis of IOMNPs in organic solvents by thermal decomposition of Fe(CO)5..................................................... 53 2.2.2.1 Procedure for the synthesis of organic nanoparticle suspensions............................................................................ 55 2.2.3 Ferrofluids in aqueous media................................................. 57 2.2.4 Procedure for the synthesis of aqueous MNPs suspensions... 61 2.3 Methods ........................................................................................... 61 2.3.1 Chemical analysis .................................................................. 61 2.3.1.1 Titration........................................................................ 61 2.3.1.2 Thermogravimetric analysis ......................................... 63 2.3.1.3 Gel permeation chromatography .................................. 63 2.3.1.4 1H-NMR ....................................................................... 64 2.3.2 Structural characterization ..................................................... 65 2.3.2.1 Fourier transform infrared spectroscopy (FTIR).......... 65 2.3.2.2 X-ray diffraction (XRD)............................................... 66 2.3.2.3 Dynamic light scattering (DLS) ................................... 67 2.3.2.4 Transmission electron microscopy (TEM) .................. 68 2.3.3 Magnetic characterization ...................................................... 69 2.3.3.1 Magnetic characterization SQUID ............................... 69 2.3.3.2 SAR .............................................................................. 70 Preface Nanoscience and nanotechnology are two related promising areas that take advantage of the novel properties that nanostructured materials exhibit. At the nanoscale the classical laws of physics are not able to explain some properties of materials, and the chemical behaviour differs from bulk materials of the same composition. This opens new opportunities to design and develop new functional materials in areas as chemistry, physics, biology, material science, electronics and medicine. Nanosystems are materials composed by different constituents with at least one of its dimensions at the nanoscale range. One of the most common structures of nanosystems are the core-shell materials, which comprehend single-core and multicore. Magnetic nanoparticles are of great interest for biomedical applications, and good candidates for being nanosystems cores. Maghemite is especially appealing because it exhibits superparamagnetic behaviour at nanoscale, is non-toxic and is less sensitive to oxidation than other magnetic particles as cobalt, nickel or iron. But it is also important to study the design of the nanosystems in order to optimize the desired characteristics for a chosen application. Normally these particles need to be coated to avoid agglomeration and degradation. The election of the material of the shell is usually based on the size of the particles and the environment they are to be used in. Furthermore, the functionalization of these core-shell systems can also be an extra benefit, so it is better to chose easily functionalized materials for the shell if the application requires it. In this thesis the production and characterization of aqueous and organic iron oxide nanoparticles and stabilized by different coatings for their application in nanobiomedicine are reported. 2 Preface In chapter 1, a brief introduction about biomedical applications of nanoparticles is reported. There are also described some especial magnetic properties that are observed in nanoparticulate materials that can be useful for the design of new core-shell materials for biomedical applications. Eventually, the purpose and goals of this thesis are exposed. The methods and the synthesis and characterization of aqueous and organic ferrofluids are described in chapter 2. These ferrofluids are mentioned later in other chapters because they are the former particles, and from them, several studies were performed and explained in chapters 3, 4 and 5. Chapter 3 is about how to obtain aqueous dispersions of high quality iron oxide nanoparticles synthesised in organic solvents. For this purpose, a ligand exchange technique is used, which consists on the substitution of the hydrophobic ligand used in the organic synthesis by a hydrophilic ligand. Chapter 4 describes the contribution to the design and development of a biosensor based on impedance measurements that are made in order to improve the signal. A multi-core-shell structure is functionalised with a siloxane ended in a carboxylic group. This group is able to bond an antibody that can interact with the capacitor of the biosensor, changing the signal. Chapter 5 describes a multifunctional multi-core-shell system that is able to combine the magnetic properties of the superparamagnetic nanoparticles of the core and the pH response of the polymer that forms the shell. This system can be used in biomedical applications with some modifications. Chapter 1 Introduction and goals 1.1 Advantages of MNPs in biomedical uses The use of magnetic nanoparticles (MNPs) in biomedicine began at the end of the 70s when they were employed as enzyme carriers (Magnogel, Dynabeads and Estapor) in bioanalysis [1, 2]. Since then, they have been used in many other biotechnological applications such as biocatalysis, bioprocessing, separation and purification. In biocatalysis, it is well known that homogeneous catalysts are more efficient than heterogeneous ones, but they are very difficult to remove from the medium after reaction. MNPs are very helpful for this task because when they are used as support for the catalysts they can be easily separated from the reaction medium with a magnet [3]. In bioprocessing, the magnetophoretic behaviour of MNPs can improve the mobility of a biosystem to perform bioseparation and target isolation under a continuous flow processing conditions [4]. For separation and purification, magnetic solid-phase extraction is a widely used technique. The typical procedure is to functionalize the target with MNPs that can interact with a magnetic adsorbent. Then, the target contained in the absorbent is recovered from the solution in a magnetic separator [5]. The physical principle behind these applications is simple: the magnetic particle is attached to a biological molecule (enzyme, cell, antibody, DNA, etc.) and the magnetic moment of the particle is used to move or fix the biomolecule with an external magnet. In the 80s, MNPs were commercially used for the first time in clinical applications, as contrast agents in Magnetic Resonance Imaging (MRI) [6]. This application is based on the effect of MNPs on the relaxation time of neighbouring water protons, especially on spin-spin relaxation time t2 [7, 8]. 4 Chapter 1. Introduction and goals An additional and interesting application of MNPs already in the market is biosensing [9]. It is important to highlight the importance of magnetic nanoparticles for detection of biomolecules and cells based on magnetic resonance [10]. Baby and col. fabricated an amperometric biosensor by deposition of glucose oxidase over a Nafion-solubilized Fe3O4@SiO2 electrode, which retains its biocatalytic activity and offers fast and sensitive glucose quantification [11]. Another exciting clinical application of MNPs is found in hyperthermia cancer therapy [12]. When superparamagnetic nanoparticles are exposed to an alternating magnetic field, they produce heat that can be used to heat cells over 4245ºC causing cellular death. Tumour cells are more sensitive to heat than healthy cells so it is expected to achieve a higher population of death tumour cells [13]. Other biomedical use of MNPs that has been further developed in the last decade is targeted drug delivery. Systemic disease treatments require a large drug doses to achieve high local concentrations that produces undesired side effects elsewhere. This can be avoided with drug targeted administration. In magnetic targeting, the drug is attached to a MNP than can be directed to the desired zone with a magnetic field. This system can be reinforced by biological specific vectors implemented in the surface of MNPs that are capable of specific recognition and binding to the target site [14]. Recently, a most promising area of application of MNPs in medicine has emerged, namely theranostics, which is based in performing therapy and diagnosis simultaneously [15]. Besides their unquestionable biomedical interest, MNPs have a large variety of industrial applications such as polymer processing through homogeneous heating or selective heating, soldering or glue-welding procedures, magnetic recording, magnetic refrigeration, magnetic printing, lubrication and sealing in vacuum systems, magnetic sensors, and others [16]. Nanoparticulate materials open a new opportunity to study at the molecular and cellular levels. Therefore, promoting fast advances in life sciences and healthcare 1.1 Advantages of MNPs in biomedical uses 5 because they are capable to pass barriers that bigger systems cannot. Small size is a great advantage of NPs, although not the only one arising from their unique properties with respect to bulk materials, which can be even more important for biomedical applications. One of these properties of MNPs is superparamagnetism. When a magnetic field is applied, the MNPs give a magnetic response, but when the field is removed, no remanent magnetization is observed. This is very important in order to avoid magnetic agglomeration when the influence of the magnetic field is taken away. The magnetic response of nanoparticles strongly depends on their morphology, crystallinity and size, therefore controlling these characteristics is of great interest to have a fine control on the MNPs properties. Most biomedical applications require specific MNPs characteristics. Ideally and for optimum use, nanoparticles of homogeneous size and uniform shape are desired. It would be interesting to have a procedure that permits the production of controlled nanoparticle diameter in the order of nanometers. Good crystallinity and phase control are also desirable tunable properties. MNPs also must have good thermal stability, biocompatibility, and an adequate magnetic moment. It is important as well that they form stable dispersions in biological fluids, such as blood. MNPs can be coated with biomolecules in order to increment the residence time in the blood circulation systems or to make them interact with a cell or a biological entity. As we mentioned before, in order to design a potential biosystem it is very important to take into account the requirements of the applications it is conceived for. Each application needs different features from the magnetic nucleus and from the coatings to obtain the optimum response of the system, as it will be commented in the following sections. 6 Chapter 1. Introduction and goals 1.1.1 MRI In recent years, medical imaging research has experienced immense improvements with the introduction of techniques such as Magnetic Resonance Imagine (MRI). In this technique, contrast agents are often needed in order to improve the diagnosis. Magnetic nanoparticles systems are good candidates for this purpose, especially, superparamagnetic iron oxide nanoparticles [17]. Superparamagnetic iron oxide nanoparticles modify the relaxation time of water protons changing the contrast image intensity of the tissue where they are present [18]. In order to avoid particle aggregation and improve stabilization, the iron oxide nanoparticles are covered by a coating. This coating may also be used to facilitate the distribution of the particles in the tissue and to add new functionalities to the system as described below. Some requirements are needed to create an ideal contrast agent system, for example, the system has to be homogeneous in size because it is important to get a uniform distribution in the tissue, and the hydrodynamic radius has to be small in order to get long blood circulation time. It is also important to control the magnetic properties. In order to be superparamagnetic, the particle size has to be smaller than 2530 nm. Moreover, the particle morphology has to be homogeneous, and the size distribution must be narrow, to achieve consistent results. The surface properties are also relevant for this application. It has been shown that surface modification with hydrophilic molecules such as PEG increases circulation times. An additional advantage of PEG coating is that it enables particles to cross cell membranes because PEG is soluble in both polar and non-polar solvents and it has high permeability in cell membranes [19]. Another possible coating is silica as shown by Taboada et al., who presented a system composed by monodisperse iron oxide and microporous silica core/shell nanoparticles of around 100 nm in diameter. This system has a high magnetization and may be particularly useful as an enhanced T2 imaging agent [20]. 