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Dalton Transactions

An international journal of inorganic chemistry
www.rsc.org/dalton Volume 40 | Number 24 | 28 June 2011 | Pages 6301–6576

ISSN 1477-9226

COVER ARTICLE Tremel et al. Synthesis and bio-functionalization of magnetic nanoparticles for medical diagnosis and treatment

Dalton Transactions
Cite this: Dalton Trans., 2011, 40, 6315 www.rsc.org/dalton

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Synthesis and bio-functionalization of magnetic nanoparticles for medical diagnosis and treatment
Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT00689K

Thomas D. Schladt,a Kerstin Schneider,a Hansj¨ rg Schildb and Wolfgang Tremel*a o
Received 21st June 2010, Accepted 13th January 2011 DOI: 10.1039/c0dt00689k The synthesis of multifunctional magnetic nanoparticles (NPs) is a highly active area of current research located at the interface between materials science, biotechnology and medicine. By virtue of their unique physical properties magnetic nanoparticles are emerging as a new class of diagnostic probes for multimodal tracking and as contrast agents for MRI. Furthermore, they show great potential as carriers for targeted drug and gene delivery, since reactive agents, such as drug molecules or large biomolecules (including genes and antibodies), can easily be attached to their surface. On the other hand, the fate of the nanoparticles inside the body is mainly determined by the interactions with its local environment. These interactions strongly depend upon the size of the magnetic NPs but also on the individual surface characteristics, like charge, morphology and surface chemistry. This review not only summarizes the most common synthetic approaches for the generation of magnetic NPs, it also focuses on different surface modification strategies that are used today to enhance the biocompatibility of these NPs. Finally, key considerations for the application of magnetic NPs in biomedicine, as well as various examples for the utilization in multimodal imaging and targeted gene delivery are presented.

Introduction
Today, the development of nanomaterials has moved beyond the discovery of totally new materials and compositions. Instead the focus has shifted to the investigation of more complex, composite systems, in which the recombination of known materials into structures of higher complexity opens new possibilities of functionality.1–5 While scientific diligence of designing new composite or hybrid materials is speeding up, industrial producers have begun merchandising the earliest discovered nanomaterials, and, in fact, are developing novel applications for them to fit the desired needs. Besides other commercial applications, magnetic nanoparticles (NPs) are intensively being investigated for utilization in many different scientific and industrial fields, ranging from catalysis to mass data storage.6,7 One of the fastest moving and most exciting research areas is the interface between nanotechnology, biology and medicine. It has been stated by numerous experts, that the application of nanotechnology in medicine, which is often referred to as “nanomedicine”, offers many exciting possibilities for healthcare in the future, and may revolutionize the areas of targeted drug delivery, disease detection and tissue engineering.8–11 An often used catch phrase in this context is “theragnostics”, which, as this
a Institut f¨ r Anorganische Chemie und Analytische Chemie, Johannes u Gutenberg-Universit¨ t, Duesbergweg 10-14, D-55099 Mainz, Germany. a E-mail: tremel@uni-mainz.de; Fax: +49 6131 39-25605; Tel: +49 6131 39-25135 b Institut f¨ r Institut f¨ r Immunologie, Johannes Gutenberg-Universit¨ t, u u a Obere Zahlbacher Str. 67, D-55131 Mainz, Germany

word construction implies, is the combination of both, therapy and diagnostics into one powerful tool. In fact, the concept of using magnetic nanoparticles to target tumor cells inside the human body, and applying them to treat cancer, was first conceived in the late 1970s.12 The key idea was to attach common anticancer drugs to small magnetic spheres outside the body before administering them to the patient. After injection into the blood stream strong external magnetic fields should concentrate the drug-loaded particles inside the tumor tissue. The authors predicted that by this approach the drug payload would be reduced significantly, and thereby the unwanted side effects associated with the systemic distribution of chemotherapeutic agents, including nausea, hair loss and a compromised immune system could be avoided. Although not yet fully in clinical use, nanomedicine has come a long way from these initial ideas, and it is proceeding with remarkable speed. Concerning diagnostics, the ability to accelerate the proton relaxation of water molecules in different tissues has been proposed to be one of the most promising features of magnetic NPs for nanomedicine, since it allows the development of novel contrast agents for magnetic resonance imaging (MRI). In fact, contrast agents based on superparamagnetic iron oxide NPs (SPIONs) have been in clinical use for almost two decades. Moreover, progress in the synthesis of magnetic NPs permits a precise tuning of their physical properties and, as a result, has led to tremendous improvements in their MRI performance. On the other hand, owing to their size, nanoparticles can penetrate cell walls13 and deliver biomolecules or drugs for therapeutic14 and diagnostic purposes.9 The use of Dalton Trans., 2011, 40, 6315–6343 | 6315

This journal is © The Royal Society of Chemistry 2011

Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT00689K

nanoparticulate (polymeric or inorganic) carriers allows the transport of hydrophobic low molecular weight drugs, to enhance their efficiency. Additionally, a feature that proves to be beneficial for the application of nanoparticulate systems is the fact that the blood vessels sustaining tumor tissue exhibit comparatively large fenestrations, and the lymph system of tumors is poorly operational. Therefore, macromolecules and nanoparticles leaking from these blood vessels can accumulate inside the tumor tissue, a phenomenon known as the EPR (enhanced permeability and retention) effect (see Fig. 1).15,16 However, one of the most prevailing issues associated with the application of these NP systems, is their behavior in vivo. For instance, the recognition and clearance by the reticuloendothelial system (RES) is a major obstacle since it reduces the circulation times of the NPs within the blood stream, and therefore, obstructs a site specific accumulation of the administered NP probes at designated areas (see Fig. 1).17 The enhanced uptake in the liver, spleen, and bone marrow is largely attributed to the macrophages residing in the tissues, which are responsible for clearing particulates and macromolecules circulating in the blood. When nanoparticles are administered intravenously, a variety of serum proteins bind to the surface of the nanoparticles, which are recognized by the scavenger receptor on the macrophage cell surface and internalized, leading to a significant loss of nanoparticles from the circulation.18 The serum proteins binding on the nanoparticles are termed “opsonins”, and the macrophages contributing the major loss of injected dose are constituents of the RES or mononuclear phagocyte system (MPS). Minimizing protein binding is the key point for developing a long circulation nanoparticle formulation.

their surface properties, including morphology, charge and surface chemistry. As a consequence, efficient strategies for the synthesis of magnetic nanoparticles and their subsequent surface modification are necessary to meet the challenges of a later application in biomedicine. This review article summarizes some recent achievements in the rapidly evolving fields of nano-biotechnology and nanomedicine. It consists of three parts; in the first section the properties and synthesis of magnetic NPs will be discussed briefly before in the second part some of the most common surface modification strategies are presented. Finally, the last section addresses the application of magnetic NPs in biomedicine.

1. Magnetic properties
Magnetic nanoparticles are among the most investigated nanomaterial systems, owing to the fact that their magnetic properties dramatically depend upon their size and their morphology. Several issues are responsible for these unique properties: finite size effects, which result from the quantum confinement of the electrons inside the material, and surface effects caused by symmetry breaking of the crystal structure at the boundaries of the particles.20–23 Concerning finite-size effects, the magnetic properties of (ferro-) magnetic nanoparticles are dominated by two key features: (1) The single domain limit and (2) the superparamagnetic limit, which both lead to individual material-dependent length scales, i.e. the single domain size and the superparamagnetic size. Both features will be discussed briefly. 1.1. Single-domain-limit A ferromagnetic bulk material usually consists of many separate areas, in which all magnetic moments of the constituent atoms are pinned in the same direction. The reason for such an arrangement arises from the fact that the magnetostatic energy (DE MS ) of the materials is lowered once a large domain is broken up into several smaller domains. However, the formation of new domain walls requires energy (E D ), and therefore, there is a size limit, below which the energy needed for the creation of a smaller domain exceeds the amount of energy gained from decreasing the magnetostatic energy. Consequently, this means that a magnetic nanoparticle with a diameter comparable to, or lower than the size of the smallest possible magnetic domain, could only consist of a single domain, which, in turn, results in a narrowing of the magnetic hysteresis curve compared to the bulk material (see Fig. 2).22,24,25 1.2. Superparamagnetic limit To understand the super-paramagnetic effect the magnetic anisotropy energy per particle (E(H)) which pins the magnetic moments of a single domain particle in a certain direction, has to be considered. E(H) is proportional to K a V (V is the particle volume), the energy barrier which stops the magnetic moment flipping from one direction to the opposite. K a V is usually much higher than the thermal energy kB T, however, with decreasing particle size K a V decreases to values equal to, or below kB T. As a result, the magnetic moments are able to overcome the energy barrier and freely flip in any direction, i.e. for kB T ≥ K a V the This journal is © The Royal Society of Chemistry 2011

Fig. 1 Passive tumor targeting due to the EPR effect. Larger particles and agglomerates are rapidly attacked by phagocytes, whereas smaller particles can travel longer through the blood vessels to reach the target tissue. Once at the tumor site, the magnetic NPs accumulate inside the tumor tissue due to the EPR effect.

In addition to that, controlling the overall size of the NPs is crucial, since particles with a mean diameter below 5 nm are usually eliminated by renal excretion, whereas larger particles (>100 nm) are taken up easily by macrophages.19 Furthermore, the ultimate fate of nanoparticles within the body is determined by the interaction with the local environment inside biological systems, which depends not only on the particle size but also on
6316 | Dalton Trans., 2011, 40, 6315–6343

27. system behaves like a paramagnet. the thermal energy of the particle decreases until kB T is lower than the energy barrier K a V (kB T £ K a V ). 6315–6343 | 6317 Fig. Then. in the presence of a paramagnetic substance the excess proton energy is released more efficiently and therefore. the magnetization in field direction reaches a higher value M z ¢¢ (M z ¢¢>M z ¢). the signal intensity relies on how fast M z is restored. inhomogeneities of the external magnetic field. and therefore.3.29. More precisely. leading to a decline of the magnetization. 2011. After excitation with a 90◦ radio frequency pulse the magnetization in the field direction M z is zero and the perpendicular in-plane magnetization M xy is maximal. leads to a faster and slower precession in the xy-plane. surface effects become more pronounced with increasing surface to volume ratio. in which the magnetic moments cannot flip freely. with which M xy is depleted. increases the particles are able to move their moments in the direction of the external field.rsc. decreasing particle size. However. an energy exchange between the spins induced by local magnetic field interferences among the spins themselves (spin–spin or transverse relaxation). in T 2 -weighted images the signal intensity Dalton Trans. with respect to the bulk value. T B can be determined by measuring the magnetization of a nanoparticle sample versus increasing temperature under an applied external magnetic field.22. Basically it is desired to shorten the relaxation times because this leads to a greater difference in signal intensity and thus to a better contrast in the MR image.26 As an example. Directly after excitation all spins are in phase and M xy is maximal.1039/C0DT00689K 1..e.30 T 2 : Transverse relaxation. In principle. including field strength. On the other hand. The symmetry breaking at the particle boundary can lead to variations in the electronic band structure. the net magnetization rises. it is also called spinlattice (or longitudinal) relaxation. This process is associated with a release of energy to the environment (the “lattice”). This reduction has been associated with different mechanisms. reported. and atom coordination and.25 Furthermore. Surface effects Surface effects depend directly on the ratio of surface spins to bulk spins. is therefore independent of the applied magnetic field. the net magnetization is zero. this phenomenon is called superparamagnetism. arising from the magnetic coils of the MRI scanner itself or the body of the patient. or the This journal is © The Royal Society of Chemistry 2011 .25 Experimentally. 40. such as the existence of a magnetic dead layer on the particle surface. their magnetic moments are distributed homogeneously.30 Just as NMR spectroscopy. or the nature of the observed tissue. as the temperature. Magnetic NPs as contrast agents for MRI MR imaging is one of the most powerful non-invasive imaging techniques in clinical use today.30 The contrast in MR images is mainly determined by three different features: the proton density.existence of canted surface spins. The time constant. lead to an additional dephasing and a faster decay of M xy with a time constant T 2 *. 1. 2 Illustration of the single domain limit. lattice constant. Basically. two components determine the dephasing process: On the one hand. above T B the thermal energy exceeds K a V . as a direct result. As a result. On the other hand. therefore. While the proton density is given by the chemical and physical nature of the tissue. there are two different types of relaxation depending on the nature of the corresponding interactions: T 1 : Longitudinal relaxation. Since the particles were “frozen” from room temperature. it is based on measuring the relaxation of protons in an external magnetic field after they have been excited with a radio-frequency pulse. As a result. the T 1 time and the T 2 time of the regarding tissue. antiferromagnetic NiO and MnO nanoparticles exhibit increasing net magnetization values and higher magnetic blocking temperatures with decreasing particle size.21 Additionally. the reason being the larger number of uncompensated surface spins. Bødker et al. The temperature at which kB T = K a V is therefore called the magnetic blocking temperature T B . Therefore. surface effects can lead to a decrease of the magnetization of small particles.29.org | doi:10. i. the particle undergoes a magnetic transition from a superparamagnetic to a blocked state.e. when the temperature is lowered for a given superparamagnetic particle. in a so called zero-field-cooled/field-cooled (ZFC/FC) experiment. M z has only recovered to a value M z ¢ if no contrast agent is applied. at which the energy is depleted (and M z increases) T 1 . for instance ferromagnetic oxide nanoparticles.28 Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. As a result. depends on different factors. an external field (generally 100 Oe) is applied and the sample is slowly heated up while simultaneously measuring the magnetization. T 1 enhancement leads to a brighter (positive) contrast in the image. In T 1 -weighted images. change the magnetic properties. On the other hand. the use of contrast agents can greatly enhance both T 1 and T 2 contrast. that the magnetic anisotropy K a of iron NPs increases with decreasing particle size. After a given time period t following proton excitation. The time constant T 2 .4. due to a higher contribution of the surface anisotropy K aS . the sample is first cooled down to a temperature well below the expected blocking temperature (usually 5 K). The loss of the transverse magnetization M xy is also associated with a dephasing of the spins precessing in the xy-plane. Since the individual atomic moments add up to one (super) moment for every particle. Over time. On the other hand. i. the ZFC curve passes a maximum at T B .20. As mentioned before. the magnetization returns into the field direction and M xy decreases.

