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Engineered Magnetic Nanoparticles for Biomedical Applications Francesco Canfarotta* and Sergey A. Piletsky
In the past decades, magnetic nanoparticles (MNPs) have been used in wide range of diverse applications, ranging from separation to sensing. Here, synthesis and applications of functionalized MNPs in the biomedical field are discussed, in particular in drug delivery, imaging, and cancer therapy, highlighting also recent progresses in the development of multifunctional and stimuli-responsive MNPs. The role of their size, composition, and surface functionalization is analyzed, together with their biocompatibility issues.
1. Introduction In the past two decades, magnetic nanoparticles (MNPs) were employed in several biomedical applications, such as drug delivery,[1–4] magnetic resonance imaging (MRI),[5–8] cancer therapy,[9–11] bioseparation,[12–14] and biosensing.[15–21] The materials used most frequently in these applications are magnetite (Fe3O4) or maghemite (γ-Fe2O3), thanks to their good biocompatibility and magnetic properties. The general requirements for biomedical application are mainly related to their magnetization value, surface coating, and size. These parameters strongly affect both the magnetic behavior of the particles and their stability, biocompatibility, and biodistribution. MNPs with diameter 10–100 nm are preferred for the use in vivo sinceas they do not have rapid renal clearance that occurs for NPs smaller than 10 nm, and internalization by reticuloendothelial system that occurs for NPs >200 nm.[10] The functionalization of MNPs surface is advisable to improve their biocompatibility, prevent agglomeration, and protect magnetic core from oxidation. Ideally, MNPs should have active targeting capabilities to allow their accumulation in the desired tissue/cells to be cured or imaged. To date, the big challenge for MNPs is related to the lack of synthetic route for obtaining functionalized monodisperse particles with high yield and by means of a reproducible process that may be scaled up without additional purification step. So far, only few formulations were approved for clinical applications such as Lumiren, Combidex, and Feridex, respectively for bowel, lymph node, and liver imaging.
F. Canfarotta, Prof. S. A. Piletsky Cranfield Health Cranfield University Cranfield Bedfordshire, MK43 0AL, UK E-mail: [email protected]
DOI: 10.1002/adhm.201300141
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In this work, we describe synthesis of iron oxide NPs and their functionalization, together with the most widespread biomedical applications such as imaging, cancer therapy, and drug delivery.
2. Synthesis of Magnetic Iron Oxide Nanoparticles
Generally, two basic approaches are used for the synthesis of MNPs: the “bottomup,” consisting of constructing nanoparticles starting from molecules or atoms, and “top-down,” consisting of disrupting bulk material into the final desired product. The bottom-up approach generally allows a better control for generating uniform particles. The monodispersity is especially important for in vivo application. For instance, in hyperthermic cancer therapy, a narrow size distribution is crucial to obtain homogeneous heat production. The size, shape, and surface characteristics of MNPs can be controlled by the synthetic route, which has its specific advantages for biomedical applications (Table 1). Among the methods used for controlled synthesis of MNPs are electron beam lithography, spray pyrolysis, and deposition from gas phase.[22–25] However, the yield of the produced particles in general is quite low and the manufacturing procedure expensive.[26] Other chemical techniques produce higher yields, these include coprecipitation technique,[27–32] microemulsion method,[33–34] sol–gel reaction,[35] hydrothermal process,[36–40] and thermo-, photo-, and sonolysis methods.[41–44] The most popular procedure is coprecipitation of Fe2+/Fe3+ salts with the addition of a base in inert atmosphere (so-called Massart method). The size of the particles can be tailored by changing pH, temperature, nature, and Fe2+/Fe3+ ratio. Unfortunately, this process often leads to polydisperse MNPs, not suitable for biomedical applications. Moreover, particles synthesized through coprecipitation technique are usually negatively charged, leading to potential agglomeration issues. The crucial factor for producing monodisperse MNPs lies in controlling kinetic factors during their synthesis.[45] This can be achieved by fast introduction of the reagents in a hot solution of surfactant, which leads to the generation of many nuclei.[46] Otherwise, it is possible to mix the reagents at low temperature, and then slowly heat the mixture. Particles will grow by Ostwald ripening, according to which small particles undergo dissolution and redeposition onto bigger NPs. Termination of particle growth can be achieved by rapid decrease of the temperature of reaction mixture.[45] The particle size can also be reduced
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by bubbling nitrogen in the reaction mixture, which in addition decreases potential oxidation issues of the magnetite.[47–48] Furthermore, by increasing pH, ionic strength, and mixing rate, smaller and more monodisperse MNPs can be obtained.[44] MNPs with controlled size can be produced by thermal decomposition of nitrates, carbonates, and organometallic compounds such as metal acetylacetonates or cupferronates, which leads to quite high yield. Important parameters for controlling size and shape of the particles are the organometallic compounds–surfactant ratio, the reaction time, and the use of additives such as cyanuric acid.[49] The presence of the last one can lead to the formation of hexagonal nanostructures (Figure 1).