1.1 Advantages of MNPs in biomedical uses 7 1.1.2 Magnetic hyperthermia The amount of heat released by MNPs under an alternating magnetic field is a promising tool for biomedical applications based on hyperthermia [21] and on thermally assisted drug delivery [22]. The use of superparamagnetic nanoparticles for hyperthermia purposes was first introduced by Jordan et al. in 1993 [23]. Further studies have demonstrated that magnetic hyperthermia could be an alternative to current therapeutic approaches for cancer treatment, by inducing tumour regression in combination with other toxic agents, or by causing necrosis of cancerous cells [24-26]. Hyperthermia is also producing a strong enhancement of radiation damage. For instance, a local heating of 43.5ºC for 1 hour yields an approximate enhancement ratio of 5 [27]. According to Rosenzweig, a ferrofluid subjected to an alternating magnetic field produces a power dissipation that comes from the orientational relaxation of particles having thermal fluctuations in a viscous medium [28]. From the point of view of the study of hyperthermic behaviour of magnetic iron oxide nanoparticles some work has still to be carried out in order to confirm Rosenzweig theoretical predictions. The heating capacity of MNPs is quantified by the specific absorption rate (SAR), which accounts for the heating power per mass unit of dissipating material. This magnitude depends on the MNPs characteristics, such a phase composition, particle shape, magnetic anisotropy, mean size and size distribution, as well as on the alternating magnetic field parameters, such as amplitude and frequency [29]. With respect to the particle size, Rosenzweig reported a very sharp maximum of SAR at 14 nm in case of magnetite nanoparticles. For the case of maghemite and magnetite nanoparticles, widely used due to their biocompatibility, small particles in the order of 1015 nm in diameter are desired because they lie well below the critical size of single domain particles. In this size range, the heat dissipation is due to the thermal relaxation of magnetic moments [29], the heating power decreases strongly with the size dispersion [28]. The heating capacity is greatly influenced by nanoparticle environment, and the degree of particle aggregation. Therefore, a protecting nanoparticle coating is highly important for hyperthermia applications. 8 Chapter 1. Introduction and goals 1.1.3 Biosensors Sensing was the first application of magnetic nanoparticles in biomedicine and this area continue to rapidly evolve with very important developments and future perspectives in this area. An example is the work of Han et al., who have reported extensively on the use of functionalized magnetic microand nanoparticles for biosensing. These authors have proposed a sensing system based on the absorption of magnetic particles by hybridized DNA that alters the sensor resistance and generates electrical signals that can be directly measured. In this system, the cells are first treated with a biotinylated primary antibody or ligand. Then, they are magnetically labelled with streptavidin coated magnetic beads. Under an electromagnetic field, the magnetic labelled cells are retained in a column containing a magnetically soft material, whereas the unlabeled cells will just flow through [30]. Another effective material for biosensing is that composed by core/shell iron oxide MNPs coated with gold as is reported by Wang et al. [31]. This system has been used as a solid support for goat anti-human antibody IgM, which could be immobilized on the surface of a surface plasmon resonance biosensor. 1.1.4 Drug delivery Nowadays nanobiosystems are of great interest in drug delivery. Novel nanoparticles are designed to alter their structure and properties during the drug delivery process to make them most effective for distribution [32]. This alteration can be achieved through the incorporation of materials that are able to respond to physical or biological stimuli, including changes in pH, redox potential or enzymes. The idea of employing MNPs for drug delivery was proposed by Widder and Senyei in 1978 [33, 34]. The basic argument is that therapeutic agents are encapsulated or attached to MNPs. These particles may have magnetic cores protected with a stabilizing material like polymers or other kind of coatings which can be functionalized. They may also consist of porous polymers containing MNPs precipitated within the pores [35-37]. Advantages of the use of these devices are conjugation with biological ligands and also the possibility to increase the circulation time of MNPs in the blood stream [38]. 1.1 Advantages of MNPs in biomedical uses 9 1.1.5 Targeting Ideally, the treatment of local diseases should be local therapy, but when the disease problem is inaccessible to common local treatments, a systemic treatment is often used. This requires the administration of large amounts of drugs with the consequent side effects. The use of drug targeting allows reduction of drugs quantities that diminishes the side effects and increases the treatment efficacy. Although magnetic targeting has been successful in a number of studies, there are only a small number of clinical trials. Lubbe et al. carried out a clinical study in mice and rats where two forms of treatment were studied [39]. Epirubicin, a well-know drug that is widely employed for chemotherapy, was attached to MNPs by electrostatic interaction, and the mechanical occlusion of the tumour with high concentrations of ferrofluid was studied as well as the targeted delivery of epirubicin with low nanoparticle concentration. As in many other related in vivo studies, the particles not attached to the tumour were accumulated in the liver and spleen and not harmful effects on organ functions were observed. Novel strategies are being developed for applying magnetic fields and MNPs which could lead to new treatments. Rapid developments in particle synthesis have enabled the use of new materials for more efficient capture and targeting. In order to design an efficient biosystem, it has to be taken into account that the nanoscale dimensions of particles should allow them not only to pass through the blood vessels but also to penetrate cell membranes when necessary [40]. Chemotherapeutic agents require internalization and slow drug release, gene therapy demands positive interaction with the nucleus, radiotherapeutic systems requires cellular internalization. The maximum size that can be used for a biosystem is 1.4 µm in diameter to avoid capillary occlusion. A great number of chemical methodologies have been used for the conjugation of targeting molecules with the biosystem surface. The primary goal is to bind the targeting molecule without compromising its functionality once attached. For example, if an antibody is bonded to the particle surface and its active site is covered, 10 Chapter 1. Introduction and goals it may loose its capacity to bind a target [41]. Some of the most interesting candidates for being targeting molecules are tumoral markers for brain and gene therapy, such as siRNA or Tat that facilitate intracellular delivery [42]. Another interesting possibility of targeting consists of magnetic targeting. In magnetic targeted drug delivery, the systems formed of coated MNPs loaded with anti-cancer drugs are injected into the body via the blood circulatory system. An external magnetic field is used to localize the biosystem at the tumour site and the drug can then be released from the system via enzymatic activity or changes in physiological conditions such as pH or temperature and be taken up by tumour cells [43]. Alexiou et al. showed that magnetic targeted drug delivery caused complete tumour remission in rabbits without any negative side effects and allowed a drug dose reduction of 20% of the usual dose [14]. 1.2 Magnetic properties of nanoparticles All materials can be classified by their magnetic response to an external magnetic field. This response is related to the magnetic interactions of the constituent atoms and the crystalline structure of the material [44]. The main types of magnetism include diamagnetism, paramagnetism and ferromagnetism. Antiferromagnetism and ferrimagnetism are considered to be subclasses of ferromagnetism. In Figure 1.1, it can be observed the different possible orientation of magnetic moments. Below a certain magnetic ordering temperature, ferromagnetic materials exhibit parallel alignment of permanent magnetic moments resulting in a large net magnetization that can remain in the absence of a magnetic field. Two characteristics, albeit not exclusive, of ferromagnetic materials are their spontaneous magnetization and the existence of hysteresis. In ferrimagnets, the moments of adjacent atoms or ions are in an antiparallel alignment, but they do not cancel to each other. 1.3. Core-Shell structures of MNPs used in medicine 17 This requirement restricts severely the type of compounds used for the magnetic nucleus and the shell. Figure 1.5 Components of biological systems. From this general scheme, the structure and size of MNPs can vary depending on the desired application. Thus MNPs for "in vitro" applications in biosensors or in magnetic separation must have a size large enough to reach a sufficient magnitude for the magnetic moment or to be confined in liquid permeable chambers. In these applications, the size of the magnetic nucleus is usually in the range from 10 nm to 50 nm in diameter and the total size is between 0.1 µm and 10 µm. However, in in vivo applications the size must be small enough to permeate biological barriers, and therefore typical sizes of MNPs for MRI are 2 to 50 nm for the core and 7 to 400 nm for the entire particle. 18 Chapter 1. Introduction and goals For instance, in magnetic hyperthermia therapy, only MNPs in a narrow size range contribute to the SAR for a given frequency of the alternating field. Other relevant structural factors are: internal structural disorder [1], aggregation [60], and interparticle separation [61]. In order to find an optimum magnetothermal behaviour, it is necessary to have a system in which these factors can be varied independently, while keeping a narrow size distribution. 1.3.1 Materials for the core The most common magnetic materials are composed by Fe, Co, Ni, their oxides and their alloys. Actually, the minerals magnetite (Fe3O4) and maghemite (  -Fe2O3) are typical examples of ferrimagnetic materials. It is well known that maghemite and magnetite are biocompatible materials [62]. Maghemite has the advantage over magnetite that it is more stable because it is the oxidized form of magnetite. So among the magnetic compounds,  -Fe2O3 is the one that provides the best expectations for use in living organisms together with a fine magnetic performance. In fact, there are many iron oxide-based particles in the market for biological applications. For example, superparamagnetic contrast agents used for MRI consist of maghemite-magnetite cores encapsulated in a polisacharide of dextran family or other coatings like Endorem (nanoparticles of 4-15 nm in diameter coated with dextran, with a total hydrodynamic diameter of 150 nm), Sinerem (nanoparticles of 4-15 nm coated with dextrane, hydrodynamic diameter: 30 nm), and MION-46 (nanoparticles in the order of the 20 nm of hydrodynamic diameter coated with dextrane) [63]. Apart from iron oxide nanoparticles, other magnetic nanoparticulate materials have been employed in biomedical applications. Recently, gadolinium hexanedione nanoparticles of about 140 nm in diameter, fabricated in microemulsions [64] have shown greater image enhancement ability than commercial gadolinium molecular products, and they are non-toxic for human stem cells. MnxZn11-xFe2O4 nanoparticles have been investigated as hyperthermia agents with the aim to improve heating temperature, SAR and biocompatibility [65]. Also in this field, monodisperse metastable Fe-Ni MNPs, synthesized by chemical reduction, showed tuneable Curies 1.3. Core-Shell structures of MNPs used in medicine 19 temperatures. This is particularly important because it opens the possibility of selfregulated heating of cancer cells, as the Curie temperature sets an upper limit to heating preventing the damage to neighbouring healthy tissue [66]. Magnetic nanorods composed of Ni and Au, synthesized by electrodeposition into a porous alumina membrane, have been proposed for biomolecular separation [67]. Moreover, biofunctionalized FePt MNPs (3-4 nm) have been successfully used for rapid detection of gram-positive bacteria at very low concentration [68]. Therefore, we can conclude that there is good variety of magnetic nanomaterials susceptible for being employed in biomedical applications. In the work described here and in spite of the moderate magnetic performance of iron oxide MNPs, we decided to use them because of their biocompatibility advantage over other materials. 1.3.1.1 Iron oxides: An introduction Iron oxides are the result of iron metal in contact with the atmosphere, which contains oxygen. So, iron oxides exist in the earth as long as both iron and oxygen have been on the planet. Iron oxides present a large variety of crystal phases. There are 16 known iron oxides, hydroxides and oxide-hydroxides [69]. Among them, only magnetite (Fe3O4) maghemite (-Fe2O3) and lepidocrocite (-FeOOH) are ferrimagnetic, the rest being antiferromagnetic or weakly ferromagnetic. Magnetite is black, ferrimagnetic, and contains both Fe(II) and Fe(III). The structure is an inverse spinel, and it was one of the first minerals being studied by XRD. It has a face-centered cubic unit cell with an edge length of a = 0.8394 nm, and 8 formula units. The formula can be written as Fe(III)[Fe(II)Fe(III)]O4, and the structure can be seen as an inverse spinel where octahedral (Oh) sites are occupied by both Fe(II) and Fe(III) ions, whereas tetrahedral (Td) sites are occupied by Fe(III), see Figure 1.6. This mineral is frequently non-stoichiometric with a deficient Fe(III) sublattice. 