that the surface characteristics of magnetic NPs (i. However. such as MFe2 O4 (M = Mn. In this respect. In the first phase. since these properties are strongly dependent on the dimensions of the nanocrystals. Co. However.84 The first research efforts associated with the formation of monodisperse colloidal particles were carried out in the 1940s. making them promising candidates for contrast enhancing purposes.rsc.1. Furthermore.org | doi:10.49–52. other nanomaterials are also objects of increasing scientific interest as MRI contrast agents. the monomer concentration increases gradually until it reaches the point of supersaturation (S).9 Unlike X-ray contrast agents for computer tomography that rely on the direct absorption of X-ray radiation. the whole nucleation and growth process can be divided into three phases (see Fig. Mn2+ or Fe3+ ). Although superparamagnetic iron oxide NPs (SPIONs) show a strong T 2 effect. that. the synthesis of uniformly-sized NPs (i. lead to a faster recovery of M z (see above). is very much higher for micellar coated compared to polymer-functionalized particles using the same size nanoparticles. Simple metal nanoparticles such as iron..57 as well as metal oxides. In phase II. is called relaxivity (r1 = 1/T 1 and r2 = 1/T 2 . homogeneous nucleation throughout the entire reaction solution can take place any time.e. but also for their many possible technological applications.86 Although this model was actually developed for sulfur hydrosols and oil aerosols. i. fat.52. or by labeling Fe3 O4 NPs with Gd-complexes. there are numerous reports in which they can also be applied as effective agents for the shortening of the T 1 relaxation time.74–79 NiPt. proteins) and the unpaired electrons. As a consequence. a large number of nuclei are formed simultaneously This journal is © The Royal Society of Chemistry 2011 Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. However. Zn). Consequently. If no seeds (like dust particles or small crystallites) are present. and hence.25 As mentioned in the previous section. at a given time t) resulting in a darkening of the corresponding MR image compared to images where the contrast agent is absent. superparamagnetic NPs have a much higher magnetic moment (compared to free ions 100–10. nucleation and growth.g. Consequently. In fact. size distribution. without further nucleation. In general. solutions of metal ions. numerous strategies have been developed to synthesize them with different compositions and homogeneous size distributions. either by applying special measurement techniques. and thus in a faster dephasing of the in-plane spins.61–64. T 1 agents require a low r2 /r1 ratio. It is in fact surprising.43–48 2. iron oxide NPs are already in clinical use as T 2 contrast agents. However. like Gd3+ . superparamagnetic MnO nanoparticles have 6318 | Dalton Trans. arises from the creation of local magnetic fields. not only for their fundamental scientific interest. the supersaturation finally reaches a critical value (Sc ). like Fe3 O4 . 6315–6343 recently attracted considerable attention as positive (T 1 ) contrast agents.e. On the other hand. The measure.58–60 g-Fe2 O3 . 2.70. whereas for T 2 agents it should be large). a narrow size distribution is necessary to efficiently exploit the magnetic properties for MRI. synthesis in (reverse) micelles. The most favorable synthetic approaches usually comprise co-precipitation.80. The degree of performance is strongly dependent on the size.35–38 Apart from iron oxide NPs. only homogeneous nucleation is possible. most commercially available contrast agents consist of paramagnetic substances with a large number of unpaired electrons and high magnetic moment (e. the effect of (super)paramagnetic substances in MRI is oblique. since the energy barrier for the initiation of a homogeneous nucleation event is considerably high. Here. which results in increased field-fluctuations. For example. the main factors that determine this process. This change depends on interactions between the protons of the tissue (water. by which the performance of MRI contrast agents is evaluated.83 have been prepared by these methods. i.e. degree of ligand loading) also play a crucial role for the MRI contrast enhancing abilities. the magnetic moment in the “inner sphere” (T 1 ) or the “outer sphere” (T 2 ) of the contrast agent.58.52.28 cobalt oxides.27 mixed metal oxides with spinel-type structure. has been intensively investigated.81 and CoPt3 82.39–42 However. which means that the system contains enough energy to overcome the energy barrier (see Fig.53 manganese oxides. the effect of MRI contrast agents is based on influencing the resonance properties of the tissue by changing the local magnetic field inside the body. 2011. it can easily be transferred to nanoparticulate systems. T 2 agents need to have a much larger magnetic moment than T 1 agents. this effect is strongly related to the ability of water molecules to enter the periphery of the agent. During that time. the presence of a contrast agent leads to a faster dephasing of the spins that precess in the xy-plane.71–73 or intermetallics such as FePt.1039/C0DT00689K . the monomer concentration can further increase (phase II). on the other hand. 3). Therefore.e. and thus the magnetic moment. 2).e. General considerations A key issue for the preparation of monodisperse magnetic nanoparticles is to understand the basic formation mechanism of nanocrystals in solution. per mol of agent). and thermal decomposition and/or reduction of appropriate metal–organic precursors.is determined by the decrease of M xy . it was shown by Weller and coworkers. Additionally. by reducing the particle diameter. Eventually. is essential to produce colloids with a narrow size distribution. LaMer and coworkers revealed. nature of surface ligands. Synthesis of magnetic nanoparticles For more than 30 years. i. The impact of T 2 agents. although many different synthetic strategies are known today (see above). gadolinium-based NPs have been extensively investigated as positive (T 1 ) contrast agents. hydrothermal synthesis.g. therefore. the ratio of T 2 to T 1 relaxivity (r2 /r1 -ratio) can also be used to estimate the contrast enhancing potential (e.65. composition and crystallinity of the NPs. nanomaterials exhibit physical and chemical properties that are quite different from their bulk counterparts. that a short nucleation burst followed by slow controlled growth. in particular r2 *. the preparation of nanocrystals.000-fold). paramagnetic T 1 contrast agents withdraw the excess energy after proton excitation. monodisperse with a standard deviation of the size distribution s £ 5%) is of key importance. crystalline materials with dimensions in the range between 1– 100 nm.53–55 cobalt56 and nickel.66–69 or NiO. which would initiate heterogeneous nucleation. the comprehensive understanding of nanoparticle generation and the structure of the nuclei are still very limited. 40. M xy drops faster (M xy ¢<M xy ¢¢.85.31–33 As already stated in the previous sections the particle size and composition mainly determines the magnetization and. According to the LaMer model. will be discussed briefly.34 The authors demonstrated that the transverse relaxivity.

respectively. since the surface free energy is much higher in this size range. the high reproducibility and high yields that can easily exceed multi kilogram amounts.92 The effects of several organic anions. The most common techniques to prevent these unwanted features use surfactant molecules to stabilize even the smallest NPs in solution.1039/C0DT00689K Fig. or even completely amorphous. 2011. the This journal is © The Royal Society of Chemistry 2011 Fe2+ /Fe3+ ratio and the pH. are usually poorly crystalline. such as carboxylate and hydroxy carboxylate ions. the monomer concentration drops drastically below a point where no further nucleation is possible. (a) Mn-ferrite NPs (with permission from ref. results in uncontrolled growth and particle agglomeration.89.3. making the NPs stable in acidic and basic media over long periods of time.91. and therefore. thiols and carboxylates.50. 40. not only. and. providing. and composition can be produced in a highly reproducible fashion Since Fe3 O4 nanoparticles are subject to oxidation and dissolution in an aqueous environment.97 However. but also long-term stability in non-polar solvents. 91). This is achieved by placing the mixture inside a sealed high-pressure reaction vessel (autoclave). the NPs will end up having an exceptionally narrow size distribution. in phase III. caution must be taken and the statements must be judged critically. requiring a subsequent annealing step. which. all nuclei grow at the same time. 6315–6343 | 6319 . and other metal (M(II)) salts (e. although it is feasible to apply most of these considerations to nanoparticle models. Finally.90 2. which makes particle agglomeration unavoidable. morphology.25 The major drawback.. These molecules usually consist of a long hydrophobic hydrocarbon chain with a functional endgroup. consequently. In some cases the solvent is heated Dalton Trans. which cover the surface of the nanoparticles during the synthesis. 3 LaMer plot illustrating the separation of nucleation and growth during the synthesis of monodisperse NPs. oleic acid is by far the most often used stabilizing agent for magnetic NPs.88.93–95 The products of coprecipitation reactions.g. Co-precipitation Fig.87 In fact.58. 4).64. once all parameters are set.88. Since magnetization and blocking temperature strongly depend on nanoparticle size. cannot be considered to be constant. conducting the co-precipitation reaction at higher temperatures (50– 100 ◦ C) automatically leads to condensation of the precipitated metal hydroxides to form crystalline metal oxides. a narrow size distribution is essential to fully exploit these properties in subsequent applications. as a result. in a process that can best be described as a nucleation “burst”.2. Solvothermal/Hydrothermal synthesis Solvothermal/hydrothermal strategies have been used to produce a large variety of nanomaterials. Partial control over the size distribution can be achieved by addition of organic stabilizing agents. 4 Magnetic NPs synthesized by co-precipitation of metal salts in basic aqueous solutions. and mixed ferrite (MFe2 O4 ) nanoparticles is the co-precipitation of iron(II)-. and. As a consequence. particles of different size. it should be noted at this point.100–107 The key idea behind these processes is to bring the reaction mixture to temperatures well above the boiling point of the respective solvent. nitrates) in aqueous solution by addition of a base (see Fig.org | doi:10. temperature. since their growth histories are identical. the high ratio of surface atoms to bulk atoms in small NPs leads to an extremely high driving force of these particles to minimize the surface energy. on the formation of iron oxides or oxyhydroxides have been studied extensively. (b) Co-ferrite NPs (with permission from ref. Therefore. chlorides. protection against oxidation. Other authors have reported that the use of various organic salts like sodium citrate or tartrate can control the dispersity of the product (see Fig. One of the most preferred methods to produce magnetite (Fe3 O4 ). that strongly attaches onto the surface of the regarding nanomaterial. maghemite (g-Fe2 O3 ). a hydrophobic shell is formed around each particle. is a poor control of the size distribution. However. 92) (c) Fe3 O4 NPs (with permission from ref.98. however.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. 93). A better dispersibility in water can be obtained by subsequent acidification of the particle surface with nitric acid or by addition of tartrate ions. amines.99 2. that this theory has been derived for microparticle systems and. On the other hand. Typical functional endgroups include phosphines.92 The major advantages of this method are. 4b). iron(III). controlled oxidation to the more stable maghemite (g-Fe2 O3 ) is often conducted as a follow-up step during nanoparticle synthesis.rsc. sulfates. like polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA). S and Sc are supersaturation and critical supersaturation.96 Depending on the metals salts used as starting materials. especially those performed at or near room temperature. it is especially interesting for industrial scale applications.