[50] In thermal decomposition technique, the temperature control plays an important role in the final homogeneity of the MNPs produced. This fact strongly depends on the composition of magnetic particles. For iron-based NPs, a temperature range between 150 and 350 °C is the most employed. Typically, each route has its optimum temperature, depending on the presence of additives, the heating rate, and the reaction time. In general, thermal decomposition allows to produce metallic NPs having magnetization values higher compared with metal oxide NPs. However, this method allows only the production of organicsoluble MNPs, which require to be engineered in order to be applied in the biomedical field. Moreover, this approach needs high temperature (≈300 °C) in order to achieve high yield and the control of particle size is quite difficult. A promising technique for producing highly monodisperse iron oxide NPs is the hydrothermal synthesis. The latter is usually carried out in autoclaves or reactors, which allow a better control over reaction parameters such as temperature and pressure. By means of hydrothermal processes, monodisperse iron oxide NPs have been synthesized and coated with several stabilizing agents such as oleic acid, oleylamine, and surfactants.[26] Magnetic particles produced in supercritical water have also been reported.[51] Supercritical water shows low dielectric constant and high self-dissociation, which allow iron oxide to be precipitated without the need of strong bases, as required in the other techniques. Narrow size distribution can be obtained also by microemulsion technique, in which two immiscible solvents are stabilized by a proper surfactant. Water-in-oil (W/O) systems have been widely employed. In this method, water is dispersed in hydrocarbon phase with subsequent formation of
Francesco Canfarotta received his M.S. degree in Pharmaceutical Chemistry and Technology from the University of Palermo in 2010. During his academic studies, he worked on the synthesis of amphiphilic copolymers for drug delivery applications. He joined Cranfield University in 2011 as Marie Curie research fellow. His current Ph.D. research studies focus on molecularly imprinted polymer nanoparticles for diagnostic applications, including fluorescent and multifunctional nanosystems based on optical detection and cell biocompatibility studies. Sergey Piletsky received his Ph.D. in Bioorganic Chemistry from the Institute of Bioorganic and Oil Chemistry, Kiev in 1991. In 1998, he joined Cranfield University and became Professor in 2002. The aim of his research is the development of a new generation of molecularly imprinted polymers that could be used as an alternative to enzymes, antibodies, and receptors in sensors and affinity separation.
dwarf droplets stabilized by surfactant, which also limits the particle growth. After addition of base and iron salts, the droplets undergo coalescence and breaking leading to precipitation of MNPs.[52] The size of these droplets affects the final size of MNPs, therefore type and concentration of surfactant must be carefully chosen. Microemulsion technique can be used also for
Table 1. Comparison of the most frequently employed methods for the synthesis of iron oxide nanoparticles. Precipitation method Size and magnetization values
5–60 nm; 20–80 emu g−1
Main parameters affecting pH, temperature and FeII/ particle size FeIII ratio
Microemulsion technique
Hydrothermal processes
Gas deposition procedure
4–20 nm; 30–60 emu g−1 20–200 nm; 10–40 emu g−1 5–900 nm; 30–80 emu g−1
5–50 nm; 20–50 emu g−1
Type and amount of surfactant
Sol – gel method
pH, temperature and dispersion composition
Advantages
High yield; fast and simple Size control; quite narrow Size control; useful for the synthetic procedure size distribution synthesis of composite particles
Disadvantages
Poor shape control; risk of Presence of surfactant; particle oxidation; broad low yield; organic solvent size distribution needed
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Presence of unreacted components
Temperature, pressure, reaction time
oxygen content of the gas phase, heating time, pressure
Size and shape control; narrow size distribution
Narrow size distribution
High temperature; impossible in situ functionalization of the particle surface
Low yield; high temperature; expensive instruments
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Figure 1. Scheme of the formation of prismatic iron oxide NPs by thermal decomposition, together with their scanning electron microscopy images. Adapted with permission.[50] Copyright 2013, Royal Society of Chemistry.
producing silica- and poly(N-isopropylacrylamide)-coated MNPs suitable for thermo-dependant biomedical uses.[53–54] Microemulsion technique allows a good control over the particle size and a narrow size distribution, but residues of surfactant or organic solvent need to be washed away before medical application. In the sol–gel technique, hydrolysis and condensation of alkoxides lead to the formation of dispersion of oxide particles. Critical parameters to be controlled in this method are pH, temperature, and composition of the particle dispersion.[55] This procedure is a simple way to obtain silica-coated MNPs potentially useful for biomedical and in vivo applications. The main advantages of this method are the good control over the size and the possibility to incorporate other molecules within the silica matrix. One problem may be the low magnetization values achieved with this method. Recently, new environmental-friendly synthetic routes were performed using microorganisms.[56] Two different approaches can be used in the microbial synthesis of MNPs. The first one is called biologically induced mineralization and leads to extracellular production of magnetite crystals under anaerobic conditions. In this case, parameters as temperature, pH, pO2, and pCO2 of the culture solution affect the final product.[57] The second approach is the biologically controlled mineralization (BCM) in which the crystals are produced intracellularly.[58] The BCM route is used by the so-called “magnetotactic bacteria” for the synthesis of iron oxide NPs, whose size and morphology were found to be specie specific.