20 Chapter 1. Introduction and goals Figure 1.6 Crystalline structure of magnetite (left) and maghemite (right). Maghemite is brown-red, ferrimagnetic, and all of the iron ions in the structure are trivalent. The structure is also a inverse spinel, thus, to compensate the higher charge in the structure, cation vacancies are created. Each cell of maghemite contains 32 O2ions, 21 Fe(III) ions and 2 vacancies. The 8 Fe cations occupy Td sites and the other are distributed in the Oh sites, as shown in Figure 1.6. The vacancies are confined in the Oh sites. The unit cell edge is 0.834 nm, slightly shorter than that of magnetite. The oxidation of magnetite takes place by migration of Fe(II) cations from the inside to the outside of the particle, creating the vacancies (□). Then, Fe(II) cations are oxidized to Fe(III) at the surface generating maghemite [70]. The resulting formula structure can be written as follows: [Fe8 3+]T d[Fe8 3+Fe8 2+]Oh O32 → [Fe8 3+]T d[Fe8 3+Fe3+ 5.33□2.66]Oh O32 In the case of magnetite, Fe(III) ions are equally split between Oh and Td, they are coupled antiferromagnetically, and hence do not contribute to the magnetic moment. The magnetic moment of magnetite is therefore due to the contribution of Fe(II) ions occupying Oh sites, which have a moment of 4 µB. The sublattice of Fe(III) in the Td sites has magnetic moment of 40 µB (8 cations x 5 µB) and the sublattice made of Fe(II) and Fe(III) in Oh sites has 72 µB (40 µB from Fe(III) and 32 µB Fe(II)). The total magnetic moment of the two combined sublattices is 32 µB. The saturation 1.3. Core-Shell structures of MNPs used in medicine 21 magnetization of bulk magnetite is 84 emu/g at room temperature, and the Curie temperature is 850 K. [Fe3+ ↑]T d [Fe2+ ↓, Fe3+ ↓]Oh O4 In maghemite the magnetic structure consists on two sublattices corresponding to the Fe(III) located on Td and Oh sites. The spins in each sublattice align ferromagnetically, but the spins between the sublattices align antiparallel. Ferrimagnetism arises from decompensation between the number of Fe(III) cations in each sublattice. The sublattice that consist on Fe(III) in Td sites is 40 µB and for Oh sublattice is 66.7 µB (40/3 cations in Oh sites). The total magnetic moment is 26.7 µB. The saturation magnetization of bulk maghemite is 74 emu/g at room temperature, and the Curie temperature is in the range of 820986 K. Maghemite has a phase transition to hematite at 800 K. [Fe3+ ↑]T d [Fe3+ 5/3 ↓, □1/3]Oh O4 1.3.1.2 Synthesis of iron oxide MNPs The preparation of iron oxide nanoparticles can be realized through bottom-up or top-down methods. The top-down approach is based on solid phase procedures. In the first known method for the production of iron oxide nanoparticles, large iron oxide materials are broken down to smaller particles by means of mechanical milling. But this method has a long preparation time and generates very high size dispersion [71]. In a bottom-up process, small building blocks such as atoms or clusters are assembled into nanoparticles. This approach includes chemical synthesis in solution and gasphase routes [72, 73]. Various synthetic strategies for the preparation of MNPs have been investigated, including chemical co-precipitation [74], microwave heating [75], microand nanoemulsion [76], sol-gel [77], hydrothermal routes [78], high temperature decomposition [79], sonochemical reactions [80], and laser pyrolysis [81]. Emulsion methods use two immiscible liquids, an organic solvent and an aqueous solution, in which small droplets are formed. The dispersion is achieved by 22 Chapter 1. Introduction and goals mechanical mixing that forms a non-stable droplet dispersion, however the addition of surfactants improves both stability and homogeneity. Organic and aqueous solvents often contain several dissolved components that allow nanoparticles formation, consequently this system behaves as a microor nano-reactor, depending on the droplet size. The temperature and surfactant concentration permit size and shape control of the obtained nanoparticles because they control the droplets size. This procedure is inexpensive and rapid but cumbersome for large-scale production [76]. The co-precipitation procedure consists on the treatment of a mixture of different metal precursors with a base in an aqueous medium. Complete precipitation can be obtained at a pH between 8 and 14. The main advantage of co-precipitation is that a large amount of nanoparticles can be synthesized. However the control of particle size distribution is limited because kinetic factors are the only controlling factor of the growth process [74]. Microwave co-precipitation allows a fast heating of the reaction mixtures, especially those containing water, so the formation of MNPs occurs almost immediately. This leads to very small particle sizes and narrow size distributions. This method offers the additional benefit of very short reaction times [75]. The main drawback is agglomeration, so an additional size-sorting process is needed. As in microwave heating processes, sonication of a liquid also produces rapid heating, but the precipitation mechanism goes through acoustic cavitation, which is the formation, growth, and collapse of a bubble in an irradiated liquid. This generates a transitory localized hot spot, with an effective high temperature (more than 700ºC) and a short lifetime (less than 1s). The chemical reactions take place inside the bubbles. One advantage is that no surfactant is used. The disadvantages are that amorphous materials are always obtained whenever a volatile liquid is used, and cooling rates are large. Nevertheless, the sonochemical decomposition of metal carbonyls in alkene solvents has been widely used to prepare iron metallic nanoparticles [80]. 1.3. Core-Shell structures of MNPs used in medicine 23 Hydrothermal reactions are performed in reactors that permit high pressure and temperature conditions. Most of the solvents in these methods are polar, such as water, methanol or isopropanol, but also other organic solvents have been used. Two main chemical routes are employed to obtain iron oxide nanoparticles in hydrothermal methods: hydrolysis/oxidation, and neutralization of mixed metal hydroxides [78]. In the well-known polyol process, a metallic precursor is dissolved in polyethylenglicol or pyrrolidone solvent and the solution is stirred and heated to reach the boiling point to get metal nanoparticles that are later oxidized. By controlling the precipitation kinetics, well-dispersed iron oxide MNPs with well-defined shape and size can be obtained [82]. In liquid aerosols routes, particles are obtained by spraying a solution into a series of reactors where the aerosol droplets evaporate. If the key step is drying, the technique is called aerosol evaporation, and when a thermolysis process is involved, it is called spray pyrolysis. Most of the pyrolysis techniques used to produce iron oxide nanoparticles start with an Fe(III) salt in a solution containing an organic reducing agent. This method is simple, rapid and continuous, but the obtained particles are often difficult to coat [81]. Sol-gel methods consist on the hydrolysis and condensation of alkoxide-based precursors at low temperature. This method starts from a chemical solution (sol) that acts as the precursor for an integrated network (or gel) of discrete particles. Typical precursors are metal alkoxides, such as tetraethyl orthosilicate (TEOS) or alkoxysilanes, and metal chlorides, which undergo a series of hydrolysis and condensation reactions in aqueous media. One of the advantages of sol-gel methods is the low reaction temperature that enables small particles to be grown. The main drawback is that the resulting particles are not homogeneous in size and shape [77]. The method allowing higher level of monodispersity and size control in iron oxide nanoparticles production (see Figure 1.7) is probably thermal decomposition of iron organic precursors, such as Fe(Cup)3, Fe(CO)5, or Fe(acac)3, in organic solvents, and in the presence of surfactants. The particle diameter can be tuned from 4 to 20 nm, 24 Chapter 1. Introduction and goals and the hydrophobic particles can be transformed into hydrophilic ones by adding a bipolar surfactant [79] and [83]. However, this process must be improved, especially in terms of reactants safety and high temperatures required, in order to be suitable for industrial preparation. Figure 1.7 Iron oxide nanoparticles obtained by thermal decomposition of metallorganic precursors. Nanoparticles growth in confined spaces is another methodology to achieve size control in nanoparticles. Superparamagnetic nanoparticles can be obtained by controlling the gelation time of a mesoporous material and an iron oxide precursor [84]. Mixtures of TEOS and alcoholic solutions of iron nitrate generate iron oxide nanoparticles homogeneously dispersed in the silica matrix [85]. Polymers can act as confined spaces as well, and the synthesis of tuneable size nanoparticles can performed using these conditions [86-88]. Reverse micelles are shown to be a suitable procedure to achieve nanoparticles with a tailored size, which can be controlled by modifications in the reaction temperature, the concentration of the precursor, or the water:surfactant ratio [89-91]. 1.3. Core-Shell structures of MNPs used in medicine 25 1.3.1.3 Strategies for the control of size in nanoparticles In order to obtain MNPs, direct precipitation methods are of great interest because a certain control of particle size can be achieved by adjusting the conditions of reaction. In batch precipitation systems, the supersaturation decreases as nucleation and growth occurs, and so does the growth rate, thus limiting the maximum size. Low solubility, fine mixing of reactants and temperature are also factors that may help to control the size and size dispersion in these systems. A typical example of direct precipitation method is the popular Massart method. There, iron oxide nanoparticles are precipitated by addition of a base to a solution of Fe(II) and Fe(III) salts in a 1:2 ratio, at a pH between 8 and 9.6. The size varies with the strength of the precipitating base [92], so that a stronger base leads to larger particles (NH3: 6 nm, CH3NH2: 10 nm, NaOH: 19 nm). This strategy is not sufficient to ensure a narrow size distribution that is usually in between 20% and 30%. A higher control can be obtained with the ratio of reactants in the reaction medium. This is very efficient in organic solution precipitation, but it is far less effective in aqueous media due to the strong solvation capacity of water. The size distribution can be further narrowed by size-sorting procedures, such as centrifugation [93], size-selective precipitation [95], magnetic separation [95], field-flow fractionation and size-exclusion chromatography [96]. The size dispersion associated to direct precipitation methods can be largely avoided by the use of additives that adsorb on the particle surface and inhibit particle growth and aggregation. This is especially the case of surfactants that have a part that binds to the particle surface and another part with a high affinity for the solvent. So they serve as growth restrainers and colloidal stabilizers at the same time. They are widely used to control the average particle size and size distribution in chemical synthesis routes. The surfactant nature and its concentration are important parameters that have an influence in particle size. Turning back to metal organic decomposition methods, the interaction between Fe atoms and the functional group of the surfactant produces small particles when this interaction is strong. The group of Cheon made a comparative study with two surfactants, trioctylphosphine (TOPO) and dodecylamine (DDA) [97]. The alkylamine ligand used in this study seems to bind weakly to the 26 Chapter 1. Introduction and goals metal centers on the surface of nanocrystals, while the TOPO ligand forms a much stronger bond to the crystal surface due to its high oxophilicity. A weakly binding ligand can reversibly coordinate to the metal sites on the surface, and further growth is possible when sufficient amounts of alkylamine ligand are available. The Fesurfactant interaction determines the decomposition temperature and the number of nucleus. The molar ratio surfactant/precursor is also important, the size increases as the quantity of surfactant increases, as it was shown in a study of Yu et al. [98]. They have reported that when the ratio moves from 1:3 to 1:8 the particle size becomes 4 times greater. However, there is an upper size limit that cannot be overcome by varying the solvent temperature, the concentration of surfactants and the concentration of precursor. In order to increase the size limit Hyeon et al. proposed a variation of the method that consisted on using an iron oleate complex as the growth source. In this way, they synthesized monodisperse iron nanoparticles of 20 nm with controlled size by the additional incremental growth of the previously prepared nanoparticles, in a seed mediated process [99]. The same group has obtained nanoparticles of a diameter of 50 nm using FeCl3 as iron precursor and sodium oleate [100]. 