101). like Na2 HPO4 and Na2 SO4 .105. solvothermal processing allows many inorganic materials to be prepared at temperatures well below those required by traditional solid-state reactions. Co.. Microemulsions. in which a mono-layered film of surfactant molecules stabilizes each microdomain of both liquids. or non-ionic.25. (b) Mn3 O4 NPs prepared from KMnO4 in ethanol–water (with permission from ref. Commonly used surfactant molecules are either ionic. The preformed a-Fe2 O3 nanorings could easily be converted into gFe2 O3 and Fe3 O4 by reduction in a hydrogen/argon atmosphere. With this understanding. Preparation within micelles Another strategy that is often applied for the synthesis of magnetic nanoparticles uses micelles as “nanoreactors” in (reverse) microemulsions.above its critical point. 5d). because the solubility and reactivity of metal salts and complexes is high enough. A vast variety of different nanoparticles have been synthesized using (reverse) micelles. even under more moderate subcritical conditions. In any event.27 reported the successful synthesis of 6–14 nm MnO and NiO nanoparticles by decomposing manganese cupferronate Mn(C6 H5 N2 O2 )2 and nickel cupferronate Ni(C6 H5 N2 O2 )2 in the presence of trioctylphosphine oxide (TOPO) as capping agent under solvothermal conditions. 6315–6343 . 2. Ghosh et al. in most cases it is not necessary to heat the solvent above its critical point.108–110 The solvothermal synthesis of magnetic metal oxide nanoparticles can be performed starting from different metal precursors. and in contrast to conventional emulsions. the addition of stabilizing molecules can control the particle morphology and significantly narrow the size distribution. microemulsions are thermodynamically stable isotropic dispersions of two immiscible liquids. at 220 ◦ C. g-Fe2 O3 and Fe3 O4 were prepared by hydrothermal treatment of FeCl3 with certain additives. where it becomes a supercritical fluid.g. larger nanoparticles of hausmannite (Mn3 O4 ) and nanorods of manganese oxidehydroxide (MnOOH) were synthesized using KMnO4 as precursor in a solution of water and ethanol (see Fig.118 After completion. consisting of octane and hydrazine as reducing agent in water.103 Monodisperse magnetite nanoparticles with an average size of 39 nm were synthesized by co-precipitation of ferrous Fe2+ and ferric Fe3+ ions in water with tetramethylammonium hydroxide (N(CH3 )4 OH) at 70 ◦ C followed by hydrothermal treatment at Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. are the very high crystallinity of the resulting nanomaterials. 5 Magnetic NPs synthesized by hydro-/solvothermal reactions. Since the size and size distribution of the synthesized nanoparticles strongly depends on the diameter of the micelles. By definition. 102) and nearly monodisperse NiFe2 O4 NPs prepared from the metal nitrates at a water–toluene interface with oleic acid as capping agent (with permission from ref. cyclohexane).4. like cetyltrimethylammonium bromide (CTAB) or sodium dodecylbenzenesulfonate (NaDBS). Mn) and g-Fe2 O3 were obtained at the water–toluene interface under conventional and microwave-assisted hydrothermal conditions using metal nitrates and chlorides and oleic acid as capping agent (see Fig. the size of the NPs could be controlled by variation of the reaction conditions. where the aqueous phase is dispersed as microdroplets (typically 1–50 nm in diameter) in a non-polar hydrocarbon phase (e. Supercritical fluids exhibit very high viscosities and are able to dissolve compounds that have a very low solubility under ambient conditions.99. Similar to the co-precipitation process. a straight proportional relationship between the effective micelle radius and the molar water-to-surfactant ratio w 0 is evident. In some cases an additional cosurfactant is also applied.106 The use of hydrothermal techniques also allows the formation of unusual morphologies.Fe2 O3 nanoplates synthesized from iron nitrate precursors in ethanol (with permission from ref.104 However. that the size of the reverse microdroplets can be adjusted by varying the molar ratio of water to surfactant:116 w 0 = [H2 O]/[S] With this equation. 100). were employed to create This journal is © The Royal Society of Chemistry 2011 Fig. efficient control over the micelle radius is crucial to obtain high quality nanocrystals. (c) ZnFe2 O4 NPs synthesized from zinc granules and ferric chloride in aqueous ammonia (from ref. ranging from metallic or intermetallic materials to quaternary metal oxides. 40. 2011. In a stirred reverse microemulsion the micelles continuously collide. monodisperse NPs of MFe2 O2 (M = Ni.1039/C0DT00689K 250 ◦ C. fuse.112.92. 103).91. It has been shown in the past. Therefore. 5a) by reacting a solution of Fe(NO3 )3 ·9H2 O in absolute ethanol with PVP as stabilizing agent at 240 ◦ C. (a) g . The main advantage compared to other “low-temperature” procedures.100 reported the solvothermal synthesis of nearly monodisperse g-Fe2 O3 nanoplates (see Fig.113–115 A special kind of dispersion is the waterin-oil microemulsion. with CTAB as surfactant and 1-hexanol as co-surfactant. but it can also be accelerated by heating the organic solvent to reflux. Here. a reaction system containing two or more precursors in individual aqueous droplets will eventually lead to intimate mixing of the reactants and initiation of the reaction. The aqueous droplets themselves are surrounded by a stabilizing monolayer of surfactant molecules. like Igepal CO-520 or Triton-X 100.101. Nanorings of a-Fe2 O3 .111 On the other hand.96. and break up again. Fe3 O4 nanoparticles with tunable sizes were prepared under hydrothermal conditions using FeCl2 ·4H2 O and ammonia. like coprecipitation and sol–gel processing.117 The nanoparticle formation itself is usually conducted at room temperature. Lu et al.rsc. 6320 | Dalton Trans.org | doi:10. Detailed descriptions of the theory of solvothermal/hydrothermal processes can be found in recent review articles. the product can be isolated by breaking up the micelles with different solvents such as methanol or acetone followed by filtration or centrifugation. a precise control of the micelle size is possible.107 As a further example. Furthermore. 5b).97 Among the metal complexes cupferronates are the most frequently used compounds.

this decomposition does not necessarily lead to the formation of nuclei. all reactants are mixed at room temperature and subsequently heated at a well defined heating rate. Furthermore. Zn. The initially formed Fe nanoparticles were subsequently oxidized by addition Dalton Trans. of the spinel structure are among the most important magnetic nanomaterials and have been widely used for various applications. Cu.g. The size of the particles could be controlled from 3–17 nm by adjusting the precursor/surfactant ratio. the concentration of these clusters increases until it reaches a critical point of supersaturation (see Fig. (e) Two. As the temperature rises. In some cases. because a precise separation of nucleation and growth would be difficult. heating rate. one of the most cited approaches for the synthesis of intermetallic NPs.1039/C0DT00689K fcc-structured nickel nanoparticles. which show promising properties for magnetic resonance imaging (MRI). a combination of reverse microemulsion and microwave assisted synthesis was recently used to produce manganese-based nanoscale metal–organic frameworks. (c) HR-SEM image of a ª 180 nm thick layer of assembled 4 nm Fe52Pt48 nanocrystals. oleylamine and 1. zero-valent precursors.119 Ternary 3d metal oxides. Fortunately. the average size of the resulting nanoparticles can be controlled by adjusting the molar ratio of water to surfactant. Once the reaction mixture has reached a temperature that allows sufficient growth. for the reproducible synthesis of highly crystalline magnetic nanoparticles with a narrow size distribution. the large scale synthesis of uniform magnetite (Fe3 O4 ) nanoparticles was achieved by precipitation from ethanolic FeCl2 /Fe(NO3 )3 solution in xylene with NaDBS under reflux conditions. 2011.and (f) three-dimensional superlattices of monodisperse MnO NPs prepared from Mn oleate. n-hexanol. the degradation of the precursor molecules rather leads to the formation of molecular clusters that can be viewed as monomers for the generation of NPs.and three-dimensional superlattices (see Fig. the preparation of metal nanoparticles followed by mild oxidation is also applied in some cases. further size selection techniques.2-hexadecandiol in octyl ether. the reduction of metal salts with suitable reducing agents. etc. However. is the preparation of monodisperse FePt NPs and their self assembly into two. such as metal carbonyls are preferably used. Mn(NO3 )2 and Fe(NO3 )3 ) in toluene with NaDBS as surfactant. For the synthesis of metal NPs.122 In contrast to that. metallic NPs show higher magnetization values than metal oxides. water and cyclohexanol with (NH4 )2 CO3 and NH4 OH as precipitants. monodisperse maghemite nanoparticles have been synthesized by injecting iron pentacarbonyl (Fe(CO)5 ) into a hot solution of oleic acid.5. 6a–d). are obtained when metal–organic precursors are decomposed or metal salts are reduced in high-boiling non-polar solvents and in the presence of surfactant molecules.54 On the other hand. the separation of nucleation and growth according to the LaMer concept (see above). 6 (a) and (b) TEM images of three-dimensional assemblies of 6 nm as-synthesized Fe50 Pt50 NPs. Sun and Murray were among the first to synthesize metallic nanoparticles by a variety of methods.org | doi:10. like the quaternary CoCrFeO4 could be synthesized by a similar approach using aqueous solutions of CoCl2 . The first method is based on the simultaneous formation of many nuclei when the precursor is injected.131 were able to prepare monodisperse e-Co nanoparticles by rapid pyrolysis of dicobalt octacarbonyl ([Co2 (CO)8 ]) in the presence of a surfactant mixture containing oleic acid.129. Mg. no considerable growth occurs. long chain amines and thiols are generally used as surfactants to stabilize the as-prepared particles from oxidation and agglomeration. Yet. As a result.127.50. can also lead to the formation of metal nanoparticles. like hydrides or alcohols. In fact. heating time.121 A more exotic example was given by Xu et al. the temperature and the injection time. who prepared BaFe12 O19 nanocrystals in a microemulsion system consisting of CTAC. Ni. 40. For nanoparticles synthesized in this way the standard deviation of the size distribution (s) is This journal is © The Royal Society of Chemistry 2011 usually around 10%.118 Additionally. a large number of nuclei are formed.132 The size of the particles could be controlled by varying the chain length of the alkyl group in the trialkylphosphines.85 can easily be achieved by controlling the basic experimental parameters like reaction temperature. like repetitive precipitation. 6315–6343 | 6321 . if not impossible.124 In the second procedure.). 2). Co. The key issue for the production of monodisperse nanoparticles. until the decomposition temperature of the precursor is reached. CrCl3 and Fe(NO3 )3 in toluene with NaDBS. Alivisatos et al. Among magnetic nanomaterials.126 Some of the most popular precursors and additives for the synthesis of magnetic nanoparticles will be discussed in the following.120 and. e-Co NPs by injecting superhydride (LiBEt3 H) into a hot solution of CoCl2 in octyl ether with oleic acid and trialkylphosphines as surfactants. Precursors with cationic metal centers are preferably used for the synthesis of magnetic metal oxide nanoparticles. however.rsc.130 For instance. lauric acid. 74).125 In most cases particle growth can be stopped by rapidly reducing the temperature. MFe2 O4 (M = Mn.123 2. For instance.g. A common approach for the synthesis of spinel ferrites comprises the combination of aqueous solutions of metal nitrates (e. e. or Cd. since the temperature only increases slowly. however. Thermal decomposition and/or reduction By far the best results. and concentrations of precursors and surfactants..50 The required short nucleation burst can basically be initiated in two ways: either by injecting a cold precursor solution into a hot mixture of solvent and surfactants.. (d) HR-TEM image of 4 nm Fe52Pt48 NPs (with permission from ref. even more complex metal oxide systems.128 Fatty acids.73. Fig.53. can lower s below 5%. and trioctylphosphine. iron NPs were prepared by a similar method. or by heating up a mixture of all components at the same time in a controlled fashion. as mentioned before.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. all particles grow at the same rate leading to a homogeneous size distribution.

Furthermore. 3. as-prepared nanoparticles are insoluble in water and a direct application of these unmodified particles into aqueous solutions would inevitably lead to particle aggregation and precipitation. not only a high colloidal stability in aqueous environment.53 However. The high chemical reactivity of the NP surface could pose health risk to patients.28. Furthermore.28 3. Therefore. since the NPs could already influence reaction pathways on the cellular level.62 and acetylacetonates61 have evolved as the most common precursors for the preparation of metal oxide particles. their vulnerability towards oxidation becomes more pronounced with smaller particle sizes. it is necessary to exchange the hydrophobic protection shell by hydrophilic ligands.137. Co and Ni (and their alloys) are extremely sensitive to air. these nanoparticle aggregates would pose an immediate threat to health. that during the heating procedure a complex of the metal ion and the surfactant is formed. all known surface modification approaches result in a core–shell-like structure. 3.rsc. 6322 | Dalton Trans. since they would clog up small blood vessels and obstruct a free blood circulation. metal organic compounds. that the use of surfactant molecules is essential to prevent the particles from aggregation and rapid oxidation. but also prevent opsonization by serum proteins under physiological conditions. a much better control over the reaction process was obtained. All the worse.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.1039/C0DT00689K of trimethylamine oxide ((CH3 )3 NO) as mild oxidant. Due to their exceptionally narrow size distribution they self-assemble to form two-dimensional and even threedimensional superlattices. 2011. Fig. Apart from surface passivation. Additionally. Amphiphilic micelles As explained in the previous section. surface modification with surfactant micelles. the colloidal stability of the NPs under physiological conditions is another important issue.2. a new strategy was recently proposed.org | doi:10. As an example. like Fe. which needs to be accomplished after their preparation. pure metal NPs. In this way. As described above. 6315–6343 This journal is © The Royal Society of Chemistry 2011 .134–136 This section will focus on some of the most common strategies for the protection and stabilization of magnetic nanoparticles (see Fig. 7).64. 40. as-prepared magnetic NPs are covered with a shell of hydrophobic surfactant molecules. that guarantee. Surface modification General considerations The previous section demonstrated that there have been numerous ground breaking developments in the synthesis of magnetic nanoparticles over the past decades. with the magnetic NPs being the core and some kind of inert organic or inorganic material being the protecting/stabilizing shell.. such as oleic acid or oleylamine. the synthesis of monodisperse and highly crystalline nanoparticles requires non-hydrolytic synthetic routes and the use of hydrophobic surfactants that efficiently cover the particle surface. It has already been pointed out. and in fact. Especially. the necessity of an efficient coverage of the nanoparticle surface becomes obvious when considering a potential use in biomedical applications. Inspired by the idea. precious-metal or carbon coating (which will not be discussed here). 6e and f show monodisperse manganese oxide (MnO) NPs synthesized by decomposition of a manganese oleate precursor in 1-octadecene.133 The latter is often referred to as the “stealth effect”. unprotected inorganic nanoparticles are prone to opsonization. the most important issue. which needs to be addressed if an application in medical or biological respect is anticipated. However. even if particle agglomeration could be prevented.138 The functional group of these Fig. such as formates. a defense process initiated by the innate immune system.73 By directly using a preprepared metal–surfactant complex. is to provide the nanoparticles with a sufficient degree of stability. Therefore. if applied directly to a living organism.1. 7 Different strategies for the surface modification of magnetic NPs. bi-functionalor polymeric ligands or silica are the most prevailing methods. highly crystalline nanoparticles of different metal oxides with exceptionally narrow size distribution (s £ 5%) were synthesized.63 acetates. in which certain serum proteins attach to the particle surface and initiate phagocytosis. Interestingly.