3. Magnetic Properties of Iron Oxide Nanoparticles Five types of magnetic behaviors can be described: ferromagnetism, antiferromagnetism, ferrimagnetism, diamagnetism, and paramagnetism. For biomedical applications, the most important phenomenon is paramagnetism. The magnetic properties of materials mainly depend on their magnetic susceptibility (χ), which is the ratio between the induced magnetization (M) and the applied magnetic field (B0). Diamagnetic materials 162
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have a very small susceptibility (from 10−6 to 10−3), whereas for paramagnetic materials it is on the order of 10−6 to 10−1. Ferromagnetic, ferrimagnetic, and antiferromagnetic materials are magnetic even without the application of an external magnetic field (EMF) and because of that they are not employed in biomedicine. In general, when magnetic materials are exposed to an EMF, their magnetic moments align in parallel to such EMF. The magnetic moment is due to electrons in the partially filled inner shell and for iron it is due to four unpaired electrons in its 3d orbitals. The nanometer sizes crystals can be considered independent single-domains or “magnetic domains”.[6] Whereas the paramagnetism is a property of the material, the superparamagnetism depends on the size and on the temperature. Unlike paramagnetic materials, superparamagnetic substances maintain no remnant magnetization after that the EMF is removed: in this way, the risk of NPs aggregation is reduced. The most widely used superparamagnetic NPs in biomedicine are made of iron oxide, and are named SPIO (superparamagnetic iron oxide) NPs. The general formula of SPIO structures is Fe23+O3M2+O, with M2+ divalent metal ion (iron, cobalt, manganese, nickel, or magnesium). In order to achieve superparamagnetism for iron oxide crystal, the convenient size of MNPs should be under 30 nm.[59] Iron oxide particles smaller than 15 nm are also named ultrasmall SPIO (USPIO). However, particles too small (80%)
[105–113]
Silica
Improves water-dispersibility. Further functionalization. Drug/gene delivery
UPCT without toxic effects: 200 μg mL−1 (if silica shell is endowed with amino groups)
[114–117]
Gold
Protects against core oxidation. Further functionalization. Applications in imaging and hyperthermic cancer therapy. Exploitation of the surface plasmon resonance effect.
UPCT: 3 × 10−3 M (cell viability ≈90% after 24 h using AuMNPs coated with polyglycerol)
[118–126]
Improve colloidal stability. Functionalization with carboxyl groups. Employed as boiling-point elevating agents
Enhancement of particle uptake. Cellular biocompatibility of fatty acids-coated MNPs still not well studied.
[127–198]
Drug loading capabilities.
Improves biocompatibility and cell uptake. UPCT without toxic effects: 1.2 mg mL−1
[199–203]
Coating/functionalizing agent
Polyacrylic acid
Fatty acids
Gelatin
Chitosan
Antibacterial and mucoadhesive properties. Applications in hyperthermic cancer therapy and magnetofection.
Dextran
Enhances blood circulation time. Further functionalization. Approved for clinical use in imaging.
a)UPCT:
Improve biocompatibility and enhances cell internalization. UPCT without toxic effecs: 4 mg mL−1
[207–215]
upper particle concentration tested.
of repulsions: electrostatic and steric. The former employs ionic agents to coat particles. Cationic molecules (alkylammoniums) stabilize better in neutral and acidic solutions, whereas anionic molecules such as citrate and phosphate are suitable for stabilization in neutral and basic conditions.[5] The use of small molecules as stabilizers does not change the hydrodynamic radius of the particle, which is good for in vivo applications, as smaller particles are better internalizated and excreted. But this kind of stabilization is very sensitive to ionic strength and pH of the particle dispersion. In the sterical stabilization, compounds such as polymers or proteins are used to create a physical barrier between particles, thus reducing the NP interactions. This method is more versatile as apart from the achieved stabilization, it also allows NPs to be functionalized. Moreover, coating agents are useful to prevent oxidation of magnetite (Fe3O4) by oxygen into maghemite (γFe2O3), which leads to changes in the magnetic properties.[68] Several materials have been used as coatings for stabilization or functionalization purposes
164
Improves biocompatibility. UPCT without toxic effects [131, 132, 204–206] 123.52 μg mL−1
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(Table 2), ranging from organic compounds such as polymers and surfactants, to inorganic materials such as silica and gold. For instance, synthetic polymers such as poly(vinylpyrrolidone), poly(vinyl alcohol), poly(ethylene-co-vinyl acetate), poly(lacticco-glycolic acid) (PLGA), poly(ethyleneglycol) (PEG), and their copolymers or natural polymers such as dextran, gelatin, chitosan, and pullulan have been widely employed. PEG remains the most popular coating agent, which can adopt “mushroom” or “brush” conformation, depending on density of the coating.[69] The brush conformation onto NPs generally enhances their circulation time, shielding the NPs from the opsonins. As illustrated in Figure 3, PEG coating can be achieved either by physical adsorption of PEG molecules onto the NPs or by covalently attachment of monofunctional or bifunctional PEG. In the latter case, NPs can be endowed with specific molecules such as targeting ligands, drugs or fluorescent tags. Carboxylterminated PEG have been used for PEGylation of MNPs by thermal decomposition method.[70] The results showed an
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Figure 3. A) Low-density mushroom configurations and B) high-density brush-type arrangements. Reproduced with permission.[69] Copyright 2011, Future Medicine Ltd. C) Different strategies for nanoparticle PEGylation. Adapted with permission.[71] Copyright 2011, Wiley.