1.3.2 Materials for the shell The stabilization of the iron oxide MNPs in aqueous solutions and organic media is crucial to obtain magnetic colloidal ferrofluids that are stable against aggregation even under the presence of a magnetic field. The stability of a magnetic colloidal suspension can be controlled reaching the equilibrium between attractive and repulsive forces. Four kinds of forces can contribute to the interparticle potential in the system. Van der Waals forces induce strong short-range isotropic attractions. The electrostatic repulsive forces can be partially screened by adding a salt to the suspension. Moreover, steric repulsion forces have to be taken into account. For magnetic suspensions, magnetic dipolar forces between two particles must be added. Because of the growing interest of in the use of iron oxide nanoparticles for biomedical applications many efforts have been employed to render them stable in biological fluids. As we mentioned before, the method of precipitation from organic solutions is the most adequate to obtain samples with good size control, narrow size distribution 1.3. Core-Shell structures of MNPs used in medicine 33 Silica can be obtained by a large variety of methods. The most common involves exposing silicon to oxygen. When silicon is exposed to air under ambient conditions, a very thin oxide layer is formed on the surface. In order to grow well-controlled layers of silicon dioxide, higher temperatures and alternative environments are used. However, the most interesting method to synthesize silica is that of sol-gel. This method is a wet chemical route; the sol evolves gradually towards the formation of a gel network containing both a liquid phase and a solid phase. In basic solutions, the particles may grow to sufficient size to become colloids. Then the sol evolves towards the formation of a phase gel. In the case of the colloid, the number of particles in an extremely dilute suspension may be so low that the removal of a significant amount of solvent may be needed for the gel properties to be recognized. This can be accomplished in a number of ways. The simplest method is to allow time for sedimentation to occur, or by centrifugation to separate the solid from the solvent. The removal of all the solvent requires drying. Chemically, the synthesis of SiO2 involves hydrolysis and condensation of a precursor, such as tetraethyl orthosilicate (TEOS), in an appropriate solvent, such as ethanol or water, with or without the use of a catalyst. SiOEt)4 + H2O + OH- → (OEt)3Si(OH) + ROH (OEt)3Si(OH) + H2O → SiO2 + 3EtOH As these reactions proceed, a network of Si-O-Si bonds is formed and the viscosity of the solution increases due to the gel formation. Finally, the gel condenses and the particles precipitate. Many efforts have been dedicated to obtain silica coated MNPs using this approach. The group of Philipse has prepared them by precipitation of sodium silicate followed by hydrolysis of tetraethylorthosilicate (TEOS) in basic conditions [136]. Another strategy to obtain well coated nanoparticles is microemulsion. Yang et al. achieved this and went a bit farther by entrapping biological molecules inside the porous of the silica shell [137]. Aerosol methods also provide good results in magnetic nanoparticle silica encapsulation, as Tartaj et al. have demonstrated [138]. Yi et al. 34 Chapter 1. Introduction and goals prepared a soluble hybrid material consisting of MNPs and silica by a modified Stöber method [139]. The Stöber method for the preparation of monodisperse spherical silica particles, see Figure 1.9, in basic solution was published in 1968 [140]. Figure 1.9 TEM micrograph of silica particles obtained by the Stöber method. The use of silica as a coating permits also the tuning of the magnetic properties of the MNPs [141]. Besides, another advantage of silica is the presence of the silanol groups on the surface that can easily react with a wide variety of ligands carrying a diversity of functionalities. In this way, enzymes, proteins and other molecules that are useful for different biomedical applications can be anchor to the particle surface. Furthermore, besides surface functionalization, this material can be produced with a mesoporous structure, i.e. MCM-41 or SBA-15, with a pore size large enough to house MNPs and/or biological molecules [142]. These materials are amorphous but show a hexagonal arrangement of pores with a size that can be varied by adjusting the synthesis parameters. The group of Yang has used materials of this kind, SBA-15, to transport drugs such as ibuprofen [143]. Even though silica offers many potential advantages as a coating of MNPs, it also represents a controversial issue concerning the use of silica in biomedical applications with regard to toxicity. Some studies have found negative toxic effects of silica in living organisms; they have proven that silica nanoparticles cause lung fibrosis in rats, and toxic effects and alterations in the protein expression in HaCaT cells [144, 145]. 1.3. Core-Shell structures of MNPs used in medicine 35 However, others contrast these findings, for example, a study on this subject has been presented by Rondeau et al. [146], they concluded that it is indigestible, without nutritional value, but no toxic. When silica is ingested orally, it passes unchanged through the gastrointestinal tract, leaving no trace behind. Actually, a study on volunteer persons over 15 years showed that high levels of silica in water appeared to decrease the risk of dementia. This study found that with an increase of 10 milligram per day of the intake of silica in drinking water, the risk of dementia dropped by 11%. 1.4 Purpose of the thesis and goals The work presented in this thesis can be classified in three main areas: (1) monodisperse ferrofluids for biomedical applications; (2) superparamagnetic beads for a biosensor; and (3) MNPs encapsulated in a polymer for drug delivery. All of them share the same fundamental perspective, which is to control the synthesis and properties of functionalized nanoparticles based on their magnetic properties. Each area of research is summarized in the following sections and a detailed discussion is presented in the following chapters. 1.4.1 Ferrofluids for biomedical applications Our main objective in this area was to obtain aqueous dispersions of high quality iron oxide nanoparticles with a coating that could be easily functionalised and used in biomedical applications. Therefore, the nanoparticles were produced in organic solvents by thermal decomposition of organometallic precursors. Then these nanoparticles were transferred to aqueous medium. Finally, they were coated with a silica shell. For this purpose, we have followed a ligand exchange technique that consists on the substitution of the hydrophobic ligand used in the organic synthesis by a hydrophilic ligand. The exchange ligand was a compound consisting on a hydrocarbon chain having a silica precursor at one end and a group with a strong affinity for the surface of iron oxide particle, namely an iron coordination group. The silica precursor group was always triethoxysilane. Several iron coordination groups have been comparatively studied in this work: imidazole, phosphate and carboxylate. 36 Chapter 1. Introduction and goals The length of the hydrocarbon chain has been also systematically varied. Stable aqueous dispersions have been obtained with both short and long chain lengths. For short chain length, the best performance was obtained with N-(3-triethoxysilylpropyl)- 4,5-dihydroimidazole and its imidazole iron coordination groups. Finally, in order to improve the biocompatibility of the nanoparticles, some of the samples were successfully covered with polyethylenglicol by reacting the silica coated nanoparticles with a PEG-alkoxysilane derivative. 1.4.2 Superparamagnetic beads for a biosensor A second thrust of research in this dissertation involves the contribution to the development of a biosensor based on the changes in the impedance produced by iron oxide nanoparticles encapsulated in silica spheres when they interact with the capacitor plates. As we mentioned before, iron oxide nanoparticles obtained by the thermal decomposition method have better qualities than those obtained in aqueous media, but depending on the application for what they are desired it can be better to use the nanoparticles synthesized in aqueous media. After careful consideration and focusing on the final application, we synthesized MNPs by an aqueous method and subsequently we encapsulated them in silica spheres by a modified Stöber method. Afterwards, the nanospheres were functionalized with a siloxane that ends in a carboxylic group. Then, the spheres were attached to the capacitor plate surface by the sandwich technique [147]. In parallel, a plate was coated with a capture antibody, the same antibody that we have used to functionalize the nanosphere and was bonded by the carboxylic group. Then, an antigen was added to the suspension of nanospheres that binds to the antibody. The interaction of the system with the capacitor plates was investigated by means of an Enzyme-Linked Immuno Sorbent Assay (ELISA). In addition, the measurement of the impedance of the capacitor before and after the treatment has permitted us to determine the sensibility of this biosensor. 1.4. Purpose of the thesis and goals 37 1.4.3 MNPs encapsulated in a polymer for drug delivery The main objective is to design a multifunctional system able to combine the properties that will make it suitable to be used for therapy and diagnostics. We have developed in this work a multi-responsive system and it consists on a pH-responsive polymer that form microspheres with encapsulated MNPs that allows the magnetic core to enable externally controlled actuation under magnetic induction. Magnetic measurements have shown that the iron oxide nanoparticles are superparamagnetic and therefore are able to undergo a local increase of the temperature when an oscillating magnetic field is applied. Poly(4-vinylpyridine) is a pH-responsive material that can provide new opportunities for the organic-inorganic system and it is a widely studied polymer in our group. 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Mannweiler. Mechanism of oxidation of magnetite to maghemite. Nature, 217:118–1121, 1968. Chapter 2 Experimental and methods 2.1 Introduction In this chapter materials and methods are described, as well as the experimental procedures for the synthesis of the MNPs that are used in the work described in next chapters. These samples are prepared in both aqueous and organic solvents. As the formation mechanisms of the particles are different depending on the reaction media, a brief summary of mechanisms is given in order to explain the main differences between the MNPs, and reveal the characteristics that can be expected for the particles obtained in aqueous media and organic media. The morphological, chemical and physical characterization of six different ferrofluids is also included in this chapter. 2.2 Experimental Depending on the application of MNPs, it is not only the size what is important. Many times the synthesis conditions need to be tuned so that the resulting nanoparticles have uniform size, and homogeneous shape. Moreover, the chemical composition and the crystal structure of these materials are desirable because they influence the behaviour of the ferrofluids. The reproducibility of the synthesis is also relevant in order to get ferrofluids with the same characteristics. Eventually, nanoparticle agglomeration must be avoided, so that the final product can be easily dispersed in a solution [1]. 50 Chapter 2. Experimental and methods As we have previously explained, there are different procedures to prepare MNPs, however, we will focus here on aqueous media and on thermal decomposition of organic precursors in organic media. Depending on the application of the ferrofluid we choose, we will select the synthetic procedure that implies better conditions for that specific application. 2.2.1 A survey of methods for the production of ferrofluids in organic media The production of ferrofluids in organic media has been widely studied. High temperature decomposition of organic precursors in organic media leads to monodisperse nanoparticles, but what is also important is that the method permits to tune the mean size of the nanoparticles. Moreover, the high growing temperature used in this method yields nanoparticles with high crystallinity. There are several methods based on the thermal decomposition of organic precursors, as summarized in Table 2.1. Several factors have to be considered in the synthesis process: nature of the solvent, temperature of reaction, types of precursor and surfactant and their concentrations, and even the speed at which the reactants are added, [25]. The use of metalorganic precursors and organic solvents in the production of inorganic nanoparticles was introduced in the middle 90s, when Murray et al prepared highly crystalline CdSe nanoparticles in this way [14]. Later, Alivisatos et al applied this method to the production of γ -Fe2O3 with variable particle size [6]. They used iron cupferron complexe Fe(Cup)3 (Cup: N-nitrosophenylhydroxylamine, C6H5N(NO)O-) as precursor in trioctylamine solvent, and they could vary the size of the nanoparticles from 4 to 10 nm by increasing the quantity of Fe(Cup)3 injected and the reaction temperature. Since then, many other groups have used this methodology to synthesize nanoparticles of several compounds in a wide range of sizes. 