The use of polymers for this purpose is reasonable. and biotin. which offer different functional groups. Consequently. there are reports where particle binding of polymeric ligands on Fe3 O4 nanoparticles was achieved via carboxylic.150. the nature of these groups depends on its affinity toward the metal constituent of the core material. 2011. such as peptides. that were developed in the following years. amines. such as the nasal mucosa. In this context. Dai and coworkers recently developed a similar PEGmodified phospholipid surface coating for graphite-protected FeCo NPs with a high magnetic moment. Another example. 6315–6343 | 6323 Fig. Polymeric ligands One of the most established methods to stabilize magnetic NPs in water or buffered solutions is provided by the use of polymers.1039/C0DT00689K to the outside. phosphate. like CTAB or oleic acid. uses the molecular structure of the hydrophobic layer for the incorporation of additional surfactant molecules. The hydrophilic endgroup of the amphiphile makes the material polar and fully dispersible in water.152–154 Furthermore. whereas the hydrophilic PEG chains point to the outside creating a hydrophilic shell. which offers. This journal is © The Royal Society of Chemistry 2011 .160–165 In addition. in 2009. by shifting the equilibrium they can be removed by washing and diluting.org | doi:10. in 2007 and Lee et al.143 The structure of these polymers usually consists of individual lipophilic and hydrophilic polar components. such as poly(maleic anhydride-alt-1-octadecene)-PEG block copolymer. chemical attachment to the NP surface can be achieved by using different functional groups. however. use amphiphilic polymers as surface coating ligands. dextran coated iron oxide nanoparticles can be cross-linked using epichlorohydrin.stabilizing agents are strongly attached to the particle surface by a coordinative bond. but also protection from oxidation and aggregation.169 In fact. NPs prepared in this way exhibit only moderate long-term stabilities and tend to agglomerate at higher salt concentrations. offer much stronger attachment to the nanoparticle surface.171 Dopamine is another anchoring group which Dalton Trans. there are many different amphiphilic polymers commercially available today. i. which would result in particle agglomeration and precipitation. the cationic trimethylammonium moieties point to the outside.rsc. poly(glycolic acid).155–158 PEGs have been extensively used as stabilizing materials for many NPs used on biomedical applications. that possess more than one anchoring group.139. 8). particularly in long circulating in vivo imaging systems. proteins. and on the other hand.3. PEG. one of the simplest approaches to enhance the solubility of hydrophobic NPs in aqueous solution. and particle aggregation is suppressed by steric repulsion. (b) Coating procedure: the hydrophobic lipid tails are incorporated between the alkyl chains of the capping molecules on the NP surface. creating repulsive forces and stabilizing the particles in suspension. thiols.148 Again. the non-polar hydrocarbon chains are bound to the hydrophobic graphite shell to form a stable double layer. leads to much larger hydrodynamic sizes than that of the starting material hampering an efficient application in living organisms. Suitable polymers include poly(analine). which leads to a complete encapsulation of the core and its original coating. perhaps the most common representative biocompatible polymer.159 For example. For example.151..142 Many of the existing strategies for NP phase transfer. poly(methylidene malonate).e. gold nanoparticles show a high affinity towards thiol groups.34. not only colloidal stability. like carboxylic acids. to increase the hydrophilicity of various inorganic NPs. “multidentate” polymers.166 Other examples for cross-linked polymer functionalized iron oxide nanoparticles include the use of chitosan and poly(ethyleneglycol)-co-fumarate.170. tetradecylphosphonate and PEG-2tetradecyl ether. Unfortunately. which uses a similar strategy for monodisperse MnO NPs. have been established in recent years.139–141 During this procedure.167.168 A variety of methods. or oligonucleotides The presence of multiple surface bilayers.43. According to Pearson’s concept of hard and soft acids and bases (HSAB). that use special chelating ligands. was demonstrated by Hyeon et al. poly(ethylene imine) and their copolymers. This surfactant addition strategy was first developed for the preparation of water-soluble quantum dots and was later also successfully applied for dispersing magnetic NPs in aqueous media. since they have the advantage that numerous functional groups can be applied on a single macromolecule. Their hydrophobic tails interact with the nonpolar surface ligand of the nanoparticles (see Fig.149 Related methods used a variety of amphiphilic polymers.144–147 Here. poly(lactic acid). that supports a resistance to protein adsorption and an ability to bypass the RES and natural barriers. or amphiphilic PEG-phospholipids. poly(pyrrole). the lipids act as the nonpolar constituent of larger amphiphiles. whereas the long alkyl chains point into the periphery. the possibility of cross-linking between the polymer chains leads to more rigid structures on the NP surface. the long alkyl chains of the CTAB molecules are integrated in between the C-chains of the oleate/oleylamine molecules due to attractive van-der-Waals interactions. 3. has received great attention due to its nonfouling property. the surfactant molecules are in a dynamic equilibrium with their unbound counterparts. 8 (a) Chemical structure of a PEG-phospholipid. The nanoparticles are assumed to remain encapsulated by the original protecting ligand shell that is never broken up. since the amphiphilic bilayer is only connected to the NP core by considerably weak van-der-Waals interactions. for the immobilization of various biomolecules. whereas manganese and iron oxide nanoparticles favor oxygen containing ligands. In principle. 40. including polystyrene–polyacrylic acid (PS-PAA) block copolymers. while the PEG chains are exposed Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.136. phosphonate and sulfate groups. creating an inert layer. and subsequently equipped with free amino groups to allow further modification.151 Additionally.

184 phosphonates. or fluorescent dyes (see Fig. As the name implies. the presence of large agglomerates cannot be excluded. Lattuada and coworkers used this method to design water soluble Fe3 O4 nanoparticles. These ligands consist of an anchor group conjugated to a hydrophilic linker carrying a functional endgroup. Furthermore. and excellent biocompatibility. anionic or neutral endgroups.172 in fact.186 and catechols. permits the successive substitution of the pentafluorophenol group by dopamine and other functional groups.174–179. 10 Surface modification of magnetic NPs using bi-functional ligands. However. stability. such as poly(pentafluoro phenylacrylate) (PPFA). Fig. different ligands could be used to grow polymer coatings with. 10). like 2. The NPs were coated with poly(lactic acid). A key requirement for a successful exchange of the hydrophobic capping agents is the complete dispersability of the NPs and the ligand molecules in the same solvent. due to stronger (mostly ionic) interactions between the bidentate (or. which impedes a suitable application for biomedical purposes. An early release or uptake of the nanoparticle/molecule system due to other surface reactions. 2011. than that of the former hydrophobic stabilizing agent. 9).rsc. 40.173 whereas thiols187 are preferred for metallic and intermetallic NPs.1039/C0DT00689K shows a high affinity towards metal and metal oxide surface.185. This journal is © The Royal Society of Chemistry 2011 . A major drawback for the use of polymers for the surface modification of magnetic nanoparticles is the fact that the presence of multiple anchoring groups facilitates the bonding to more than 6324 | Dalton Trans. PEG chains.173. Since the binding of the surface ligands is an equilibrium process with continuous attachment/detachment of the chelating groups.182 As an example.183 or tetramethylammonium hydroxide. in some cases. cell targeting and multimodal imaging. followed by esterification through acylation. the use of reactive ester polymers. 9 (a) Chemical structure of a typical multifunctional polymeric ligand.152 In that way. bi-functional molecules are generally composed of two parts: an anchor group. Typical anchor groups for metal oxide nanoparticles include carboxylates. multidentate) functional group and the metal-/metal oxide surface. A different approach to functionalize magnetic nanoparticles with polymers is enabled by atom transfer radical polymerisation (ATRP).183. Bi-functional ligands A key factor for in vivo tracking and targeting applications is a high stability of the bonding between functional molecules and the nanoparticle surface.4.45 one nanoparticle at a time. could be detrimental to the application or possibly to the patient. (b) Illustration of a magnetic NP modified with a multifunctional polymer.org | doi:10. Metal oxide nanoparticles functionalized with such polymers containing several dopamine moieties were used in different applications ranging from protein separation. 3. The co-polymer contains 3-hydroxytyramine (dopamine) as anchor group. (PEG). there is a non-neglectable possibility that one polymer chain simultaneously covers several particles.81 Owing to the multidentate coordination of the chelating moieties. which efficiently binds to the surface of the nanoparticle and a hydrophilic part which allows stability in aqueous media. the bi-functional ligand exchange strategy principally provides much better colloidal stability of the magnetic NPs under physiological conditions. One of the most often used methods for surface modification of magnetic nanoparticles is the exchange of the hydrophobic stabilizing ligands by hydrophilic bifunctional molecules (see Fig. including amines. and free amino groups for further functionalization. a fluorescent dye.. Compared to the amphiphilic micelle coating.180. the bonding strength of these polymers is generally very high. Although initial attempts to modify hydrophobic nanoparticles were carried out with simple bi-functional molecules. it has evolved as a very popular binding ligand. 6315–6343 Fig.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.181 With this method. the key requirement for an efficient attachment on the surface is a stronger binding strength of the anchor group.5-dihydroxybenzoic acid. which enabled the addition of halide moieties to transform the nanoparticle surfaces into macro initiators. owing to its outstanding solubility. As a consequence. polymers are grown on the surface of nanoparticles after previous treatment with an initiator.188 PEG has evolved as the most often used hydrophilic linker. either cationic.

208. which leads to dissolution of metal ions and. Sun et al. Furthermore.198 The use of two different binding groups is reasonable. eventually. allows the use of these core@silica nanomaterials for diagnostic and therapeutic purposes (e. the classical ¨ Stober synthesis. that occurs naturally in the adhesive protein of some marine mussels. giving the fact. various approaches have been exploited to fabricate silica coatings for colloidal nanoparticles. Cheng et al. and therefore. Once coupled to the humanized IgG1 monoclonal antibody Herceptin.197 Since the dopamine anchor groups were conjugated with a PEG linker. Wang et al. Here. are only soluble in non-polar solvents. Especially. The presence of pores on the surface permitted the slow diffusion of cisplatin inside the hollow structures.000 g mol-1 ) on uptake in macrophage cells. the cisplatinloaded hollow NPs were able to target SK-BR-3 breast cancer cells. Especially. where only one of them was used. other catechol anchor groups were also extensively investigated and found to be more reliable for biomedical applications.000. where two different anchor groups were used for the attachment of bi-functional ligands was provided by Hong et al.5. Additionally. whereas the opposite is the case for the thiol group. The particles themselves were prepared by controlled oxidation of Fe NPs. the surface of the magnetic particles was functionalized with a dopamine.55 The same group investigated the influence of the PEG-chain length (HOOC-PEG(n) -COOH. MRI).213 Over the years. these strategies have shown good results with various nanomaterials. The advantage of using silica as a coating material is based on its exceptional stability. catechols are known to be liable to oxidation under aerobic conditions. however. and it was confirmed. the above mentioned strategies are not applicable on these NPs. it has to be assumed that this protecting organic ligand shell around the nanoparticle is not densely packed. the binding interaction of dopamine with the surface of iron oxide NPs has been thoroughly investigated in the past. 40.197.4-dihydroxyphenylalanine (L-DOPA). reported a new approach for the targeted delivery and controlled release of the anticancer drug cisplatin using PEGylated porous and hollow Fe3 O4 NPs. forming dark and insoluble polymers. the need to start from water-soluble NPs.191 The authors revealed. These advantages make silica an ideal. Based on Pearsons’ hard and soft acids and bases (HSAB) concept.205 Additionally. the application of both ligands showed much better results compared to studies. and cell culture medium.. and thus.218 Although. 11). also reported the modification of monodisperse iron/iron oxide core/shell NPs with a dopamine-PEG (DOPAPEG) ligand.org | doi:10. An example. 3. as an example. Furthermore. silica coatings can equip magnetic NPs with many additional benefits. a group of naturally occurring compounds that show antifungal. 6315–6343 | 6325 . and therefore.212. high-quality monodisperse NPs are protected by a hydrophobic surfactant layer. M w (PEG) = 600. to which the drug molecules could easily be attached. there have also been critical remarks concerning the application of dopamine as anchor group for metal oxide NPs. the reason for this behavior is simply the circumstance. is the limitation of these processes to aqueous solutions. low-cost material to tailor the surface properties of various nanomaterials.205. ionic solutions. an amino acid. to the degradation of the nanoparticle itself. that the strong attachment was due to an improved orbital overlap of the five-membered ring and a reduced steric effect on the iron complex which can be obtained in a bidentate coordination. indeed show a high affinity toward the iron sites.189–191 It is a derivative of L-3. the downside.206 Apart from dopamine. and anticancer properties. From a technical point of view the easy regulation of the coating process. Consequently.173.2-diols.217 or the use of silane coupling agents. special focus has been addressed to catechols. for intermetallic FePt NPs. dopamine is one of the most considered high affinity binding groups to stabilize metal oxide NPs in water and physiological environment.200 but only a weak attraction toward platinum. However. i. In a similar way.203 However.g. e.192–195 Indeed. which Dalton Trans. while acting simultaneously as a dispersing agent for electrostatic colloids. FePt NPs coated with a protective iron oxide shell were stabilized with a dopamine-PEG ligand. and. the free carboxylic acid groups of the ligands permitted further conjugation of biomolecules.172.207 Careful selection of the different functionalities allowed close control of the hydrodynamic diameter and the interfacial chemistry of iron oxide nanoparticles. including water. In this report the PEG chains were terminated with free amino groups. processability combined with chemical inertness. the use of dopamine-PEG ligands can also be applied for more complex nanostructures. the potential applicability increases significantly. as well as the slow release of the drug under physiological conditions. Silica coating The surface modification strategies presented in the previous sections relied on the coordinative attachment of organic ligands carrying functional groups to the metal atoms on the particle surface.178. The ligand was synthesized by a simple EDC/NHS coupling reaction of dopamine with a PEG-dicarboxylic acid. controlled porosity. that catechols. permits diffusion of water molecules to the surface. The modified Fe/Fe3 O4 core/shell NPs were stable in water and phosphate buffer solutions. 6.196. once it is attached to the surface of iron oxide NPs. and optical transparency are the most important factors. such as high biocompatibility and the possibility of subsequent functionalization (see Fig. some authors reported on rather rapid degradation of dopamine due to a surface reaction.rsc. as well as other 1.g. that macrophage uptake decreased with increasing PEG chain length (M w (PEG)). The NPs were stable in various aqueous media. 20.211 Thus. followed by acid etching to create porous This journal is © The Royal Society of Chemistry 2011 Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. 2011.1039/C0DT00689K Fe3 O4 nanocrystals with spherical cavities in the middle. to PEGylated Fe3 O4 NPs. Hence. that both molecules on their own would only ensure poor binding strength on FePt. coupled chromones.199.173.198 In a similar way.000.Among the above mentioned anchor groups.e.214–216 the formation of biosilica coatings. cations and positively charged molecules can be linked covalently to the polymeric silica layer at silica water interfaces under basic conditions. antiviral.209 In addition. the authors demonstrated that the drug could be released by variation of the pH value. 3.202 The resulting particles exhibited high cytoxicity due to the presence of the FePt core and strong T 2 MR contrast enhancement due to the presence of an iron oxide shell.210. especially in aqueous media. the functionalized iron oxide NPs were readily soluble in aqueous solution and stable over long periods of time.and a thiol-containing PEG ligand. An alternative method. antihypertensive. the silica layer provides steric and electrostatic protection for the cores.201.204 However. As already stated above.