increase in the particle solubility and blood circulation time upon increase of the molecular weight of the PEG employed. Other works showed that the use of two polymer layers or copolymers can improve biocompatibility and/or drug delivery efficiency.[72,133,134] For instance, MNPs coated with β-cyclodextrin and pluronic polymer showed greater hyperthermic effect and improved MRI efficiency compared with MNPs only coated by β-cyclodextrin.[134] Furthermore, by joining together two different polymers new properties can be achieved, arising from the specific features of each polymer. For instance, PEG–polyethyleneimine (PEI) copolymer shows both DNA binding affinity (PEI property) and stealth behavior (PEG property).[135] In another example, MNPs were coated with a triblock copolymer made of PEG and two methacrylic acid derivatives for delivery of an anticancer drug (adriamycin).[136] The particles were also coated with a folate-conjugated block copolymer for targeting purposes. The drug was released at acidic pH values due to the carboxylate protonation of the methacrylic moieties which lead to the break of the ionic bonds between drug and polymer. Moreover, PEG coating improves the particle biocompatibility, enhancing their cellular uptake. It should be noted that the thickness of the polymer layer is crucial, as it may not be capable of preventing oxidation of MNPs if the coating is too thin. A good method for controlling the thickness of the polymer layer is given by the atom transfer radical polymerization.[137–139] Moreover, amphiphilic polymers have been employed both to bestow NPs with aqueous dispersibility properties and to conjugate biomolecules onto the MNP’s surface. Typically, –COO− groups are widely exploited to improve water solubility. The use of amphiphilic polymers is straightforward and fast (i.e., ligand-exchange method), considering that it simply relies on hydrophobic interactions. For instance, a poly(maleic anhydride)-based polymer modified with organic molecules and hydrophobic side chains was synthesized and used to coat several types of nanoparticles (quantum dots [QDs], gold, and iron oxide NPs).[140] The advantage of such a polymer relies on the fact that the maleic anhydride portions react spontaneously with molecules bearing amino terminal groups, without the need of any additional compound for the coupling reaction and generating at the same time free carboxylic groups useful to enhance water dispersibility. Poly(maleic anhydride)-based polymers were successfully conjugated with PEG or glucose to confer biocompatibility to gold-coated MNPs.[141]
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Polysaccharides such as alginate, gum Arabic, starch chitosan and dextran, and polyamino acids have been successfully employed as coating agents.[131] Chitosan increases the particle biocompatibility and enhances particle adhesion to cells because the positively charged polymer coating interacts electrostatically with the negatively charged glycocaly present on the cellular surface. However, polysaccharides show low mechanical resistance and the microemulsion technique cannot be employed for the in situ particle coating as polysaccharides are difficult to solubilize in organic solvents. Furthermore, nonpolymeric stabilizers such as fatty acids have been successfully employed to improve MNPs stability in organics.[142] By means of ligand-exchange approach, the oleic acid moieties present onto the surface of such particles can be replaced with other capping agents bearing hydroxyl groups useful for further derivatization.[143.] MNPs can also be embedded in phospholipid bilayers (liposomes), allowing enhanced uptake in cells for drug and gene delivery.[144] Silica coating is another method for improving stability, biocompatibility, and surface functionalization of MNPs by exploiting silanol groups, which can easily react with silane coupling agents and alcohols.[114,145,146] (3-Aminopropyl)triethoxysilane, aminopropyl trimethoxysilane, and (3-mercaptopropyl) triethoxysilane are the most frequently employed agents used for introducing functional groups for further derivatization. The silica matrix can be used for drug loading, thus employing silica-coated MNPs both as diagnostic and therapeutic agents.[145,147,148] Silica coating is usually performed by hydrolysis and condensation of tetraethoxy silane (Stöber method), by microemulsion or sol–gel processes. Depending on the synthetic parameters (pH in particular), either silica NPs embedded with several MNPs or single coated MNPs can be obtained. The thickness of the silica layer can be controlled down to 2 nm.[149] Li et al. encapsulated RNA molecules within mesoporous silica MNPs, which showed in vivo tumourtumor inhibition.[115] The silica coating has also a protective role preventing interactions between magnetic particle and molecules linked on its surface. For instance, the attachment of a dye molecule to iron oxide NPs may lead to quenching of the fluorescence emission of such a dye, due to short-range energy transfer phenomena. This problem can be overcome by embedding the dye within a polymeric or silica matrix, thus increasing the distance between dyes and MNPs. Moreover, PEG-silanes can be used to make hydrophobic oleic acid-coated MNPs easily