2.2 Experimental 51 Table 2.1: Methods for the production of ferrofluids in organic media Precursor Solvent Surfactant Reaction T Particle size Reference Fe(Cup)3 Trioctylamine - 250-300ºC 4-10 nm [6] Co(acac)2·4H2O Diphenylether Oleic acid Trioctylphosphine 100-300ºC 1-15 nm [7] Pt(acac)2 Fe(CO)5 Octyl ether Oleic acid Oleylamine 298ºC 4 nm [8] Fe(CO)5 Octyl ether Oleic acid or lauric acid 298ºC 4-16 nm [9] Fe(acac)3 Phenyl ether Oleic acid Oleylamine 265ºC 4-20 nm [10] Fe(CO)5 Decalin Polyisobutene 170ºC 2-10 nm [11] FeCl2·4H2O FeCl3·6H2O Diethylene glicol N-methyl diethanolamine 210-220ºC 6-17 nm [2] FeO(OH) 1-octadecene Oleic acid 350ºC 20-30nm [12] FeCl2·4H2O FeCl3·6H2O 1-octadecene Oleic acid 300ºC 3-50 nm [4] Fe(acac)3 Dibenzylether Decanoic acid 200ºC 3-50 nm [13] In 2001 Murray and his group described the synthesis of Co and FePt nanoparticles based on high temperature decomposition of organic precursors. In order to obtain Co nanoparticles they decomposed cobalt acetylacetonate in diphenylether, and oleic acid and trioctylphosphine as stabilizers at 200ºC. Tailoring the ratio of the concentration of reagents to that of surfactants, and the temperature, they control the nanoparticles size in a range of 115 nm [7]. In other paper they 52 Chapter 2. Experimental and methods described the synthesis of 4 nm FePt nanoparticles. They used Pt(acac)2 and Fe(CO)5 as metal precursors and decomposed them in octyl ether with oleic acid and oleylamine as surfactants at 300ºC [8]. The same year, Hyeon and col. reported a two step synthesis of maghemite nanoparticles with a size range of 416 nm. In a first step they add Fe(CO)5 to a mixture containing octyl ether and oleic acid or lauric acid at 100ºC, then they rose the temperature to 298ºC for 1h. In the second step they oxide the sample with (CH3)3NO. The nanoparticles were highly crystalline and monodisperse [9]. In 2002 Sun et al got 4 nm Fe3O4 nanoparticles by decomposition of Fe(acac)3 in phenyl ether with 1,2-hexadecanediol, oleic acid, and oleylamine under nitrogen at 265ºC. To make larger Fe3O4 nanoparticles, they used a seed-mediated growth method [10]. Another variation of the high temperature decomposition method is to employ polymers instead of surfactants for the control of the particle size and to avoid nanoparticle agglomeration. Butter and col. reported the preparation of Fe nanoparticles with a 2 to 5 nm size in dilute solutions of decalin containing polyisobutene at 170ºC [11]. In 2004, Caruntu et al. described a method based on high temperature hydrolysis of chelate iron alkoxide complexes in solutions of the corresponding alcohol, diethylene glycol, and N-methyl diethanolamine. They obtained the chelate iron alkoxide from FeCl2·4H2O and FeCl3·6H2O and diethylene glycol, then a NaOH solution was added and leave to react for 5 h. They were able to produce nanoparticles from 6 to 17 nm [2]. In the same year, Yu was the first to report the synthesis of iron oxide nanoparticles with a particle size larger than 20 nm using non-toxic hydrated iron oxides as iron precursor in octadecene and oleic acid [12]. The next year, Jana et al. used an analogous procedure to obtain magnetite from 3 to 50 nm but in this case they started from iron chlorides instead hydrated iron oxides [4]. In 2010, Guardia and col. reported the effect of decanoic acid as a surfactant on the synthesis of iron oxide nanoparticles by thermal decomposition of Fe(CO)5 in dibenzyl ether. They achieved nanoparticles ranging from 5 to 30 nm [13]. 2.2 Experimental 53 2.2.2 Synthesis of IOMNPs in organic solvents by thermal decomposition of Fe(CO)5 In this work, we have used the synthesis method proposed by the group of Hyeon that yields highly crystalline and monodisperse γ-Fe2O3 nanoparticles [9]. As it can be seen in Figure 2.1, this procedure, which allows a variation of the particle size by controlling the experimental parameters, is based on the thermal decomposition of an iron precursor in the presence of a surfactant and subsequently the product is oxidized. The iron precursor is Fe(CO)5, the solvent is dioctyl ether (OE) or dichlorobencene (DCB), the surfactant is oleic acid, and the oxidant is trimethylamine oxide. Figure 2.1 Iron oxide organic synthesis scheme. Mechanisms of reaction: The formation of iron oxide nanoparticles in the Hyeon method occurs in several steps: Step 1. Decomposition: In a first stage the Fe(CO)5 complex is decomposed according to the following global reaction: Fe(CO)5 → Feo + 5CO (1) According to Redl et al. the decomposition of the complex occurs already at 170oC, liberating all CO that is no longer observed in IR [15]. After decomposition of the precursor the iron seems to be in the form of Fe-oleate coordination compounds [16]. Step 2. Nucleation and growth. After an initation time, nucleation of Fe nanoparticles occurs. Fe-oleate → ↓ Fe NPs (2) 54 Chapter 2. Experimental and methods Fe-oleate complexes are very stable in solution and consequently initation times in the presence of oleic acid are large and the supersaturation at the burst of nucleation is very high, thus favouring monodispersity, as shown by Casula et al [17]. Nucleation of Fe nanoparticles occurs at 300oC when the temperature is increased after decomposition of Fe(CO)5. However, even when the reactants are added by rapid injection at 280300ºC, initation times can be as long as 1 h. Using ligands weaker than oleate, such as oleylamine, or no ligand at all, results in shorter nucleation times and broader size distributions. The initation time and subsequently the size dispersion are also dependent of the temperature and of the reactants concentration. Step 3. Oxidation. In the presence of a mild oxidant, trimethyl amine oxide, iron nanoparticles are oxidized to -Fe2O3. Fe NPs + (CH3)3NO → -Fe2O3 (3) The process of oxidation is not straightforward, but it goes through intermediate phases, first wüstite (FeO) and then magnetite (Fe3O4) [5]. Therefore, it is very important to ensure a complete oxidation to avoid a mixture of iron oxide phases in the final ferrofluid. Particle size can be varied from 4 to 16 nm by controlling the experimental parameters. Particles in the order of 20 nm can be obtained by seeding the reaction media. Figure 2.2 shows a TEM micrograph of an organic ferrofluid prepared in this way. 2.2 Experimental 55 Figure 2.2 Transmission electron micrograph of an organic ferrofluid. 2.2.2.1 Procedure for the synthesis of organic MNPs suspensions We have synthesised several ferrofluids in organic media that were later used in chapter 3 and 5 in the production of aqueous suspensions for biomedical applications. As pointed out above, the Hyeon method was used. The experimental set-up is shown in Figure 2.3. The organic solvent is placed in a three neck rounded flask together with the surfactant. A refrigerant is adapted to the central neck, and it is topped with a bubbler. The other two necks are closed with septa. In one of them a thermocouple is placed through the septum in order to control the reaction temperature. The last neck is used to inject the reactants. The procedure can be described as follows. An argon flux is passed through the experimental set up in order to remove the air. The temperature is raised to 100ºC and then the iron pentacarbonyl is injected through the septum. Then the temperature is raised gradually until the solvent boiling point. The reaction is left to react till the colour changes from yellow to black, from 2 to 48 hours, depending on the carbonyl/surfactant ratio. Iron nanoparticles were formed in this process. In order to obtain iron oxide nanoparticles the system is cooled to room temperature for oxidation. The oxidation is carried out by either employing a mild oxidant such as trimethylammonium oxide or by atmospheric air at high temperature. 56 Chapter 2. Experimental and methods Figure 2.3 Experimental set up. Five different organic ferrofluids were produced. The reaction conditions are summarized in Table 2.2, indicating the solvent, the precursor, the oleic acid and the oxidant quantities. Table 2.2: Synthesis of an organic ferrofluid stabilized by oleic acid Sample Chapter Solvent Fe(CO)5 Oleic acid Trimethylamine oxide A0 2 20 ml OE 0.4 ml 3.41 g 0.68 g A1 3 40 ml DCB 0.8 ml 2.2 g 1.36 g A2 3 30 ml OE 0.6 ml 3.4 g air A3 3 20 ml OE 0.4 ml 2.2 g air A4 5 40 ml DCB 0.8 ml 2.2 g 1.36 g OE = dioctyl ether; DCB = dichlorobencene 2.2 Experimental 57 2.2.3 Ferrofluids in aqueous media The process of iron oxide precipitation in aqueous media is really complex. Iron(III) ions are initially in the form of [Fe(H2O)6]3+ octahedral units [18]. These units link to each other by hydrolysis reactions generating protons, and the pH of the solution decreases to a level at which hydrolysis stops. When a base is added to the solution, the pH increases rapidly, and the hydrolysis process is restarted until the pH reaches a new equilibrium value, which depends on the iron concentration and the hydrolysis ratio, n = [OH]/[Fe]. Continuous hydrolysis results in the formation of dimers, then trimers and then linear iron polymers [18, 19] that become branched as n increases, as it can be seen in Figure 2.4. The precipitates are finally formed from these iron polymers by condensation [18, 20]. Figure 2.4 Chains of Fe(H2O)6]2+ octahedra. Magnetite can be formed by addition of an alkali solution to an aqueous solution containing Fe(III) and Fe(II) in a molar ratio of 2. As explained above the hydrolysis of Fe(III) ions occurs readily at acid pH forming amorphous hydrated iron (III) oxides. Above pH=7, the Fe(II) ions also precipitate, first as green rust (Fe(OH)2), and then as magnetite by redisolution of Fe(II) and Fe(III) intermediate oxides. The overall reaction can be expressed as: 58 Chapter 2. Experimental and methods [Fe(H2O)6]2+ + 2[Fe(H2O)6]3+ + 8OH → Fe3O4 + 4H2O Magnetite is sensitive to oxidation transforming into maghemite. In aqueous solution is frequent to obtain mixtures of both phases, but oxidation in air is not the only way to transform Fe3O4 in -Fe2O3. Various electron or ion transfers are involved depending upon the pH of the suspension. Fe3O4 + 2H+ → -Fe2O3 + Fe2+ + H2O The oxidation of magnetite to maghemite is a topotactic transformation. That implies migration of ferrous ions from the interior to the surface through the lattice framework, creating vacancies to maintain the charge balance. A typical crystallization process involves two stages: 1) a short burst of nucleation when the concentration of the species reaches critical supersaturation, and 2) a slow growth of the nuclei by diffusion of the growth units to the particle surface. To produce monodisperse iron oxide nanoparticles, these two stages must be separated. This means that nucleation should be avoided during the period of growth like in a LaMer precipitating system, see Figure 2.5. In this system the particle size decreases with the concentration of reactants [21]. In iron oxide aqueous systems, the precipitation occurs by condensation of polymeric precursors, which size will increase with the iron concentration in solution. Thus, one may expect that the size of iron oxide particles precipitated in aqueous solutions should increase with the concentration of iron in the medium, as it is actually the case. 2.3. Methods 65 protons must not be allowed to interfere. Deuterated solvents especially for use in NMR are preferred, like deuterated chloroform, CDCl3. 1H-NMR spectra of most organic compounds are characterized by chemical shifts in the range +12 to -4 ppm and by spin-spin coupling between protons. The integration curve for each proton reflects the abundance of the individual protons. 1H-NMR spectra of the polymers were recorded at 300 MHz in CDCl3 solution at room temperature using a BRUKER ARX-300 spectrometer. 2.3.2 Structural characterization Some of the characterization techniques for nanomaterials differ from the characterization methods for macroscopic materials. Actually, the synthesis and study of nanomaterials have been rapidly developed since we have the appropriate methodologies to observe them. Since the transmission electron microscopes were developed the observation of nanoparticles and nanomaterials was easily achieved. Nowadays, we are able to study the morphology and the size, but also the structure of these materials with a transmission electron microscope tool, the selected area electron diffraction. Moreover, chemical information about the samples can be obtained by means of another transmission electron microscope tool, the electron energy loss spectroscopy. Due to these applications, the transmission electron microscope is a very helpful device for nanoscience. Otherwise, it is also useful the utilisation of classical characterization tools as infrared, dynamic light scattering or X ray diffraction, that are cheaper and more accessible. 2.3.2.1 Fourier transform infrared spectroscopy The main aim of infrared (IR) absorption spectroscopy is to measure how a sample absorbs light at different wavelength. Chemical bonds have specific frequencies at which they vibrate corresponding to energy levels. Resonant frequencies can be related to the strength of the bond and the mass of the atoms. Thus the frequency of the vibrations can be associated with a particular bond type. In order to analyze a sample a monochromatic light beam is directed to the sample and how 66 Chapter 2. Experimental and methods much of the light is absorbed is measured. This step is repeated for each distinct wavelength. Fourier transform infrared spectroscopy (FTIR) is a less intuitive way to obtain information. This technique is based in the impact of a beam containing many different frequencies of light at once to the sample, and measures how much of that beam is absorbed by it. Then, the beam is modified to contain a different combination of frequencies, giving a second data point. This process is repeated many times. Afterwards, a computer takes all these data and gives the absorption at each wavelength. The samples were measure in a Spectrum 100 FTIR, its optical module contains a Helium Neon laser, which emits visible, continuous wave radiation at a wavelength of 633 nm and a maximum power of 1 mW. This system enables to collect data over a total range of 7800 to 370 cm-1 with a best resolution of 0.5 cm-1. 