orientated.238 In most approaches. leading to a possible degradation through metal ion leaching. makes them promising candidates for future cancer diagnosis and therapy. the thickness of the silica shell can easily be controlled.org | doi:10. discrete and monodisperse mesoporous silica nanoparticles consisting of a single Fe3 O4 nanocrystal core and a mesoporous silica shell were prepared (see Fig. whereas the outer mesoporous (m) silica shell not only offers a high surface area for the derivation of numerous functional groups. artificial liposomes were suggested as carriers of proteins and drugs for the treatment of various diseases. 6326 | Dalton Trans. are constantly and rapidly exchanged between different droplets. Already in the 1960s. around which the silica shell is polymerized. Furthermore. biomacromolecules or even functional nanoparticles. nano-biotechnology and nanomedicine are offering numerous exciting possibilities for the future. 12). which limits the size of the nanocomposites to diameters larger than 50 nm.224–230 Another advantage of silica coating of magnetic NPs is the opportunity to create a mesoporous shell.. has become quite popular recently.223 As microemulsions are thermodynamically stable systems (as opposed to emulsions). 13a–e). Subsequently. The silica coating procedure is then initiated by adding the desired silanes as precursors. as well as a reduction of quenching effects.Fig. accessible mesopores and high colloidal stability are highly valuable for biomedical applications. The hydrophobic NPs and surfactant molecules are dispersed in a non-polar solvent. Unfortunately. both the content of the droplets and the surfactant molecules at the interfaces.232–236 Hyeon and coworkers designed magnetically separable high-performance biocatalysts by cross-linking enzyme molecules on the surface of the Fe3 O4 @SiO2 nanoparticles. The advantage of this practice is a prolonged stability and higher efficiency of the dye inside the shell compared to simple attachment on the outside of the nanocomposite.231 The approach is shown in Fig. since research activities in this scientific area are proceeding with great speed. The reason for this is an extremely effective protection of the dye molecules towards oxidation and photobleaching. Biomedical applications The use of nanotechnology in biology and medicine has led to tremendous breakthroughs in the past decades. 11 Illustration of a multifunctional silica coated magnetic NP. along with their potential use as a drug delivery vehicles. the SiO2 coated magnetic NPs are retrieved by addition of methanol. the template is removed either thermally or by chemical treatment yielding highly ordered mesoporous matrices. core–shell magnetic mesoporous silica microspheres with strong magnetic responsivity.141 The integrated capability of the coremesoporous shell structured NPs as MR and fluorescence imaging agents. most synthetic routes for the preparation of mesoporous silica coated NPs.239 and since then Fig.. The nonporous silica interface layer (n) is thought to protect the magnetite from leaching. CTAB is used as templating agent to form long tubular structures. are based on templating procedures in aqueous solution. 2011. the authors demonstrated that the Fe3 O4 @nSiO2 @mSiO2 microspheres still showed superparamagnetic behavior. who incorporated a thin non-porous silica layer between the magnetic core and the mesoporous shell (Fe3 O4 @nSiO2 @mSiO2 ) to provide additional protection. This hampers potential in vivo applications of these particles.98 An interesting feature of silica coating is the possibility to incorporate fluorescent dyes into the SiO2 matrix. Subsequently. 12). Upon addition of aqueous NH4 OH micelles are formed in which the NPs are entrapped. 13f–h. Since microemulsion techniques are not ¨ applicable in this case. Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs.rsc. The great advantage of this approach is the possibility to dissolve as-prepared hydrophobic nanocrystals in the oil phase before entrapping them inside the micelles (see Fig. is based on a microemulsion synthesis. Additionally. 40. and yet. 12 Synthetic scheme showing the silica coating procedure in a reverse (or water-in-oil) microemulsion.219–222 Microscopically they consist of small heterogeneous domains of water in a surrounding hydrophobic phase (oil) separated by a surfactant monolayer. like drugs. thereby facilitating chemical reactions involved in particle synthesis. but also provides a large accessible pore volume for the adsorption and encapsulation of various agents. In fact. where micelles or inverse micelles are used to confine and control the coating of silica on the core of the inner nanoparticle (see Fig. By optimizing the synthetic conditions it is even possible to capture only one NP per micelle. the highly porous structure of the silica shell permits an easy diffusion of water molecules to the core particle. 6315–6343 This journal is © The Royal Society of Chemistry 2011 .237 In a similar fashion. However. Stober-type methods have to be used.1039/C0DT00689K 4. the condensation of tetraethoxysilane (TEOS) leads to the formation of SiO2 on the NP surface. An improvement of the core-mesoporous-shell approach was reported by Deng et al. After complete reaction.

there have been remarkable advances in nanomaterials synthesis and functionalization. Design considerations As pointed out in the previous sections. in vivo imaging. 6315–6343 | 6327 .g.243 This is due to a more targeted localization inside the desired area of the body (e. Dalton Trans. the basic principles of nanoparticle design for biomedical applications.rsc. biomaterials. the overall NP size. there are over 40 FDA approved nanomedicine products on the market and even more are currently in clinical trials. 40.8. One of the greatest challenges associated with the use of magnetic NPs in vivo is the ability to circumvent or overcome the different biological barriers. but also the final distribution within the body. therapy of various diseases (like cancer. cardiovascular and neurological diseases). 4. (d) and (e) TEM images of Fe3 O4 @mSiO2 NPs (with permission from ref. In fact.. tumor tissue).10 most of them being used for drug delivery applications.8. 13 Encapsulation of magnetic NPs with mesoporous silica. not only the circulation time inside the blood vessels. while at the same time. not only limited to drug delivery. morphology. the targeting efficiency This journal is © The Royal Society of Chemistry 2011 of nanoparticulate probes is often limited by the precocious recognition and elimination by the reticuloendothelial system (RES). morphology. (b) and (c) TEM images of hollow mSiO2 NPs. Moreover.11. followed by active cellular uptake of the nanoparticle probe. and surface chemistry of magnetic NPs. as well as the biodistribution and clearance of magnetic NPs from living organisms will be discussed briefly. and tissue engineering. early clinical results suggest that nanoparticle therapeutics can show enhanced efficiency compared to conventional drugs. negative side effects can be reduced significantly.2.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. In this respect.241 Up to now. magnetic properties and surface features can be tailored individually to meet the particular challenges of in vitro and in vivo biomedical applications. The application of nanotechnology is. (f) Reaction scheme depicting the experimental process to create Fe3 O4 @nSiO2 @mSiO2 microspheres. which today allow a precise engineering of the composition. size distribution. size. (g) TEM image showing the thin nSiO2 layer between Fe3 O4 and mSiO2 . 141). the fundamental interactions with biological systems. 14).240.org | doi:10. and (h) the porosity in the mSiO2 shell (with permission from ref. however. various organic/inorganic nanostructures are currently being applied or show promising potential in many different biomedical areas including in vitro diagnostics. owing to their MR enhancing properties.242. or hollow mesoporous silica particles. nanotechnology has had a major impact on the development of drug delivery systems. (a) Reaction scheme illustrating the synthetic procedure to create mesoporous silica (mSiO2 ) coated Fe3 O4 NPs. Before magnetic NPs can be used for biomedical applications.1039/C0DT00689K Fig. they require thorough consideration concerning the assembly of different components (see Fig. Both. As an example. 231). charge and surface chemistry are crucial properties that determine.1. 2011. either by passive or active cell targeting.10. the utilization of magnetic nanoparticles for simultaneous real-time in vivo monitoring of drug delivery is also an area of intense interest.244–246 In the following.7.

which will cleave under an external stimulus (e. which recognize certain kinds of cells inside the body. the NPs will slowly travel outside the blood vessels into the surrounding extracellular matrix (ECM). optical).257 Proteins with high concentrations and high association rate constants will initially occupy the nanoparticle surface. and eventually. a faster diffusion of macromolecules and NPs into the ECM is observed in tumor tissue. the blood vessels. The NPs need an inert and hydrophilic shell that protects them and offers both. compared to the extravascular space. certain drugs may also be attached to the composite to allow a specific treatment in a spatially confined area. Furthermore. 3. On the one hand.249 However. Finally. the surface chemistry can change significantly by the adsorption of proteins. regarding the blood circulation time. 6315–6343 After multifunctional NPs are intravenously injected.1039/C0DT00689K Fig.248. 5. which is defined as the overall size of the particle including the solvent shell. and. At this point. a feature commonly referred to as enhanced permeability and retention (EPR) effect (see Fig.255 This dynamic layer of proteins (and/or other biomolecules) adsorbs to nanoparticle surfaces immediately upon contact with biofluids. Additionally. photosensitizers to produce reactive oxygen species (ROS) upon optical excitation. 2011. unless they are protected by an inert hydrophilic shell. the surface charge of the magnetic NPs also plays a crucial role in cellular uptake and blood half-life. they travel along the blood stream through the cardiovascular system. On the other hand. eventually entering the lymphatic system.259–261 However. Moreover. that particles with sizes larger than 200 nm are quickly attacked by phagocytotic cells of the spleen. the size of the NPs must be sufficiently small to avoid rapid splenic filtration. Certain fluorophores that permit a subsequent detection by optical methods may also be included.263 This journal is © The Royal Society of Chemistry 2011 . strong negative charges on the particle surface are also unfavorable. there is a constant exchange of blood substances through the capillary microcirculation. pH.5 nm are rapidly removed through the kidneys (see Fig. 9). could guarantee a safe delivery of the payload to the desired region.2.17 For these NPs. demonstrating the importance of an efficient shielding of the NPs (“stealth effect”).258 These interactions may prove unfavorable possibly resulting in an inefficient uptake or even overload of SPIONs depending on the presence of certain proteins in the blood plasma that may act synergistically with the behavior of NPs. resulting in recognition and elimination by Kupffer cells and other tissue macrophages. immediate opsonization and/or phagocytosis is reduced. Once in the extravascular space. This corona may not immediately reach equilibrium when exposed to a biological fluid. since they lead to increased liver uptake.133 The role of protein–nanoparticle interactions has been described using the concept of the nanoparticle–protein “corona”.250–252 In fact. 1). are leaky. However. whereas particles with an average diameter below 5. was demonstrated by Papisov et al.254 On the other hand. heat. that sustain a tumor. which is regulated by concentration gradients. and higher affinity.org | doi:10. This leads to a preferred accumulation of the NPs in the interstitial space inside the tumor. stability in biological systems and long circulation times in the cardiovascular system..253 How great the impact of this non-specific interaction actually is.g. Additionally. or gold NPs which produce heat upon NIR illumination. Passive cell targeting Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. on which all other constituents will be assembled. 40. some NPs are taken up by the cells of the tissue and others are retained in the interstitial fluid.262 This effect originates from the intrinsic vascular characteristics of tumor tissue and the lack of an efficient lymphatic recovery system in solid tumors. The composition of the protein corona at any given time will be determined by the concentrations of the over 3700 proteins in plasma256 and the kinetic on and off rates (or equilibrium binding constants) of each protein for the particular nanoparticle.particles tend to non-specifically stick to the surface of cells. 1). but may also dissociate quickly to be replaced by proteins of lower concentration. the uptake of the nanocomposites into the tumor cells. Their hydrodynamic size. this attribute was already thoroughly discussed in the previous section. is strongly correlated to the ability of the nanoparticle to overcome the initial biological defense system and penetrate through the vascular barriers. Since after injection the local NP concentration in the blood is very high. 14 Illustration of a multifunctional magnetic nanoparticle (adapted from ref.g. it has to be estimated that NPs with a near neutral surface charge exhibit prolonged blood circulation times. they are the key factor that ensures the detection of diseases inside the body. these processes are comparatively slow. In contrast to the vasculature in healthy tissue. they should be large enough to circumvent renal clearance. because they represent the platform. During blood circulation. specific targeting ligands. 1.244 Therefore.9. particles of the appropriate size range are subject to opsonization. can be attached to the surface of the nanocomposite to ensure a selective delivery to the target tissue. 4. e. that positively charged polymers and small 6328 | Dalton Trans. 4.246. In this respect special responsive linker molecules. depending on their physical properties (magnetic. slower exchange.247 It has been shown. as long as the gradient persists. on the other hand. with large fenestrations and without an effective clearance by the lymphatic system.rsc.9. the overall size of the multifunctional magnetic NPs must also be considered. Thus. it can be speculated that NPs undergo two main processes that are important determinants of their cellular uptake: transient NP– protein binding (in culture media in vitro or body fluids in vivo) and NP–protein interaction with cell surface/membrane macromolecules. enzymatic cleavage).15. The NPs themselves can act as the active part of the nanocomposite probe. 6. it has been shown. responsive moieties can also be included that will react to external excitation. 2.