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dispersible in aqueous media, by means of the ligand-exchange technique.[71] Unfortunately silica is not stable under basic conditions and precise control over silica deposition on MNPs is difficult to achieve. Gold is another coating agent used due to its capability to protect the iron core from oxidation and allow further functionalization. Furthermore, gold coating can be exploited in cancer therapy due to its ability to convert energy into heat by alternating magnetic field, radiowaves, or near-infrared light.[150,151] This targeted heat generation is useful against tumors resistant to chemotherapy, or for improved therapeutic efficacy when combined with radiotherapy or classic anticancer drugs. Usually, the deposition of gold layer is carried out by reduction of Au(III) on the surface of iron oxide NPs. Other authors employed sonochemical methods to produce iron/gold core/ shell NPs with narrow size distribution. For instance, Tamer and Gündogˇdu synthesized monodisperse gold-coated iron oxide NPs (12.5 ± 3 nm) first by coprecipitation of Fe (II) and Fe (III) mixture, then by sonication of the MNP dispersion obtained in the presence of HAuCl4 and NaBH4.[152] Recently, carbon has been used as coating agent due to its high thermal, chemical stability, and biocompatibility. MNPs coated with carbon show higher magnetic moment compared with their corresponding oxides because they are usually in the metallic state.[153] However, the synthetic methods for obtaining carbon-coated MNPs often produce agglomerated particles. Moreover, the carbon coating is often inhomogeneous and a fine control over the coating thickness still has to be achieved. 4.2. Biocompatibility and Biodistribution The particle size and shape affect particle toxicity and their internalization within cells. This latter process is also affected by the particle surface coating. In physiological conditions, any type of NPs is likely to undergo a covering process by the biomolecules (mainly proteins) present in the serum. Because such a protein corona is the outmost layer, it can affect the particle interactions with cells. This fact has been proven to be independent of the particle core, considering that both organic (polystyrene) and inorganic (QDs and gold NPs) nanosystems showed such a corona effect.[152] The presence of a polymeric layer surrounding the particle core can reduce such a protein corona effect. Recently, polymer -coated iron oxide NPs showed better stability in biological fluids compared with particles covered by low molecular compounds (citric acid).[153] In general, the MNP cytotoxicity is also related to the production of reactive oxygen species, probably via Fenton or Haber– Weiss reactions, which may lead to protein and lipid oxidation as well as DNA damage. Thus functional coating is required to minimize toxic effects and to aid intended application. The large surface-to-volume ratio of the NPs compared with bulk systems leads to higher potential cytotoxicity and agglomeration issues, which are also affected by the particle size and surface coating. A recent study conducted on PVA-coated iron oxide NPs demonstrated that the biocompatibility of the NPs can be enhanced by increasing the polymer/iron mass ratio (r value).[154] It has been proven that the best size for in vivo applications should be
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Engineered Magnetic Nanoparticles for Biomedical Applications Francesco Canfarotta* and Sergey A. Piletsky
In the past decades, magnetic nanoparticles (MNPs) have been used in wide range of diverse applications, ranging from separation to sensing. Here, synthesis and applications of functionalized MNPs in the biomedical field are discussed, in particular in drug delivery, imaging, and cancer therapy, highlighting also recent progresses in the development of multifunctional and stimuli-responsive MNPs. The role of their size, composition, and surface functionalization is analyzed, together with their biocompatibility issues.
1. Introduction In the past two decades, magnetic nanoparticles (MNPs) were employed in several biomedical applications, such as drug delivery,[1–4] magnetic resonance imaging (MRI),[5–8] cancer therapy,[9–11] bioseparation,[12–14] and biosensing.[15–21] The materials used most frequently in these applications are magnetite (Fe3O4) or maghemite (γ-Fe2O3), thanks to their good biocompatibility and magnetic properties. The general requirements for biomedical application are mainly related to their magnetization value, surface coating, and size. These parameters strongly affect both the magnetic behavior of the particles and their stability, biocompatibility, and biodistribution. MNPs with diameter 10–100 nm are preferred for the use in vivo sinceas they do not have rapid renal clearance that occurs for NPs smaller than 10 nm, and internalization by reticuloendothelial system that occurs for NPs >200 nm.[10] The functionalization of MNPs surface is advisable to improve their biocompatibility, prevent agglomeration, and protect magnetic core from oxidation. Ideally, MNPs should have active targeting capabilities to allow their accumulation in the desired tissue/cells to be cured or imaged. To date, the big challenge for MNPs is related to the lack of synthetic route for obtaining functionalized monodisperse particles with high yield and by means of a reproducible process that may be scaled up without additional purification step. So far, only few formulations were approved for clinical applications such as Lumiren, Combidex, and Feridex, respectively for bowel, lymph node, and liver imaging.