2.3.2.2 X-ray diffraction Powder x-ray diffraction (XRD) is a method for determining the localization of atoms within a crystal, in which a beam of x-rays interacts with the crystal powders and diffracts into specific directions. From the angles and intensities of these diffracted beams, the positions of the atoms in the sample can be determined, as well as their chemical bonds, their disorder, the nanoparticles average size and various other information. Relative to other methods of analysis, powder diffraction allows for rapid and non-destructive analysis of multicomponent mixtures without complicated sample preparation. The positions, corresponding to lattice distances, and the relative intensity of the peaks are indicative of a particular phase and material, providing a "fingerprint" for comparison to the standards database Joint Committee on Powder Diffraction Standards (JCPDS). The XRD patterns of the dry nanoparticles samples were obtained using a D-Max Rigaku diffractometer equipped with a CuKa1 radiation source. The dry powders were 2.3. Methods 67 separated from the as prepared organic suspensions by addition of acetone, washed three times with acetone, and dried in air. 2.3.2.3 Dynamic light scattering The potential uses for metal oxide nanoparticles are often size dependent. Dynamic light scattering is a physical technique that can be used to determine the size distribution of small particles in suspension. The basic principle in this technique is the measurement of the scattering of an incident monochromatic and coherent beam of light in to a sample (composed by particles in suspension) with time. DLS measures Brownian motion of the particles in solution and relates this to the size of these particles. The particle size distributions are often reported in terms of volume, number or scattering intensity but usually produce different interpretations of the results, despite the data come from exactly the same sample. When comparing size results obtained by DLS with electron microscopy, it must be noticed that the best corresponding DLS size distribution for comparison is the number distribution for us, because in microscopy techniques particle are counted and sorted into histograms from the images. The DLS measurements were preformed on a Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Diluted suspensions of the samples were irradiated with red light (HeNe laser, wavelength λ= 632.8 nm) and the intensity fluctuations of the scattered light (detected at a backscattering angle of 173º) analysed to obtain an autocorrelation function. Samples were measured in disposable polystyrene cuvettes at a temperature of 25ºC. Data were acquired in automatic mode, ensuring enough photons were accumulated for the result to be statistically relevant. The software (DTS v 5.03) incorporates a "data quality report" that indicates good quality for all data obtained, and provides both the size mean and polydispersity index (PdI), employing the size distribution data obtained by intensity. Measurements were conducted in triplicate. 68 Chapter 2. Experimental and methods 2.3.2.4 Transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique based on a beam of electrons that is transmitted through a sample. The electrons interact with the sample as they pas through it and an image is formed. The image is magnified and focused in an imaging device, as a fluorescent screen, on a photographic film, or registered in a digital camera. TEMs are capable of imaging at a significantly higher resolution than light microscopes. At smaller magnifications TEM image contrast is due to absorption of electrons in the material, due to the thickness and composition of the material. At higher magnifications complex wave interactions modulate the intensity of the image, requiring expert analysis of observed images. Alternate modes of use allow us to observe chemical properties, crystal orientation and electronic structure as well as the sample image. Electron energy loss spectroscopy (EELS) is a technique that can be performed inside a TEM and permits to obtain maps of elements of a sample. In order to achieve that, a sample is exposed to electron beam energy. The electron paths are slightly deflected and detected so the energy loss can be measured. The inner shell ionizations are especially useful for determining the elemental components of the sample. Selected area electron diffraction (SAED) is a crystallographic technique associated to a TEM microscope. The electron beam hits the sample, and the electrons are diffracted to particular angles, allowing the characterization of the crystalline structure of a selected area. The result is an image that consists of a series of spots called SAED patterns that are a projection of the reciprocal lattice. This technique is similar to x-ray diffraction but the area of analysis is in the nanometer scale. Sample preparation is a necessary prerequisite for TEM. In order to be transparent to an electron beam, the samples must be thin, typically about 100 nm or less depending on the average atomic number of the material. Particulate materials sample preparation normally consists on transferring a suspension of the particles in a solvent to a carbon coated grid, and letting the solvent to evaporate. Our TEM samples were 2.3. Methods 69 prepared by putting a drop of the as prepared suspension on a carbon coated copper grid and then dried in air. The grid was then placed under a Philips CM-30 instrument. The images were taken at an acceleration voltage of 300 kV. Another microscope that we have used is a Hitachi 800MT with Gatan Multiscan chamber working at an acceleration voltage of 200 kV. EELS spectra were obtained in a Gatan Image Filter (GIF2000) coupled to a Jeol 2010F microscope. 2.3.3 Magnetic characterization The magnetic behaviour of the MNPs that compose our systems is very important in order to decide if they are suitable for the potential applications that they are conceived for. Measurements of magnetization provide us information about the sample magnetic properties, like superparamagnetism, a valuable property for certain applications requiring the particles to be single domain and to remain not aggregated after the removal of a magnetic field. But we also are interested in the heating capacity of the MNPs. The SAR of a ferrofluid depends on the magnetic field strength and the field frequency. But it also depends on the ferrofluid characteristics, as the magnetic particle size and the magnetic particle concentration. 2.3.3.1 Magnetic measurements SQUID Measurements of magnetization versus applied field at a fixed temperature provide us information about the response of the MNPs to a magnetic gradient. The measurement of the magnetization while the temperature is increasing allows us to estimate the magnetic size of the particles and also to calculate the blocking temperature of the ferrofluid, in other words, the temperature above which the sample becomes superparamagnetic. The magnetic properties of nanoparticles were determined with a commercial SQUID magnetometer (MPMS, Quantum Design). The measurements were performed on ferrofluid samples in organic and aqueous solvents. The dispersions were diluted down to a certain concentration and then frozen inside a capsule of gelatine or polycarbonate depending on the solvent. 70 Chapter 2. Experimental and methods 2.3.3.1 SAR SAR was determined by a calorimetric method using a special purpose magnetothermal setup [23] working under adiabatic conditions. Compared to current nonadiabatic installations, this setup and method allows direct measurement of sample temperature increments with negligible heat losses, overcoming the limitation of heat dissipation for SAR estimation in standard SAR equipments. 2.4 Characteristics of the organic MNPs suspensions Five different organic suspensions samples of monodisperse iron oxide nanoparticles, A0A4, have been prepared, according to the methods described above, which have been used in the following chapters as the starting materials for the preparation of biological ferrofluids. Sample A0 has been used to explore the magnetothermic effect of iron oxide nanoparticles when they are submitted to an alternating magnetic field. Samples A1, A2 and A3 have been used in chapter 3 for the preparation of monodisperse silica coated aqueous ferrofluids for biomedical applications. And sample A4 has been used in chapter 5 for the preparation of polymer coated aqueous ferrofluids for drug delivery. Here we present the results of the structural characterization of these samples. The main structural parameters are shown in Table 2.3. 2.4. Characteristics of the organic MNPs suspensions 71 Table 2.3: Structural parameters of the organic ferrofluids Sample Structure Diameter (nm) (TEM) Hydrodynamic diameter (DLS) A0 98.7% Maghemite 1.3 % Magnetite 11.6 ± 1 14 nm A1 Magnetite & Maghemite 5.9 ± 1 11 nm A2 Magnetite & Maghemite 9.6 ± 1.5 15.5 nm A3 97.2 % Maghemite 2.8 % Magnetite 13.5 ± 2.5 11 nm A4 99.97% Maghemite 0.03 % Magnetite 5.7 ± 1  Fig. 2.8 shows the x-ray diffraction patterns of dry powder samples separated from the as prepared organic suspensions by addition of acetone as described in section 3.2.2. All the patterns show the typical broad peaks of nanoparticles of iron oxide spinel structure at angles 2 close to 2.95, 2.52 2.09, 1.70, 1.61, 1.48 corresponding to (2 2 0), (3 1 1 ), (4 0 0), (4 2 2 ), (5 1 1) and (4 4 0) reflections of maghemite crystal structure. No other peaks are observed apart from these ones, confirming the phase purity of the samples. 72 Chapter 2. Experimental and methods Figure 2.8 XRD of A0, A1, A2 and A4 powder samples. 2.4. Characteristics of the organic MNPs suspensions 73 The crystal structure of sample A3 was determined from electron diffraction patterns that are also consistent with maghemite crystal structure as shown in Fig. 2.9. By the titration method we find that this sample is composed by 97.2 % of maghemite. Figure 2.9 Electron diffraction pattern of sample A3 showing rings corresponding to interplanar distances: 2.99, 2.51, 2.10, 1.65, and 1.50 A that are close to the main reflections in maghemite structure: (220), (311), (400), (422), (511), (440) (2.95, 2.52, 2.09, 1.70 and 1.48 A). Figure 2.10 TEM image of the as prepared iron oxide nanoparticle samples A0, A1, A2, A3 and A4. 74 Chapter 2. Experimental and methods TEM images in Fig 2.10 show, in all cases, electron-dense spherical nanoparticles covered with a layer of light material corresponding to the oleic acid coating. Figure 2.11 Histograms of TEM nanoparticle size distributions of A0A4 samples. 2.6.Magnetothermic properties of the organic MNPs suspensions 81 Figure 2.16 Heating steps at f= 109 kHz: experimental data with a smooth field. The dotted lines are extrapolation of the T drifts after ac-field application. As it can be observed in Table 2.4, the SAR of a ferrofluid, measured at 315 K, 3 kA/m and 109 kHz was found to double as the ferrofluids concentration was decreased by a factor 4. Summarily, in our case, four times more concentrated sample implies half SAR value, that is, half the nanoparticle mass can be used to obtain the same temperature increase, because the concentration of the sample promote interactions between particles. 82 Chapter 2. Experimental and methods 2.7 Characteristics of aqueous prepared ferrofluids Ferrofluid B1 was synthesised as we described before. In Figure 2.17 it can be seen the TEM micrograph of the sample, the shape of the nanoparticles is irregular and the size dispersion is wide, but we can estimate an average size of 5 nm (Figure 2.18). Figure 2.17 Transmission electron micrograph of ferrofluid B1. Figure 2.18 Histogram of iron oxide aqueous nanoparticles. From Figure 2.17 we can say that the particles are agglomerated, moreover it confirmed by DLS (Figure 2.19). The XRD pattern, in Figure 2.20, shows that the sample is composed by iron oxide inverse spinel structure, as it can be maghemite and 2.7. Characteristics of aqueous prepared ferrofluids 83 magnetite. We can also indicate that the peaks are very broad due to that the sample is composed by small nanoparticles. Figure 2.19 DLS of iron oxide aqueous nanoparticles. Figure 2.20 DRX of iron oxide aqueous nanoparticles. 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Shape evolution of single-crystalline iron oxide nanocrystals. Journal of the American Chemical Society, 126:1950–951, 2004. [27] R. Rosensweig. Heating magnetic fluid with alternating magnetic field. Journal of Magnetism and Magnetic Materials, 252:370–374, 2002. Chapter 3 Core-Shell ferrofluids for biomedical applications 3.1 Introduction As it has been pointed out in chapter 1, there is a broad range of applications for magnetic nanoparticles in biology and medicine. Consequently, the structural requirements for each application are also variable. For instance, biotechnological applications such as enzyme carriers, separation, purification, analysis, catalysis and processing need particles with a large size and high magnetic moment [1]. However, in vivo applications require a more complex particle structure. That is especially true in the case of magnetic resonance imaging contrast agents targeted drug delivery and cancer therapy [2-4]. Before going into the structural requirements for these applications, let us remind the advantages they may bring. Superparamagnetic nanoparticles (SPN) with respect to usual contrast agents have higher responses necessary to reach single cell detection [5]. Concerning cancer therapy, this technique is already in clinical phase for particles directly injected in the tumour [6], and they will be really useful when they are biologically vectorised so they can reach tumours that, due to their location and/or small size, are currently not accessible by other therapies [7]. In addition, they would avoid most of the undesirable secondary effects associated with radiotherapy and chemotherapy treatments [8]. Moreover, therapy and diagnosis with SPN could be performed simultaneously, thus entering the most promising area of theranostics [9]. Coming back to the structural requirements for in vivo applications, the first one is, of course, biocompatibility. Apart from that, there are three important issues to be 88 Chapter 3. Core-Shell ferrofluids for biomedical applications considered: 1) optimal magnetic performance, which is obtained when the nanoparticles are monodisperse; 2) high facility to penetrate biological barriers, this requires small hydrodynamic sizes, which in single-core iron oxide nanoparticles (IO) are minimized with a narrow shell; and 3) easy particles functionalization, which requires to have them coated with an adequate reactive surface. This chapter addresses these three issues. Concerning the first issue, aqueous preparation methods are simple, cheap and, therefore, the favourite for industrial production. However aqueous methods are associated with high size dispersion and interparticle agglomeration that may diminish their magnetic performance. These problems are overcome by organic methods that allow a precise control of the particle size, particle size dispersion and particle agglomeration [10]. The problem with organic methods is that, in order to control the particle growth, they use hydrophobic surfactants. That makes the resulting particles non dispersible in water. With respect to the second and third issues, the main strategies to coat and redisperse these hydrophobic particles in aqueous media are [1112]: 1) to attach amphiphilic molecules to the hydrophobic surface [1314], and 2) to coat the particles with polymeric or silica shells [1517]. The first approach yields small single-core nanoparticles as desired but the multifunctionalization requires complex chemical processing. The second approach facilitates further multifunctionalization of the particles by means of remnant surface reactive groups in the coating, such as the silanol groups on silica coatings [1820]. In the following we will consider several chemical routes to obtain aqueous suspensions of functionalized magnetic nanoparticles departing from nanoparticles produced in organic solvents. 