15a and b). eventually. (g) finally. leading to an improved diagnostic differentiation.1039/C0DT00689K Passive targeting can also be exploited through the clearance of NPs by the reticuloendothelial system (RES) (i. 15 also illustrates another common challenge of targeted (drug) delivery that needs to be addressed for NP design.264–266 However. Indeed. is the possibility to attach several (hundreds. leading to the formation of more binding sites.279 However. which is mainly due to their extremely high specificity.285 A great advantage of short peptides and other small molecules is their high bonding strength once This journal is © The Royal Society of Chemistry 2011 Fig. the NPs are internalized by formation of an endosome. (a) healthy cells only carry a certain number of cell receptors.e. and colon cancer. that small PEGylated NPs can easily diffuse through the cell membrane. For this reason.288 The typical pathway for a receptor-mediated cellular uptake of NPs is shown in Fig. in these cases. lymph nodes and bone marrow). based on magnetic iron oxide NPs.287.e.276. such as TAT peptides. will be taken up by the cancer cells. or even thousands of) targeting ligands on each NP. The release mechanisms of most delivery systems depend on processes such as diffusion. ovarian. i. and ionic concentrations. 40. Therefore. an FDA-approved mAb that binds to the HER2/neu receptor on the surface of certain cancer cells (overexpressed in breast. the development of HerceptinTM (trastuzumab). mAbs have evolved as one of the most popular targeting ligands. a longer residence time and a more site-specific accumulation of the NP probes is desired in most cases. why efficient surface functionalization strategies are of supreme importance. Fig.267–273 The first targeting agents. stomach cancer). chemical and enzymatic reactions. due to enhanced T 2 contrast. the controlled release of the NPs.221. a successful passive targeting of tumor tissue can only be achieved. an increased binding affinity. benefited from the rapid uptake of the NPs by Kupffer cells of healthy hepatic parenchyma. more cell receptors can be addressed simultaneously. if the blood circulation time of the NPs is reasonably long.282 on the other hand. healthy tissue appeared darker than diseased tissue in the MR image. peptides. it has been shown. therefore. (e) internalization of the endosome. Nevertheless. 15c–g. they are conjugated to the NP surface. renal.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. including breast. Compared to large peptides or proteins. liver. and especially the loaded therapeutic agents. or changes in various environmental factors. spleen. the first clinically approved T 2 contrast agents for MRI. Several targeting ligands. have been examined for this purpose. This is another reason. Thus.275 Up to now.280 Another popular technique for the targeted delivery of magnetic NPs to tumor cells is the use of short peptides or small molecules conjugated to the NP surface. permeation enhancers. more NPs can attach to the malignant cell. After attachment on the cell surface. (f) endosomal acidification by proton pumps leads to elevated osmotic pressure and swelling. has led to various applications of magnetic NPs for selective targeting of cancer cells. in which magnetic NPs were further functionalized with specific targeting molecules that possess a high affinity toward unique molecular signatures found on malignant cells (see Fig.289–293 As an example. there is a reduced risk of bond breaking and loss of functionality after administration of the NPs into the body. ovarian. and therefore. In principle. in cancer cells (b) these receptors are overexpressed. Dalton Trans.286. For example. which impedes a sufficient circulation and diffusion through biological barriers..266 The key issue in this case is the fact that cancer cells overexpress certain kinds of receptors compared to healthy cells. NPs carrying ligands which will specifically bind to these receptors will preferably accumulate in tumor tissue and. not only to improve the MRI detectability of various tumors in vivo282–284 but also to destroy cancer cells via hyperthermia treatment.274. 15 Illustration of active tumor targeting using magnetic NPs with cell-specific ligands. since the receptors of this vitamin can be found on the surface of many human cancer cells. The advantage. there are several possible mechanisms for the cellular uptake of NPs. 4. folic acid modified magnetic NPs have been used in the past. a major drawback of mAbs is their comparatively large size and inherent immunogenicity.277 During the last decade. can significantly increase the degree of NP uptake. comprised monoclonal antibodies (mAbs). active targeting strategies have been developed. that the passive accumulation of magnetic NPs in tumor tissue due to the EPR effect is a great benefit for the application of these NPs in the diagnosis and treatment of cancer. solvent effects.rsc. aptamers and small molecules. (c) Receptor-mediated endocythosis of the NPs: (d) formation of an endosome carrying the NPs. once the NPs have entered the interior of the cell. that were used for the specific delivery of magnetic NPs. As a result.3.281 Indeed. the endosome ruptures and releases the magnetic NPs. dissolution. Another important issue associated with the targeted delivery of magnetic NPs to tumor cells is the internalization of the particles into the malignant cell. Various strategies have been developed to accomplish this task. 6315–6343 | 6329 . including proteins. lung. Active cell targeting There is no doubt about the fact. which can be used for active targeting. including temperature.269 The most investigated ligand in this respect is folic acid.org | doi:10. pH.278. 2011.

Hu et al. As mentioned previously. Concerning cancer imaging.310–312 The basic physical phenomena leading to an enhancement in MR contrast have already been presented. there are a number of reports dealing with the design of different manganese oxide NPs and their application for MR contrast enhancement. to initiate payload release. developed silica coated Mn3 O4 NPs functionalized with folic acid and contained Rhodamine B isothiocyanate (RBITC) as fluorescent dye for optical detection. showed that un-functionalized Mn3 O4 and MnCO3 NPs can be internalized within phagocytotic cells and subsequently shuttled to endosomes and/or lysosomes. or molecular imaging. Hyeon and coworkers were the first to prepare T 1 a contrast agent based on monodisperse MnO NPs and investigate its performance in vivo (see Fig. in the early stages of the disease.1039/C0DT00689K an efficient release can be achieved by providing a positive surface charge to the NP probe. as a photosensitizer initiates the production of reactive singlet oxygen when irradiated with visible light. Recently.309 Although enormous progress has been achieved in the technological development of MRI. highly crystalline. 16). was given by Cheon and coworkers (see Fig. there has hardly been any improvement in the development of the contrast agents which are in clinical use today. magnetic NPs may also serve as probes for multimodal imaging. Enhanced binding of the RT1 functionalized SPIONs indicated a definitive specificity for the renal medulla and thus potential for disease detection.323 The following decomposition of the NPs inside the lysosome leads to the release of Mn2+ ions which act as a T 1 agent. is the possibility of uncontrolled release due to different release mechanisms depending on the location in and the character of the respective cell. On the other hand. the tremendous advances in the synthesis and functionalization of magnetic NPs discussed in the previous sections have led to a variety of new possibilities in the future design of MRI contrast enhancing probes. apoptosis. magnetic NPs have been studied thoroughly over the past few years as MRI contrast agents for applications. slow diffusion mediated processes from hollow or mesoporous NP structures.313–319 Specially designed NPs with tailored properties and cell specific surface ligands have a great potential to pinpoint biological targets.. the MnO NPs generated a positive contrast and by combination of both imaging techniques. Typical T 2 contrast agents consist of polydisperse and aggregated iron oxide NPs.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. angiogenesis. These NPs showed enhanced MRI sensitivity for the detection of cancer and enabled the visualization of small implanted tumors in mice.316 While the SPIONs produced a negative contrast.31 The changes in the magnetic relaxation which are associated with the particle self-assembly could then be detected by MRI.46 Protoporphyrin IX. especially sophisticated pulse sequences for image generation. It was also shown that a combination of SPIONs and MnO NPs can be used to track transplanted cells. However.296–299 For these purposes specific stimuli responsive chemical linkers can be tailored and subsequently inserted between the NP and the drug. such as cancer.g. cell migration. Although the use of manganese oxide NP as T 1 contrast agents is not as common yet. nanocomposite particles consisting of silica spheres decorated with Fe3 O4 NPs were synthesized by Lee et al. followed by conjugation of different cancer specific antibodies. 40.313 As an example for magnetic NPs being used as molecular sensors. Magnetic NPs as MRI contrast agents in biomedicine Superparamagnetic iron oxide NPs (SPIONs) have been used as T 2 contrast agents for over two decades. the development of multifunctional magnetic NPs has led to many improvements in the detection.294. 2011. The use of mesoporous silica furthermore offered the possibility to load. For this reason various methods have been developed that rely on physical triggers. Perez et al. or by cleavage of the linker bond by pH reduction inside the lysosome. functionalized with Herceptin and showed no severe toxicity even after several weeks.320 6330 | Dalton Trans. the nanoparticles must be monodisperse. The downside of this approach.166. but also induced apoptosis in Caki1 cells once illuminated with visible light. simultaneous imaging with opposite contrast offers the possibility for MR “double labeling” of different cell populations. or protein–protein interactions) by the reversible self-assembly of SPIONs. showed that PEGylated Fe3 O4 NPs conjugated to the monoclonal antibody rch 24. Apart from sole MRI contrast enhancement. Shapiro et al. our group reported on the development of highly water soluble MnO NPs conjugated with protoporphyrin IX as multifunctional agents for MRI and photodynamic therapy. The authors showed that the Herceptin functionalized NPs accumulated in breast cancer tissue.org | doi:10. DNA–DNA. created immunotargeted SPIONs to investigate the MHC class II expression in renal medulla..274. whereas un-functionalized control NPs also illuminated the surrounding tissue. In this respect. an anti-cancer drug into the pores of the silica sphere.322 Additionally. Hultman et al. a fluorescent dye was incorporated into the silica domain to permit both MRI and optical detection. such as cancer imaging. Additionally. that the NPs not only exhibited excellent T 1 contrast. diagnosis and treatment of solid tumors. Very recently. such as heat or radiation. in which magnetic NPs were used to detect cancer markers by MRI in vivo. 17). demonstrated the transgene expression in gliosarcoma cells in vivo using dextran coated iron oxide NPs. on the other hand. We were able to show. and slowly release. These strategies can involve bond breaking by radiative excitation. cardiovascular disease imaging.33 The authors developed novel magnetic nanoprobes by systematically tuning the spin. Yang et al.321 For this purpose.43 The particles were coated with an amphiphilic phospholipid. The combination of different materials enables the selective utilization of the This journal is © The Royal Society of Chemistry 2011 .rsc. developed biocompatible magnetic nanosensors that act as magnetic relaxation switches to detect molecular interactions (e.4.265. gene expression. which show a comparably weak performance. high magnetization values and good biocompatibility under biological conditions. Weissleder et al. In a similar way. however. such as Herceptin. 6315–6343 Another example. The incorporation of cationic polymers on NP surfaces has proved to be an appropriate method to induce osmotic swelling of the endosome caused by the “proton sponge” effect.300–308 4. effectively targeted human colon carcinoma xenograft tumors implanted in nude mice. monodisperse Fe3 O4 NPs were encapsulated in a phospholipid shell and further conjugated to RT1 anti-MHC Class II antibodies that are capable of targeting normal cells expressing specific target antigens.295 As a result the endosomal membrane is disrupted and the NPs are released into the cytosol.47 These particles accumulated selectively in cancer cells overexpressing folic acid receptors and could be traced by MRI. size and composition of metal ferrite NPs. and water soluble to provide reproducible quality.

324 A similar approach was recently reported where the authors used MnO NPs coated with human serum albumin (HSA) and which were further conjugated with a 64 Cu containing complex. both by MRI and PET. On the other hand.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. permitted the use of these hybrid NPs as dual imaging agents for MRI and positron emission tomography (PET) and. This journal is © The Royal Society of Chemistry 2011 Dalton Trans. 33). For example. Over time. combining the benefits of MRI together with the high sensitivity of PET (see Fig. 16 Specifically engineered MFe2 O4 NPs as MRI contrast agents. (b) In vivo MR detection of cancer cells using Herceptin conjugated MnFe2 O4 NPs (left) and bare cross-linked iron oxide NPs (CLIOs) (right). an increase in contrast enhancement due to preferable accumulation of Herceptin-conjugated MnFe2 O4 NPs in the tumor tissue is observed (with permission from ref. 6315–6343 | 6331 . reported that the incorporation of 124 I into the biocompatible shell of manganese ferrite NPs.. therefore. Choi et al.rsc. Upon conjugation with the antibody AntiHer2. the NPs preferably accumulated in Her2-overexpressing cancer cells.1039/C0DT00689K Fig. A higher magnetic moment results in an increase of the magnetic relaxivity and therefore.48 With this method it was possible to visualize xenografted U87MG glioblastoma cells in vivo. 2011. as confirmed by MRI and CT. Another example for multimodal imaging was demonstrated by water soluble FePt NPs that were used for in vitro and in vivo imaging by MRI and computer tomography (CT). 18). individual properties. enhanced T 2 contrast effect. 40.325 Different sized FePt NPs were functionalized with cysteamine to ensure hydrophilicity and the possibility for further biofunctionalization.org | doi:10. (a) The magnetic properties can be tuned by varying the composition of the NPs.