F. Canfarotta, Prof. S. A. Piletsky Cranfield Health Cranfield University Cranfield Bedfordshire, MK43 0AL, UK E-mail: [email protected]
DOI: 10.1002/adhm.201300141
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In this work, we describe synthesis of iron oxide NPs and their functionalization, together with the most widespread biomedical applications such as imaging, cancer therapy, and drug delivery.
2. Synthesis of Magnetic Iron Oxide Nanoparticles
Generally, two basic approaches are used for the synthesis of MNPs: the “bottomup,” consisting of constructing nanoparticles starting from molecules or atoms, and “top-down,” consisting of disrupting bulk material into the final desired product. The bottom-up approach generally allows a better control for generating uniform particles. The monodispersity is especially important for in vivo application. For instance, in hyperthermic cancer therapy, a narrow size distribution is crucial to obtain homogeneous heat production. The size, shape, and surface characteristics of MNPs can be controlled by the synthetic route, which has its specific advantages for biomedical applications (Table 1). Among the methods used for controlled synthesis of MNPs are electron beam lithography, spray pyrolysis, and deposition from gas phase.[22–25] However, the yield of the produced particles in general is quite low and the manufacturing procedure expensive.[26] Other chemical techniques produce higher yields, these include coprecipitation technique,[27–32] microemulsion method,[33–34] sol–gel reaction,[35] hydrothermal process,[36–40] and thermo-, photo-, and sonolysis methods.[41–44] The most popular procedure is coprecipitation of Fe2+/Fe3+ salts with the addition of a base in inert atmosphere (so-called Massart method). The size of the particles can be tailored by changing pH, temperature, nature, and Fe2+/Fe3+ ratio. Unfortunately, this process often leads to polydisperse MNPs, not suitable for biomedical applications. Moreover, particles synthesized through coprecipitation technique are usually negatively charged, leading to potential agglomeration issues. The crucial factor for producing monodisperse MNPs lies in controlling kinetic factors during their synthesis.[45] This can be achieved by fast introduction of the reagents in a hot solution of surfactant, which leads to the generation of many nuclei.[46] Otherwise, it is possible to mix the reagents at low temperature, and then slowly heat the mixture. Particles will grow by Ostwald ripening, according to which small particles undergo dissolution and redeposition onto bigger NPs. Termination of particle growth can be achieved by rapid decrease of the temperature of reaction mixture.[45] The particle size can also be reduced
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by bubbling nitrogen in the reaction mixture, which in addition decreases potential oxidation issues of the magnetite.[47–48] Furthermore, by increasing pH, ionic strength, and mixing rate, smaller and more monodisperse MNPs can be obtained.[44] MNPs with controlled size can be produced by thermal decomposition of nitrates, carbonates, and organometallic compounds such as metal acetylacetonates or cupferronates, which leads to quite high yield. Important parameters for controlling size and shape of the particles are the organometallic compounds–surfactant ratio, the reaction time, and the use of additives such as cyanuric acid.[49] The presence of the last one can lead to the formation of hexagonal nanostructures (Figure 1).[50] In thermal decomposition technique, the temperature control plays an important role in the final homogeneity of the MNPs produced. This fact strongly depends on the composition of magnetic particles. For iron-based NPs, a temperature range between 150 and 350 °C is the most employed. Typically, each route has its optimum temperature, depending on the presence of additives, the heating rate, and the reaction time. In general, thermal decomposition allows to produce metallic NPs having magnetization values higher compared with metal oxide NPs. However, this method allows only the production of organicsoluble MNPs, which require to be engineered in order to be applied in the biomedical field. Moreover, this approach needs high temperature (≈300 °C) in order to achieve high yield and the control of particle size is quite difficult. A promising technique for producing highly monodisperse iron oxide NPs is the hydrothermal synthesis. The latter is usually carried out in autoclaves or reactors, which allow a better control over reaction parameters such as temperature and pressure. By means of hydrothermal processes, monodisperse iron oxide NPs have been synthesized and coated with several stabilizing agents such as oleic acid, oleylamine, and surfactants.[26] Magnetic particles produced in supercritical water have also been reported.[51] Supercritical water shows low dielectric constant and high self-dissociation, which allow iron oxide to be precipitated without the need of strong bases, as required in the other techniques. Narrow size distribution can be obtained also by microemulsion technique, in which two immiscible solvents are stabilized by a proper surfactant. Water-in-oil (W/O) systems have been widely employed. In this method, water is dispersed in hydrocarbon phase with subsequent formation of
Francesco Canfarotta received his M.S. degree in Pharmaceutical Chemistry and Technology from the University of Palermo in 2010. During his academic studies, he worked on the synthesis of amphiphilic copolymers for drug delivery applications. He joined Cranfield University in 2011 as Marie Curie research fellow. His current Ph.D. research studies focus on molecularly imprinted polymer nanoparticles for diagnostic applications, including fluorescent and multifunctional nanosystems based on optical detection and cell biocompatibility studies. Sergey Piletsky received his Ph.D. in Bioorganic Chemistry from the Institute of Bioorganic and Oil Chemistry, Kiev in 1991. In 1998, he joined Cranfield University and became Professor in 2002. The aim of his research is the development of a new generation of molecularly imprinted polymers that could be used as an alternative to enzymes, antibodies, and receptors in sensors and affinity separation.