3.1. Introduction 89 3.1.1 Transferring nanoparticles from organic to aqueous media A summary of typical coating methods used for transferring NPs dispersed in an organic solvent to an aqueous medium is given in Table 3.1. Table 3.1: Phase transfer routes from organic to aqueous medium for nanoparticles Type of coating Chemical route Example Carboxylic acid Oxidation of double bond in oleic acid O3 [21], KMnO4 [22] Intercalation Pluronic F-127 [23], PMMA-PEO [24], Tetradecene [15], PEI [24], Cyclodextrine [11] In situ precipitation of nanoparticles in polymer matrixes Norbornano [25], HOOCPEG-COOH [26] In situ polymerization in the presence of nanoparticles 2-Bromopropionilesther [27] Polymers Micelle methods PMAO–PEG [13] Particle encapsulation DSP-PEG-Biotine [28] Liposomes Micelle methods PEG-PE +PC [29] Sol-gel TEOS [30] Silica Microemulsion Silsesquioxano [31] Heterogeneous nucleation Ciclohexane/igepal [32] Bridge bifunctional molecule HS(CH2)10COOH [33] Gold Reduction of Au HAuCl4 [34] 90 Chapter 3. Core-Shell ferrofluids for biomedical applications In the first method, the particle surface is modified from hydrophobic to hydrophilic affinity without removal of the oleic molecules. This is achieved by oxidation of the oleic acid double bond by a strong oxidant, such as KMnO4 or O3. The oxidation produces the breaking of the double bond and creates a carboxylic group at the end, without affecting the carboxylic group at the other chain end interacting with the iron oxide surface [21, 22]. This method has the advantage that the surface of the IO core is never altered during the process. When the coating material is a polymer, the stability of the resulting nanoparticles depends on several factors: the ratio between hydrophobic and hydrophilic parts in the polymer chain, the chain length, the anchoring bond, and the polymer conformation. The most popular chemical route for polymer coating is the intercalation of the hydrophobic part of an amphiphilic polymer with the surfactant hydrocarbonous chain [11, 15, 23, 24]. A second route is in situ iron oxide precipitation [25, 26] and a third one, in situ polymerization, which consists in using monomers as surfactants that are later polymerized to form compact polymer coatings [27]. Besides those, micelle methods based on the use of amphiphilic polymers as surfactants are simple and versatile [13]. An example of nanoparticle encapsulation into liposomes was given by Gopalakhrisnan et al. These authors prepared hybrid vesicles of CdSe quantum dots by dispersing them together with phospholipids that formed the double liposome membrane [28]. The amount of nanoparticles encapsulated inside the liposome can be adjusted by regulating the volume of the organic solvent and the ratio of nanoparticles. Another possibility is to encapsulate nanoparticles in phospholipid block-copolymers micelles. In this way, the formation of single-core nanoparticles can be achieved [29]. Inorganic coatings can also be very effective, especially silica and gold coatings. Gold is an ideal inert material and it is also biocompatible. Besides, gold surfaces can be easily functionalised with macromolecules or any other ligand having a thiol terminal group (-SH). Moreover, it confers optical properties to the material due to superficial plasmons. Gold-IO dimmer structures can be built by epitaxial growth of 3.2. Experimental 97 Synthesis of TESPDA. The synthesis of TESPDA was carried out by condensation of the amine alkyl carboxylic acid with trialkoxysilane isocyanate by means of urea bridges as represented in scheme 3. In a standard procedure, 2.15 g of 12-aminododecanoic acid (1) were dissolved in a mixture of 8 ml of acetic acid, 16 ml of ethanol, and 16 ml of chloroform, then 2.5 g of 3-cyanobutyltriethoxysilane (2) were added, and the solution was kept under stirring overnight to obtain the alkoxysilane precursor (3). Figure 3.4 Synthesis of the silane precursor. Synthesis of IO@TESPDA particles. An scheme of the procedure is shown in Fig. 3.4. 10 ml of IO@OA organic suspension were mixed with 0.3 ml of TESPDA and 15 ml of acetone. The resulting nanoparticles were separated with the help of a magnet and the solution was discarded. This operation was repeated twice. Then, the iron oxide nanoparticles were dispersed in 30 ml of hexane during 10 minutes and then treated with 1 ml of alkoxysilane solution. After 15 minutes stirring, 0.1 ml of NH4OH/ethanol (1:1 by volume) and 0.1 ml of Milli-Q water were added and sonicated during 3 minutes. The sample was well dispersed. Subsequently the sample was magnetically stirred during 4 hours. Then a quantity of 20 ml of Milli-Q water was added. The organic and aqueous phases were easily separated, the aqueous phase containing all the magnetic nanoparticles. 3.2.2 Physical and chemical characterization The characterization of the materials has been carried out using techniques described in chapter 2. Namely, XRD, TEM, TGA, DLS and SQUID magnetometer. 98 Chapter 3. Core-Shell ferrofluids for biomedical applications 3.3 Results 3.3.1 Use of short chain organosilanes with iron coordinating groups 3.3.1.1 DTESP The organic ferrofluid A1, described in chapter 2 has been used for these experiments. The ligand exchange has been performed as described in the experimental section. Figure 3.5 shows the two liquid phases before and after the coating with the phosphate organosilane. It is clear that all the particles passed to the aqueous medium after coating. The colour of the particles becomes lighter due to the silica layer around the black magnetite particles. The aqueous suspension remains stable for several days. Figure 3.5 Images of the organic and aqueous media before and after ligand exchange. In Figure 3.6 we can see some TEM micrographs of the sample dispersed in water. The particles seem to be agglomerated in assemblies of different sizes up to 100 nm. The particle size analysis for individual iron oxide nanoparticles yield an average size of 6.2 ± 0.6 nm. A histogram is shown in Figure 3.7. Comparing this size with that of the original organic ferrofluid, D=5.9 ± 1 nm (see sample A1 in chapter 2), we can see that the diameter of the particles has slightly increased after the ligand exchange. Figure 3.8 shows DLS plots of the aqueous suspension. The distribution of hydrodynamic diameters is bimodal with one population with average size DH=65 nm, 3.3. Results 99 corresponding to small aggregates and a second population with average size DH=290 nm that corresponds to larger aggregates probably formed by secondary aggregation of the smaller ones. Let’s remind that the original ferrofluid had a hydrodynamic size of 11 nm corresponding to individual nanoparticles. Therefore a particle agglomeration has taken place during the coating reaction. Figure 3.6 TEM micrographs of IO@DTESP nanoparticles. Figure 3.7 Histogram of IO@DTESP nanoparticles. 100 Chapter 3. Core-Shell ferrofluids for biomedical applications Figure 3.8 DLS of IO@DTESP nanoparticle aqueous suspensions. In a second stage the IO@DTESP nanoparticles were coated with TEOS. With that purpose, an amount of aqueous suspension of these particles was redispersed in a mixture of isopropanol and water. Then, NH4OH was added to catalyse the hydrolysis of TEOS on the surface of the silane-stabilized nanoparticles. Figures 3.9 and 3.10 show the corresponding TEM image and DLS plot after the hydrolysis of TEOS. Comparing with TEM results before coating, it is apparent that the layer of lighter material around the aggregates is broader after coating with TEOS indicating the deposition of a new layer of TEOS around the particles. Nevertheless, the DLS peak does not show any significant shift to larger sizes indicating that the average size has not changed too much in comparison with the total particle size. It is also clear from DLS plot that the coating process did not cause any particle aggregation. Aqueous suspensions of IO@DTESP@TEOS were quite stable along the time (Figure 3.11). 3.3. Results 101 Figure 3.9 TEM micrographs of IO@DTESP@TEOS nanoparticles Figure 3.10 DLS of IO@DTESP@TEOS nanoparticle aqueous suspensions. Figure 3.11 Photograph of iron oxide nanoparticles coated with TEOS stabilized in water. 102 Chapter 3. Core-Shell ferrofluids for biomedical applications In a last step the IO@DTESP@TEOS nanoparticles were coated with PEG in order to improve their biocompatibility, make then more stable in the blood stream, and make then invisible to macrophages. The choice of PEG for surface coating is founded on its protein anti-adherence properties that make it the preferred polymer coating by far for in vivo applications of nanoparticles in general. The anchoring of PEG to the surface was performed by hydrolysis of a PEG trialkoxysilane precursor following the procedure described in the experimental section. The TEM micrographs (Figure 3.12) and DLS plots (Figure 3.13) show that the particles have not experienced important changes during the process. The small size and aqueous stability of the resulting nanoparticle makes them very appropriate for biomedical applications. Figure 3.12 TEM micrographs of iron oxide nanoparticles coated with TEOS (left) and SilPEG (right). 3.3. Results 103 Figure 3.13: DLS of iron oxide nanoparticles coated by TEOS and Sil-PEG. 3.3.1.2 TESDI The procedure to obtain aqueous suspensions of magnetic nanoparticles using TESDI as ligand exchange organosilane precursor was similar to that used for DTESP. The starting material was with the sample A2, described in chapter 2, with an average diameter of 9.6 ± 1.4 nm. In a first step, the original nanoparticles were dispersed in hexane where the ligand exchange was carried out. Then, the nanoparticles were transferred to water. Figure 3.14 shows the nanoparticle dispersion in water. In a further step, the nanoparticles were coated with TEOS in isopropanol/water media. As it can be appreciated in Figure 3.15, the transferred nanoparticles could be easily dispersed in water with better stability than in the added octyl ether. 104 Chapter 3. Core-Shell ferrofluids for biomedical applications Figure 3.14 Photograph of iron oxide nanoparticles suspension after coating with TDESDI in water (right). Figure 3.15: Photograph of iron oxide nanoparticles in a two phase octyl ether/water system as prepared (left) and after coating with TEOS (right). Notice that all the silica coated nanoparticles are in the bottom water phase. In order to study the size, the shape and the state of aggregation of the nanoparticles in the aqueous suspension we analyse them by TEM and DLS. Figure 3.16 shows two TEM micrographs of the sample. The nanoparticles seem to have the same size as the former nanoparticles that is 9.6 nm, and they form aggregates. In Figure 3.17 we can see the histogram of the particle size obtained from TEM micrographs. In the second micrograph we can observe some aggregates produced by nanoparticle agglomeration that are also evident in DLS plots (Figure 3.18). 3.3. Results 105 Figure 3.16 TEM micrograph of iron oxide nanoparticles coated with TESDI in water. Figure 3.17 Histogram of the particle size distribution. 106 Chapter 3. Core-Shell ferrofluids for biomedical applications Figure 3.18 DLS of iron oxide nanoparticles stabilized by means of TESDI in water. Figure 3.19 DLS of iron oxide nanoparticles stabilized by means of TEOS. 3.3. Results 113 Above the freezing temperature, there is a sudden susceptibility increase, particularly in  ’, in the case of aqueous dispersion that is absent in the organic dispersion. This increase can be attributed to the onset of Brown relaxation that is not appearing in the organic dispersion. Thus, contrary to the case of Néel relaxation, Brown relaxation is indeed affected by the dispersing medium. The Brownian relaxation time can be expressed as:         TK V B hyd   3 B (2) where  is the viscosity of the medium, and Vhyd is the hydrodynamic volume, respectively. Thus, the differences of intensity must be related to differences of the solvent viscosity that hinder the rotation of the particles in the organic medium with respect to water. This can be of considerable importance for magnetothermic applications. 