5.. (c) Herceptin conjugated MnO NPs increase the contrast inside the tumor tissue. 40. (a) TEM images of monodisperse MnFe2 O4 NPs. Fig. MnFe2 O4 NPs. 18 Bimodal imaging using 124 I conjugated MnFe2 O4 NPs. (a) TEM images of monodisperse MnO NPs of different sizes. reliable methodologies to estimate the toxicological impact upon intended administration are still lacking. proponents and sceptics. 17 MnO NPs as T1 contrast agents. and 124 I-conjugated MnFe2 O4 NPs (with permission from ref. direct exposure to the human body is definitely intended. whereas bare MnO NPs (d) also led to an enhanced contrast in the surrounding tissue (with permission from ref.326 Despite the fact that an in vivo application of any nanomaterial requires a thorough comprehension of the kinetic processes and the toxicological impact this material exerts inside a living organism.327–330 This journal is © The Royal Society of Chemistry 2011 . (c) aqueous solutions of these NPs with different NaCl concentrations and varying pH. (d) Preparation scheme fot the conjugation of 124 I onto MnFe2 O4 NPs. (e) MRI and PET images of free 124 I. it is crucial to investigate the cellular responses upon NP exposure. 324).org | doi:10. although there are already numerous studies on cytotoxic effects of different kinds of nanoparticles. the most frequently used screening studies are based on in vitro cytotoxicity experiments. understanding the behavior and properties of magnetic NPs on the organism after 6332 | Dalton Trans. since they are simpler.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. However.45 4. to counteract any possible harmful effects. 19). we were able to combine the properties of MnO NPs and Au NPs for simultaneous MRI and optical detection by creating biocompatible Au@MnO “nano-flowers” (see Fig. as the number of different nanomaterials increases with an unprecedented speed. is essential before considering clinical use. Particularly. 43). Especially. (b) hydrodynamic size. and therefore. 6315–6343 administration. there is the predominant opinion among both. Since these NPs are intentionally designed to interact with cells. (b) T1-weighted images of aqueous MnO NP solutions. Toxicity of magnetic NPs The need to evaluate the possible risk of human exposure to different kinds of nanomaterials has become a central issue in modern materials and biomedical science. that the vast potential of nanotechnology requires immediate attention to safety issues. faster and less expensive compared to in vivo studies. since most research on the toxicology of nanomaterials has focused on the effects of nanoparticles that enter the body accidentally. in the field of nano-biotechnology.rsc. 2011. already during NP development.1039/C0DT00689K Fig.

What are the uptake mechanisms and efficacies of particles as a function of size.333. However. and melanoma cells. reported a significant reduction in cell viability in human mesothelioma cells upon addition of only 3.339 This was attributed to a collapse of the dextran shell exposing the cellular components to magnetite NPs. Particular emphasis is devoted to particle adhesion. be decreased by coating magnetite particles resulting in fewer oxidative sites that are less reactive. In fact. a size-dependent toxicity was reported for silver and gold NPs exposed to alveolar macrophages. light and moisture according to: Fe3 O4 + 2H+ → g-Fe2 O3 + Fe2+ + H2 O. the oxidation state of iron in and protein–NP interaction. Contemporary research on nanotoxicity deals with two major questions that are largely unsolved to date. the toxicity depends on various factors such as surface-coating or its breakdown products. (b) TEM image of polymer coated Au@MnO nanoparticles (with permission from ref. hydroxyl radicals) by Fenton or Haber–Weiss reactions. in spite of the cytotoxicity of uncoated and dextran-coated Fe3 O4 NP the albumin-coated particles did not lead to cell death.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. 45). quantum dots can also initiate an inflammatory response and the production of ROS. 6315–6343 | 6333 . which are a common component in many commercially available products. Fe3 O4 . 40. This resultant altered composition of the cell medium to which the cells are exposed results in the observed cytotoxicity. cell entry and impact on cell viability and metabolic pathways to gain a comprehensive understanding of the main reasons of nanoparticle mediated cytotoxic effects on cells of the blood compartment.334 Additionally. The Au core is functionalized with a red fluorescent dye tagged thiolated oligonucleotide. ionic strength and chemical composition of the blood plasma. the toxicity induced by uncoated particles was significantly reduced compared to surface-saturated SPIONs which were prepared by pre-incubating the media with the NPs prior to exposure to cells. The main goal of most toxicity studies is to cover the main events in nanoparticle mediated cellular interactions.339 Furthermore. whereas uncoated NPs caused a 50% decrease in cell viability already at comparatively low concentrations (250 mg mL-1 ). Magnetite can undergo oxidation to form maghemite in the presence of air. The authors reported that PEGylated iron oxide NPs had almost no toxic effect on fibroblast cells. the cell behavior and morphology of cells treated This journal is © The Royal Society of Chemistry 2011 with dextran-coated magnetite was different from the uncoated NPs.344 The toxicity can.345.338 Concerning magnetic metal oxide NPs (above all. can cause inflammatory responses and generation of reactive oxygen species (ROS) leading to DNA damage.177 The importance of an efficient and biocompatible surface coating was further demonstrated by Gupta et al. macrophages. A study investigating the effect of different surface coatings on cell behavior and morphology showed that dextran-coated Fe3 O4 NPs led to cell death and reduced proliferation quite similar to the interaction of cells with naked magnetite NPs. Another cytotoxicity study on a mouse fibroblast cell line showed that the uncoated particles induce greater toxicity than the polyvinyl alcohol (PVA)-coated particles. which showed more severe membrane disruptions. However. even at concentrations as high as 2 mg mL-1 .341 However. Dalton Trans. it was also shown that in vitro incubation of single-walled carbon nanotubes (SWNTs) with keratinocytes and bronchial epithelial cells. also showed hardly any toxic effect.332 Moreover.117 The low cytotoxicity of PEGylated Fe3 O4 NPs was further confirmed by other authors using different kinds of PEG.1039/C0DT00689K Fig.343 Magnetite (Fe3 O4 ) and maghemite (g-Fe2 O3 ) can show different cellular responses because of their ability to undergo oxidation/reduction reactions. mitochondrial dysfunction. Furthermore.org | doi:10. Brunner et al. Another important aspect is the stability of these SPIONs with time.153 Besides PEG. oxidative stress. recent studies revealed that TiO2 NPs. connective tissue fibroblasts. However. and changes in cell morphology. iron oxide NPs with other surface coatings.75 ppm iron oxide. magnetite has been shown to cause higher levels of oxidative DNA lesions (using the comet assay) in A549 human lung epithelial cell line in the absence of decreased cell viability as compared to maghemite owing to its potential to undergo oxidation.339. chemical composition of the cell-medium.340 The membrane disruption after exposure to albumin functionalized Fe3 O4 NPs turned out to be related to the interaction between albumin and membrane fatty acids and phospholipids. gFe2 O3 and MnO).335–337 On the other hand..331 For instance.rsc.350 The authors proposed that this high toxicity was due to ironinduced free radical production (e. 2011. most in vitro cytotoxicity studies revealed rather negligible toxic effects. epithelial cells. however. it was stated by several authors that “naked” iron oxide NPs show a higher toxic potential compared to those that were modified with an inert biocompatible coating. cellular uptake of the NPs and the production of ROS.346–348 The reason for the higher cytotoxicity of “naked” iron oxide NPs was attributed to both.342 This effect may not occur in vivo because homeostasis maintained by the liver and kidneys efficiently regulates any changes in pH. 19 (a) Schematic illustration of Au@MnO “nano-flowers” separately functionalized using a multifunctional polymeric ligand carrying a catechol anchor group and a green fluorescent dye tagged to the PEG side groups. caused by Fe-ion leaching. led to lipid peroxidation. such as cosmetics or sun screen lotions. including various polymers or silica. During this pre-incubation biomolecules from the cell medium adsorb onto the SPIONs which masks their reactive surface thereby preventing unfavorable cell–nanoparticle or serum protein–nanoparticle interactions that in turn led to reduced cellular uptake and lower toxicity.349 For instance.g. the toxicity was governed by compositional changes in the media because serum proteins can bind to the negatively charged uncoated NPs. shape and surface functionalization and which biochemical pathways are triggered that eventually cause cell toxicity in cells mirrored either by apoptosis or necrosis? An often examined parameter to assess possible harmful effects of nanomaterials inside living organisms is an inflammatory response.

6. which themselves have sizes on the nanoscale (e. and surface chemistry. Choi et al. it is also crucial to determine where nanoparticulate probes will eventually end up and how they are finally excreted from the body.org | doi:10. thermodynamically stable Mn compounds which are not prone to Mn ion-leaching should be preferred as NP platforms for in vivo applications. such as size. high doses of these NPs caused interference with the actin cytoskeleton resulting in decreased cell proliferation indicating the possibility that nontoxic doses could cause other forms of cellular stress.326 As already stated in the previous sections. This would represent a truly new paradigm in the field of nanoscale toxicology.46 Concerning free manganese ions. These NPs not only inhibited lysozyme amyloid aggregation by blocking the nucleation process but also induced depolymerisation of lysozyme aggregates by interacting with and interrupting the adjoining protein sheets. Small particles with diameters less than 5. NPs with diameters of between 5 and 100 nm and a neutral and hydrophilic polymer extended surface exhibit prolonged blood circulation.g.355 Currently there are no clinical drugs to reverse or prevent the formation of aggregates and based on this study the Fe3 O4 NPs could potentially be used as novel therapeutic agents in the treatment of protein amyloid aggregation-associated human pathologies.352 Another case of SPION-mediated cellular stress involves disruption of a cytoskeleton protein.353 However. but it may also lead to significant alterations in the pharmacokinetic profiles of the parent drug and the drug attached to the NPs. exhibited no cytoxic effect in CaKi-1 cells in concentrations as high as 140 mg mL-1 . 2011. Specifically.47 For instance. Thus. and chemically monofunctional (mostly iron oxide) nanoparticles as a function of their size. although the MnO NPs showed a slightly higher toxicity than the Fe3 O4 NPs. shape and chemical nature. In general. and in the design of nanocarriers for nanomedicine. the pharmacokinetics and biodistribution of NPs must be monitored to understand and predict their efficacy and side effects. larger particles with diameter > 200 nm are easily sequestered by the spleen and eventually removed by the cells of the phagocyte system. shape anisotropy. For this purpose it is not only important to understand the behavior and interaction of magnetic NPs with living organisms.360 This journal is © The Royal Society of Chemistry 2011 . nanoparticles could be classified in terms of their biomolecule corona. However. The majority of nanoparticle types studied so far are found to bind apolipoproteins. After administration. which have been poorly assessed up to now. However. although minor concentrations of these ions are not considered to be harmful to the human body. In fact. literature data suggest that.A further issue in this context is the dependence of the degree of NP internalization on the quality of the surface coating. Therefore. a recent study on protein amyloid aggregation (relevant for Parkinson’s and Alzheimer’s disease or type II diabetes) found the protein aggregates were significantly reduced by magnetic Fe3 O4 NPs. charge.. Apolipoprotein E is known to be involved in trafficking to the brain.354 and therefore. Magnetic NPs are usually within this suitable size range. our group demonstrated recently that both. Reduced liver metabolism and renal clearance of drugs encapsulated in the nanoparticles often result in prolonged blood circulation with an increased chance of accumulation in the target tissue.45. 6315–6343 Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. amphiphilicity. Au@MnO nanoflowers and PEGylated MnO NPs carrying protoporphyrin IX as a photosensitizer. Opsonization is a major issue that induces MPS uptake of NPs.316. the following scientific questions must be addressed: (i) How does multifunctionality and shape anisotropy impact the interaction of particles with biomembranes? (ii) What are the possible consequences for the uptake of heterogenic multifunctional nanoparticles? Do Janus particles exert curvature in cell membranes that inevitably leads to endocytosis?359 What exposure routes (basal or apical) lead to highest uptake rates? (iii) What kind of nanoparticle-mediated intracellular processing mechanisms can be identified? 4.357 Furthermore. tubulin that has been shown to be associated with the uptake of transferrin-derivatised SPION.356 On the other hand. 100 nm for chylomicron to ~10 nm for high density lipoproteins). the toxic properties of MnO NPs have hardly been investigated so far. namely multifunctional or so-called Janus particles. Biodistribution and clearance An essential topic that is often neglected in literature is the long-term fate of magnetic NPs after they have fulfilled their duty inside the body. investigated the toxic potential of Fe3 O4 and MnO NPs coated with a hydrophilic micellar phospholipid shell and revealed that.5 nm are rapidly removed through extravasations and renal clearance. Similarly it was demonstrated that magnetoliposomes can affect the actin cytoskeleton architecture as well as the formation of focal adhesion complexes and impair the cell proliferation. resulting in decreased blood circulation times.258 As apolipoproteins are known to be involved in lipoprotein complexes.351 However. The pharmacokinetic profile and the internalization of metal oxide NPs are determined by their chemical and physical properties. which mediates their interaction with cellular machinery. For instance. there may be size-dependent interactions that drive the binding of apolipoproteins to nanoparticles. these results further substantiate the importance of effective surface coating techniques for magnetic NPs in biomedical applications. geometrically isotropic. These particles possess new challenges for biological cells associated with high dipole moments. some NPs may be able to traverse the blood–brain barrier. These studies must be extended in the future to elucidate of the impact of particles with intrinsic chemical and physical asymmetry.1039/C0DT00689K Undoubtedly. NPs can enhance the delivery of drugs to certain tissues. the overall toxic potential is acceptable. De Cuyper and coworkers designed a cationic amphiphile to maximize internalization of SPION without inducing any cytotoxic effects on neural progenitor cells and human blood outgrowth endothelial cells.358 6334 | Dalton Trans. most reports on the biomedical application of MnO NPs suggest that the toxicity of this material is comparatively low. lysozyme adsorption seemed to govern both these processes. moderate liver uptake and an increased level of drug delivery as determined by radiolabeling (PET/MR). a wealth of information has been collected on the toxicity of chemically pure.rsc. and therefore the surface characteristics and surface functionalization greatly determine their pharmacokinetic profile. and chemical diversity/patchiness. 40. this may not only cause new side effects.