dwarf droplets stabilized by surfactant, which also limits the particle growth. After addition of base and iron salts, the droplets undergo coalescence and breaking leading to precipitation of MNPs.[52] The size of these droplets affects the final size of MNPs, therefore type and concentration of surfactant must be carefully chosen. Microemulsion technique can be used also for
Table 1. Comparison of the most frequently employed methods for the synthesis of iron oxide nanoparticles. Precipitation method Size and magnetization values
5–60 nm; 20–80 emu g−1
Main parameters affecting pH, temperature and FeII/ particle size FeIII ratio
Microemulsion technique
Hydrothermal processes
Gas deposition procedure
4–20 nm; 30–60 emu g−1 20–200 nm; 10–40 emu g−1 5–900 nm; 30–80 emu g−1
5–50 nm; 20–50 emu g−1
Type and amount of surfactant
Sol – gel method
pH, temperature and dispersion composition
Advantages
High yield; fast and simple Size control; quite narrow Size control; useful for the synthetic procedure size distribution synthesis of composite particles
Disadvantages
Poor shape control; risk of Presence of surfactant; particle oxidation; broad low yield; organic solvent size distribution needed
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Presence of unreacted components
Temperature, pressure, reaction time
oxygen content of the gas phase, heating time, pressure
Size and shape control; narrow size distribution
Narrow size distribution
High temperature; impossible in situ functionalization of the particle surface
Low yield; high temperature; expensive instruments
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Figure 1. Scheme of the formation of prismatic iron oxide NPs by thermal decomposition, together with their scanning electron microscopy images. Adapted with permission.[50] Copyright 2013, Royal Society of Chemistry.
producing silica- and poly(N-isopropylacrylamide)-coated MNPs suitable for thermo-dependant biomedical uses.[53–54] Microemulsion technique allows a good control over the particle size and a narrow size distribution, but residues of surfactant or organic solvent need to be washed away before medical application. In the sol–gel technique, hydrolysis and condensation of alkoxides lead to the formation of dispersion of oxide particles. Critical parameters to be controlled in this method are pH, temperature, and composition of the particle dispersion.[55] This procedure is a simple way to obtain silica-coated MNPs potentially useful for biomedical and in vivo applications. The main advantages of this method are the good control over the size and the possibility to incorporate other molecules within the silica matrix. One problem may be the low magnetization values achieved with this method. Recently, new environmental-friendly synthetic routes were performed using microorganisms.[56] Two different approaches can be used in the microbial synthesis of MNPs. The first one is called biologically induced mineralization and leads to extracellular production of magnetite crystals under anaerobic conditions. In this case, parameters as temperature, pH, pO2, and pCO2 of the culture solution affect the final product.[57] The second approach is the biologically controlled mineralization (BCM) in which the crystals are produced intracellularly.[58] The BCM route is used by the so-called “magnetotactic bacteria” for the synthesis of iron oxide NPs, whose size and morphology were found to be specie specific.
3. Magnetic Properties of Iron Oxide Nanoparticles Five types of magnetic behaviors can be described: ferromagnetism, antiferromagnetism, ferrimagnetism, diamagnetism, and paramagnetism. For biomedical applications, the most important phenomenon is paramagnetism. The magnetic properties of materials mainly depend on their magnetic susceptibility (χ), which is the ratio between the induced magnetization (M) and the applied magnetic field (B0). Diamagnetic materials 162
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have a very small susceptibility (from 10−6 to 10−3), whereas for paramagnetic materials it is on the order of 10−6 to 10−1. Ferromagnetic, ferrimagnetic, and antiferromagnetic materials are magnetic even without the application of an external magnetic field (EMF) and because of that they are not employed in biomedicine. In general, when magnetic materials are exposed to an EMF, their magnetic moments align in parallel to such EMF. The magnetic moment is due to electrons in the partially filled inner shell and for iron it is due to four unpaired electrons in its 3d orbitals. The nanometer sizes crystals can be considered independent single-domains or “magnetic domains”.[6] Whereas the paramagnetism is a property of the material, the superparamagnetism depends on the size and on the temperature. Unlike paramagnetic materials, superparamagnetic substances maintain no remnant magnetization after that the EMF is removed: in this way, the risk of NPs aggregation is reduced. The most widely used superparamagnetic NPs in biomedicine are made of iron oxide, and are named SPIO (superparamagnetic iron oxide) NPs. The general formula of SPIO structures is Fe23+O3M2+O, with M2+ divalent metal ion (iron, cobalt, manganese, nickel, or magnesium). In order to achieve superparamagnetism for iron oxide crystal, the convenient size of MNPs should be under 30 nm.[59] Iron oxide particles smaller than 15 nm are also named ultrasmall SPIO (USPIO). However, particles too small (80%)
[105–113]
Silica
Improves water-dispersibility. Further functionalization. Drug/gene delivery
UPCT without toxic effects: 200 μg mL−1 (if silica shell is endowed with amino groups)
[114–117]
Gold
Protects against core oxidation. Further functionalization. Applications in imaging and hyperthermic cancer therapy. Exploitation of the surface plasmon resonance effect.