3.4 Conclusions Phase transfer of iron oxide nanoparticles from organic to aqueous solvents has been achieved by ligand exchange of oleic acid with three different organoalkoxysilanes. The shape and size of the original nanoparticles are maintained after the phase-transfer process in all cases. The diethylphosphatoethyltriethoxysilane ligand yielded stable aqueous dispersions, but the resulting particles were agglomerated to some extent. The N-(3triethoxysilylpropyl)-4,5-dihydroimidazole ligand presented similar results but with a lower degree of agglomeration was lower, so thatthe quantity of singlecore nanoparticles was substantially increased. This can be due to the interactions between the iron oxide and the silanes functional groups. As the phosphate group interacts weaker than the imidazole with the iron oxide nanoparticle, the concentration of free silane increases and condense forming agglomerates. In both cases the single-core particles can be isolated from the agglomerated particles by filtration, magnetic separation or centrifugation. Alternatively, by means of 3-(triethoxisilyl) propylureido dodecanoic acid, we have described a straightforward procedure to transfer high quality iron oxide nanoparticles synthesized in organic medium into aqueous medium forming single core-shell nanoparticle suspensions. The phase transfer is more 114 Chapter 3. Core-Shell ferrofluids for biomedical applications efficient than in the methods described before, probably due to the fact that the long chain permits a better ordered disposition of ligands around the the nanoparticles. The aqueous magnetic nanoparticles maintain the shape, crystallinity and size distribution of the original organic nanoparticles, as well as with short chain ligands. The magnetic studies of organic and aqueous ferrofluids are consistent with the superparamagnetic behaviour of the nanoparticles. To summarize we can conclude that the first two ligand exchange procedures employing two short chain commercial silanes permit to obtain stable aqueous suspensions of iron oxide nanoparticles. The phosphateligand, produces agglomerates and the imidazol ligand a mixture of single-core nanoparticles and agglomerated nanoparticles. The main drawback of these procedures is is a low yield. The ferrofluids obtained in these ways can be useful in biomedical applications after separation of aggregates by filtration, magnetic separation or centrifugation. In the third procedure, using a long chain ligand, the transfer from organic to aqueous medium is more effective, and it yields single-core aqueous suspensions, which was one of the purposes of this work. Besides, the resulting nanoparticles can be further coated with a silica layer that can be easily functionalized by hydrolysis of biofunctional molecules having alkoxysilane groups. 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Two processes have been studied in order to develop the best methodology to get the maghemite nanoparticles internalized in the silica matrix in aqueous medium for the biosensor. The first one is the method described by Deng et al based in the hydrolysis and condensation of TEOS in isopropanol-water mixture [15]. The second method is the one described by Salgueriño-Maceira performed in an ethanol/water mixture [16]. The first synthetic procedure is based in the Stöber method with some modifications. The coating of magnetite nanoparticles with silica was carried out in basic alcohol/water mixture at room temperature by using magnetic ferrofluids as seeds. First, the magnetic ferrofluid was diluted with water, alcohol and aqueous ammonia. Then, this dispersion was homogenized by ultrasonic vibration in a water bath. Finally, under continuous mechanical stirring, TEOS was slowly added to this dispersion, and after stirring for 4 h, silica was formed on the surface of magnetite nanoparticles through hydrolysis and condensation of TEOS. 130 Chapter 4. Superparamagnetic beads for a biosensor In a typical synthesis of the second procedure, a mixture of NH4OH, H2O, EtOH, and the previously washed magnetic nanoparticles in water solution was added to an ethanolic solution of TEOS in EtOH while the solution was mechanically stirred. The hydrolysis and condensation of TEOS onto the magnetic nanoparticles was completed in 4 h. The formed nanobeads were centrifuged to eliminate excess reactants and redispersed in pure water. 4.2.2 Characterization Both procedures permit to obtain silica nanobeads, but we achieve them with different shape and morphology. By means of TEM (Figure 4.2) we can observe an average diameter of 130 nm for the spheres formed by the isopropanolic method, and 80 nm for the ethanolic procedure. It is clear that the ethanolic method permits to achieve more homogeneous nanobeads than the ones obtained by the isopropanolic procedure; however, the ethanolic method also has also drawbacks as we explain below. Figure 4.2 a) Isopropanolic method b) Ethanolic method. We carried out several experiments to probe the reproducibility of both methodologies. The results were positive, but we also found some difficulties related to the sample concentration and the presence of impurities that we can see in TEM micrographs. 4.2. Synthesis and characterization of superparamagnetic nanobeads 131 The first inconvenient that we tried to solve was sample dilution. We need more concentrate samples in order to perform the bioconjugation experiments. The obtaining of nanobeads is carried out in diluted solutions due to requirements of the synthesis. More concentrated samples can lead to form agglomerated nanobeads in the condensation of TEOS. So it was necessary to study concentration procedures that could provide non-agglomerated nanobeads. We chose centrifugation and evaporation of the solvent as possible methodologies to concentrate the sample. The first methodology that we used to concentrate the sample was to evaporate the solvent. The ethanol was easily evaporated; however, the results were not as convenient as it can be seen in Figure 4.3: the samples were agglomerated. Figure 4.3 Solvent evaporation in ethanolic synthesis. a) Before evaporation, b) After solvent evaporation. Then, we tried to concentrate the sample by centrifugation. The results can be seen in Figure 4.4 where the beads have lost quality as when we tried to concentrate them by evaporating the solvent. Before centrifuge, the nanobeads have well defined edges and spherical shape, then, after centrifuge the beads agglomerate and lose homogeneity in shape and size. 132 Chapter 4. Superparamagnetic beads for a biosensor Figure 4.4 Solvent evaporation in ethanolic synthesis. a) Before centrifuge b) after centrifuge. Moreover, although the ethanol method permits to obtain a major concentration of nanobeads, they are smaller than the obtained by the isopropanolic method. We attempted to grow the beads by employing more silica precursor in a posterior step. The results are shown in Figure 4.5. The nanobeads keep their shape and size but after centrifuge they agglomerate as well. Figure 4.5 Ethanolic synthesis. a) Before centrifuge, b) alter centrifuge. Analysis of equivalent results of the same concentration study following the isopropanolic synthesis are even more frustrated, as it can be seen in Figure 4.6. 4.2. Synthesis and characterization of superparamagnetic nanobeads 133 Figure 4.6 Isopropanolic synthesis. a) Before solvent evaporation b) after solvent evaporation. We also attempted to increase the reaction time. The silica precursor was allowed to react during 24 hours in an isopropanolic suspension of the magnetic nanoparticles. We obtain nanobeads of diameters from 150 to 200 nm. In Figure it 4.7 can be observed the results of a synthesis of 24 hours in isopropanol. When we centrifuge the sample and disperse it in water, the morphology and size of the nanobeads are maintained and they do not seem to agglomerate after elimination of the solvent by evaporation. Figure 4.7 Isopropanolic synthesis 24 h. A) Initial sample, b) Centrifugation, c) Solvent evaporation. Similarly, we carried out the ethanolic synthesis for 24 hours obtaining nanobeads of 100200 nm with more uniform morphology than the one obtained by the 134 Chapter 4. Superparamagnetic beads for a biosensor isopropanolic synthesis. When we centrifuged the sample, the particles conserved their original characteristics. Concentrating the sample by the solvent evaporation method also yielded good results. However, TEM images shown that there were impurities in the sample. When we centrifuge the sample we observe two deposits, one brown coloured on the bottom and another one, white, on the top. It can be due to the presence of maghemite nanoparticles outside the silica beads, instead in TEM pictures it can not be seen any nanoparticle neither, inside or outside, the silica nanobeads. This may be due to the high contrast of the sample in TEM. Thus, it is impossible to distinguish magnetic nanoparticles inside the nanobeads. Taken into account the obtained results, we decide to use the isopropanolic synthetic route instead of the ethanolic one and the centrifugation method to concentrate the sample. In Figure 4.8 the procedure followed to obtain nanobeads following the ethanolic synthesis and keeping it for 24 hours is shown. As in the short reaction time synthesis, the centrifuged sample presented lower impurities content. Figure 4.8 Ethanolic synthesis 24 h. a) Initial sample, b) centrifugation, c) solvent evaporation. 3.2.3 Nanobeads functionalization To functionalize the surface of the silica nanobeads with carboxylic groups we chose carboxyethylsilanetriol. In aqueous/alcoholic media and by means of the action of an alkaly, this alcoxysilane reacts with the silanol groups at the nanobeads surface and condenses leaving the carboxylic group pointing outward. In Figure 4.9 it can be observed how, by means of the hydrolysis of a silane, it is possible to anchor to the 4.2. Synthesis and characterization of superparamagnetic nanobeads 135 nanobead surface a molecule with a functional carboxylic group that is able to anchor a protein. Figure 4.9 Scheme of the silanization of the nanobeads. Once nanobeads were obtained following either the ethanolic or the isopropanolic routes explained before, the alcoxysilane was added. In both cases 0.07 ml of carboxyethylsilanetriol were added and mixed during 20 hours. After centrifugation, the sample was resuspended in water and washed several times to remove the excess of silane that was not reacted. The TEM micrographs of both preparations can be seen in Figure 4.10 before and after sililation. Figure 4.10: isopropanolic procedure. a) Before silanization, b) after silanization. The morphology of the particles is well kept, however this does not indicate the anchoring of the alcoxysilane. In order to find it out, we compare the IR spectra before and after silanization, as depicted in Figure 4.11. 136 Chapter 4. Superparamagnetic beads for a biosensor Figure 4.11 IR spectra of functionalized nanobeads (red line) and non-functionalized nanobeads (blue line). The spectra of both preparations are coincident in almost all the frequencies, except in those where the silanol bonds appear. Silanol groups are only localized on the surface of the silica beads, hence there have been a surface modification. Due to the low concentration of the carboxylic groups their characteristic IR absorptions are not observed. A study about the degree of functionalization of the nanobeads varying silane concentration was also carried out. With this purpose we perform a battery of experiments changing progressively the silane concentration, from 0.1 to 0.025 g of carboxyethylsilanetriol: A1: 0,1 g, A2: 0,085 g, A3: 0,05 g y A4: 0,025 g. The general tendency is that the silanization degree increases as the concentration of silane is augment. However for low concentrations, the results were more homogeneous, as it can be observed in Figure 4.12. 4.2. Synthesis and characterization of superparamagnetic nanobeads 137 Figure 4.12 TGA of the silanized nanobeads with different quantities of silane. The TGA technique consists of a study of the variation of weight while the temperature of the sample is increased at inert atmosphere. We can observe the total mass lose corresponding to the silane in the table. A1: 0,1 g silane 16,53% A2: 0,085 g silane 14,28% A3: 0,05 g silane 12,85% A4: 0,025 g silane 12,77% 138 Chapter 4. Superparamagnetic beads for a biosensor 4.2.4 Magnetic characterization Magnetic measurements were performed using a commercial device MPMS-XL from Quantum Design with a SQUID magnetometer. The samples StoAc1 (isopropanolic) and StoAc2 (ethanolic) were washed, centrifuged and dried to obtain light brown powders, for their posterior magnetic characterization. The diamagnetic contributions of the sample holder (-4.90·10-7 emu/Oeg capsule) and silica (-4.47·10-7 emu/Oegsilica) were calibrated independently and their contribution was subtracted from the sample experimental data. The in-phase susceptibility of samples StoAc1 and StoAc2 show evidence of a superparamagnetic blocking at temperatures TB around 25 K (see Figure 4.13). Notice that TB does not depend with the synthetic methodology used. Figure 4.13: In-phase susceptibility at 1Hz, 9 Hz, 117 Hz, 852 Hz shows superparamagnetic relaxation at TB = 25 K; Inset: out-of-phase susceptibility at 9 Hz show similar blocking temperatures for StoAc1 and StoAc2 samples. Above TB magnetic nanoparticles behave as superparamagnets so that magnetization curves should scale with H/T. The magnetization isotherms of StoAc1, shown in Figure 4.14, follow such behaviour, considering the temperature dependence of the magnetic moment displayed in Figure 4.16. However, this is not the case for the