sensitivity could be improved about 300-fold in comparison to the commercially available immunoassay used so far and allowed the prediction of disease relapse followed by adjuvant and salvage therapies at a much earlier time. Lee et al. Tumor cells express a wide variety of proteins that can be recognized by the immune system. which transfers DNA or RNA into target cells. these strategies attempt to augment protective antitumor immunity and to disrupt the immune-regulatory circuits that are critical for maintaining tumor tolerance.. anti EGFR Human anti EGFR Disease Non-Hodgkin lymphomas breast cancer acute myeloid leukaemia chronic lymphocitic leukaemia colorectal tumor Metastatic colorectal carcinoma This journal is © The Royal Society of Chemistry 2011 Dalton Trans. especially recombination technology. Established cancer therapies employ a variety of manipulations to activate antitumor immunity.4. Antibodies play an essential role in providing protective immunity to several pathogens. Genentech Inc. TLRs are type I transmembrane glycoproteins characterized by an extracellular leucine-rich repeat domain and a conserved intracellular domain. The improved sensitivity of assays using antibody-coated nanoparticles has also recently been shown by Thaxton et al.366 Here. cell sorting. gene delivery.368. sensing. the ultra-sensitive MR detection of various types of cancers should be possible. They comprise tumor-specific proteins containing mutations or fusions and also tumor-associated proteins normally expressed in a developmentally or tissue-restricted fashion. and include contrast agents for magnetic resonance imaging (MRI). The infusion of manufactured monoclonal antibodies can generate an immediate immune response while bypassing many of the limitations that impede endogenous immunity. Antibodies.365.rsc. Their discovery in 1997 by cloning and characterization of TLR4369 gave new impulses in the field of innate immunity and in the understanding how innate and adaptive immunity are tightly interwoven. enzyme immobilization. The conjugation of antibodies to nanoparticles now generates a new product that combines the physico-chemical properties of nanoparticles like thermal. bioseparation. Fe. and (iii) the systemic or local delivery of cytokines. drug carrier.364 In diagnosis.367 PAMPs are produced only by pathogens and not by host cells allowing the early distinction between ‘self’ and ‘microbial nonself’. These include (i) passive immunization with monoclonal antibodies. which include membrane-bound receptors in the cell surface or in intracellular compartments. imaging. and so forth.363 The possible applications of antibody-conjugated nanoparticles are numerous and can be divided in therapy and diagnosis. 6315–6343 | 6335 . Magnetic NPs for immunotherapy Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. Their detection is mediated by a limited number of pattern recognition receptors (PRRs). (ii) active immunization by the application of adjuvants alone or in combination with tumor antigens. This suggests that by using appropriate cancer targeting molecules. which is homologous to the cytosolic domain of the IL-1 receptor and therefore named Toll/IL-1 receptor (TIR) domain. Today. functionalized nanoparticles represent a promising tool for immunotherapy of tumors.1039/C0DT00689K With the development of DNA technology. Immune adjuvants.362 In addition.org | doi:10. Toll-like receptors (TLRs) represent a group of pattern recognition receptors with a great variety of different ligand specificities. A variety of novel strategies have been developed based on fundamental advances in our understanding of the interactions between tumors and the immune system. Many known PAMPs are typical nucleic acids or conserved components of cell wall structure from microorganisms. and the administration of tumor-targeting monoclonal antibodies has proven to be one of the most successful forms of immune therapy for cancer at the moment. magnetic hyperthermia. the applications can be divided into those using in vivo and those using in vitro experimentation. These proteins represent ideal targets for therapeutic interventions as they allow the specific detection by cells or components of the immune system. transfection (gene delivery). 20. for the detection of the prostate specific antigen (PSA) in the serum of patients after radical prostatectomy). 2011.361 Finding effective transfection methods is a major objective in gene therapy research due to rapid degradation of DNA and RNA by enzymes and their poor diffusion across cell membranes. Therapeutic applications include targeted drug delivery. During the initial phase of infection. Collectively. purification. immunoassays. eleven different mammalian TLRs Table 1 Monoclonal antibodies approved for tumor therapy in humans Product Rituximab (Rituxan) Trastuzumab (Herceptin R ) Gemtuzumab Ozogamicin (Mylotarg) Alemtuzumab (Campath) Cetuximab (Erbitux) Panimumab (Vectibix) Company Genentech Inc. Monoclonal antibodies have been approved for the treatment of several solid and non-solid tumors (see Table 1). the pathogen associated molecular patterns (PAMPs) as summarized in Fig. or soluble proteins secreted into the blood stream and tissue fluids. gene therapy.7. has become a novel approach for disease therapy. An example for a therapeutic application is the use of Au–Fe3 O4 hetero-nanoparticles coupled with Herceptin and a platinum complex as target-specific nanocarriers for delivery of platinum into Her2-positive breast cancer cells. pathogen sensing by the immune system is based on recognition of a limited number of microbial “molecular signatures”. Wyeth Averst Millennium/Ilex Partners LP ImClone Systems/Bristol-Myers Squibb/Merck KgaA Amgen Specificity Chimeric Ig anti-CD20 Humanized IgG anti-HER2 Humanized Ig anti CD33 Humanized Ig anti CD52 IgG1 . 40. Co or Ni) showed enhanced sensitivity for cancer cell detection and also made the in vivo imaging of small tumors possible. photodynamic therapy and the delivery of encapsulated molecules in vaccination strategies.33 showed that Herceptin-functionalized MFe2 O4 nanoparticles (M = Mn. or magnetic characteristics with the ability of antibodies to specifically recognize antigens on the surface of tumor cells.

378 TLR triggered activation of DCs having captured microbial antigens does not only lead to the upregulation of co-stimulatory and MHC molecules but also to a switch in This journal is © The Royal Society of Chemistry 2011 . NF-kB is consequently free to translocate into the nucleus and induce the expression of its target genes. and facilitating their migration to the inflamed tissue. neutrophils. 20 Signalling through TLR3. such as monocytes. MAL).rsc. NF-kB. They contribute 6336 | Dalton Trans. but is known to associate with these endosomal TLRs and to be required for them to signal. and ultimately results in the activation of several kinases inducing phosphorylation. thereby undergoing conformational changes required for the recruitment of the adaptor molecule MyD88 (myeloid differentiation primary-response protein 88). TICAM2)373–377 In an orchestrated interplay pathogen recognition by TLRs links innate and adaptive immune responses by inducing the expression of diffusible chemotactic factors and cell surface adhesion molecules attracting innate immune cells.1039/C0DT00689K Fig. also known as TIR-domain-containing molecule 1.371 TRIF (TIRdomain-containing adaptor protein inducing IFN-b. 2011.org | doi:10. 40. TLR8 and TLR9 in response to endosomal nucleic acids of viral origin. thereby releasing NF-kB (nuclear factor-kB). through the recruitment of tumour-necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) and receptor-interacting protein 1 (RIP1). IRF7 and IRF3 translocate to the nucleus to induce the transcription of genes encoding cytokines such as TNF. 367). Besides this MyD88-dependent signalling cascade. have been identified. TLR8 and TLR9 that are expressed in the endosome. also known as MyD88-adaptor-like protein. followed by ubiquitylation and subsequent degradation of IkB. TLR7. signalling proceeds through TIR (Toll/interleukin-1 receptor (IL-1R))-domain-containing adaptor protein inducing interferon-b (TRIF) or myeloid differentiation primary-response gene 88 (MyD88).Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. signalling in response to specific virally encoded proteins through MyD88. basophils. which interacts with the TIR domain of the receptor. MyD88 recruits TRAF6 and IL-1R-associated kinase (IRAK) and activates IRF7 and NF-kB. TRIF. respectively. MyD88 consists of a C-terminal TIR domain. UNC93B is a multiple-transmembrane-spanning protein that is predominantly located in the endoplasmic reticulum (ER). They recognize a wide range of pathogen associated molecular patterns (PAMPs) derived from microbes and viruses such as ds-RNA. Following the recognition of viral double-stranded RNA (dsRNA). 6315–6343 to a MyD88-independent activation of the NF-kB pathway and include: TIRAP (TIR-domain containing adaptor protein. TRIF and/or TRAM (TRIF-related adaptor molecule). ss-DNA. IL-6 and type I IFNs (with permission from ref. additional receptorproximal adaptor proteins have been described. also known as TIR-domaincontaining molecule 2.. eosinophils and NK cells as well as adaptive immune cells. with their genes dispersed throughout the genome. as well as TANK-binding kinase 1 (TBK1) and inducible IkB (inhibitor of nuclear factor-kB (NF-kB)) kinase (IKKi) activate interferon (IFN)-regulatory factor 3 (IRF3) and NF-kB. TLR4 (not shown) also detects viruses. and lipopolysaccharides (LPS) as well as intrinsic stress proteins. single-stranded RNA (ssRNA) or CpG-containing DNA by TLR3 or TLR7. Upon ligand binding TLRs dimerize. The Toll-like receptors (TLRs) that sense nucleic acids can operate in non-infected cells of many types to detect the production of infection in other cells.370. TICAM1)372 and TRAM (TRIF-related adaptor molecule.

6315–6343 | 6337 . 2011. Because of these features.rsc. skin. 21).org | doi:10.388 A similar approach was taken by Uto and colleagues. nanoparticles have been used to activate the inflammasome in an approach to induce potent immune responses. Here CpG oligonucleotides and ovalbumin was immobilized using biodegradable glutamic acid nanoparticles.389 Potent induction of proinflammatory cytokine release in a TRL9-dependent manner and activation of cytotoxic T cells was observed. This has been demonstrated for siRNA molecules complexed to polyethylenimine-based nanoparticles triggering DC activation in a TLR5-dependent manner.. To further improve the use of TLR ligands as adjuvants immobilization on nanoparticles represents a very attractive approach and provides a tool to limit the systemic release of proinflammatory cytokines associated with TLR ligand application in solution.380 stimulate a powerful innate immune responses by interacting with TLR9.385 for CpG oligonucleotides386. ODNs are currently evaluated for the immunotherapy of cancer. This has the disadvantage that the exact composition of the nanoparticles is difficult to control especially when functionalization using several molecules is intended. 21 Modification of manganese oxide nanoparticles with a multifunctional polymeric ligand and linkage to ssDNA. TLR ligands represent a group of molecules ideally suited for the use as immune adjuvants.177 These different nanoparticles efficiently activated the TLR9 signaling pathway (Fig. including the treatment of kidney. A solution for this problem will be the development of nanoparticles carrying different functional groups on their surface and therefore allowing a controllable immobilization of TLR ligands for innate immune cell activation. uterine and immune malignancies. Nanoparticles represent an ideal tool to combine these two stimuli in newly developed vaccination protocols. Because cancer cells often express a variety of abnormal proteins that can serve as targets for an immune response (antigens) local administration of adjuvants can induce tumor-associated inflammation and protective immunity. This journal is © The Royal Society of Chemistry 2011 However. This was achieved by the incorporation of LPS on the surface of poly(lactic-co-glycolic acid) nanoparticles loaded recombinant West Nile envelope protein. This has been demonstrated for cationized gelatin nanoparticles carrying CpG oligonucleotides and the model antigen ovalbumin. antigens for adaptive immune cell activation and Dalton Trans.Downloaded on 20 June 2011 Published on 01 March 2011 on http://pubs. For example.390 However. most approaches so far immobilize TLR ligands and antigens in an unspecific manner by adsorption or encapsulation.381–384 Therefore. The ssDNA conjugated nanoparticled trigger the immune cascade by activating NF-kB (reproduced with permission from ref. 40.1039/C0DT00689K Fig. which are present at a much higher frequency in the genomes of prokaryotes than of eukaryotes379. chemokine receptor expression and to the secretion of cytokines and chemokines finally resulting in the generation of effector responses including T helper cell and CTL responses.387 encapsulated within liposomal nanoparticles. In addition. Immunization of mice resulted in the protection against a murine model of West Nile encephalitis. optimal induction of adaptive immune responses does not only rely on efficient innate immune cell activation but requires the presentation of antigens at the same time. 387). synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs. breast.

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