UPCT: 3 × 10−3 M (cell viability ≈90% after 24 h using AuMNPs coated with polyglycerol)
[118–126]
Improve colloidal stability. Functionalization with carboxyl groups. Employed as boiling-point elevating agents
Enhancement of particle uptake. Cellular biocompatibility of fatty acids-coated MNPs still not well studied.
[127–198]
Drug loading capabilities.
Improves biocompatibility and cell uptake. UPCT without toxic effects: 1.2 mg mL−1
[199–203]
Coating/functionalizing agent
Polyacrylic acid
Fatty acids
Gelatin
Chitosan
Antibacterial and mucoadhesive properties. Applications in hyperthermic cancer therapy and magnetofection.
Dextran
Enhances blood circulation time. Further functionalization. Approved for clinical use in imaging.
a)UPCT:
Improve biocompatibility and enhances cell internalization. UPCT without toxic effecs: 4 mg mL−1
[207–215]
upper particle concentration tested.
of repulsions: electrostatic and steric. The former employs ionic agents to coat particles. Cationic molecules (alkylammoniums) stabilize better in neutral and acidic solutions, whereas anionic molecules such as citrate and phosphate are suitable for stabilization in neutral and basic conditions.[5] The use of small molecules as stabilizers does not change the hydrodynamic radius of the particle, which is good for in vivo applications, as smaller particles are better internalizated and excreted. But this kind of stabilization is very sensitive to ionic strength and pH of the particle dispersion. In the sterical stabilization, compounds such as polymers or proteins are used to create a physical barrier between particles, thus reducing the NP interactions. This method is more versatile as apart from the achieved stabilization, it also allows NPs to be functionalized. Moreover, coating agents are useful to prevent oxidation of magnetite (Fe3O4) by oxygen into maghemite (γFe2O3), which leads to changes in the magnetic properties.[68] Several materials have been used as coatings for stabilization or functionalization purposes
164
Improves biocompatibility. UPCT without toxic effects [131, 132, 204–206] 123.52 μg mL−1
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(Table 2), ranging from organic compounds such as polymers and surfactants, to inorganic materials such as silica and gold. For instance, synthetic polymers such as poly(vinylpyrrolidone), poly(vinyl alcohol), poly(ethylene-co-vinyl acetate), poly(lacticco-glycolic acid) (PLGA), poly(ethyleneglycol) (PEG), and their copolymers or natural polymers such as dextran, gelatin, chitosan, and pullulan have been widely employed. PEG remains the most popular coating agent, which can adopt “mushroom” or “brush” conformation, depending on density of the coating.[69] The brush conformation onto NPs generally enhances their circulation time, shielding the NPs from the opsonins. As illustrated in Figure 3, PEG coating can be achieved either by physical adsorption of PEG molecules onto the NPs or by covalently attachment of monofunctional or bifunctional PEG. In the latter case, NPs can be endowed with specific molecules such as targeting ligands, drugs or fluorescent tags. Carboxylterminated PEG have been used for PEGylation of MNPs by thermal decomposition method.[70] The results showed an
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Figure 3. A) Low-density mushroom configurations and B) high-density brush-type arrangements. Reproduced with permission.[69] Copyright 2011, Future Medicine Ltd. C) Different strategies for nanoparticle PEGylation. Adapted with permission.[71] Copyright 2011, Wiley.
increase in the particle solubility and blood circulation time upon increase of the molecular weight of the PEG employed. Other works showed that the use of two polymer layers or copolymers can improve biocompatibility and/or drug delivery efficiency.[72,133,134] For instance, MNPs coated with β-cyclodextrin and pluronic polymer showed greater hyperthermic effect and improved MRI efficiency compared with MNPs only coated by β-cyclodextrin.[134] Furthermore, by joining together two different polymers new properties can be achieved, arising from the specific features of each polymer. For instance, PEG–polyethyleneimine (PEI) copolymer shows both DNA binding affinity (PEI property) and stealth behavior (PEG property).[135] In another example, MNPs were coated with a triblock copolymer made of PEG and two methacrylic acid derivatives for delivery of an anticancer drug (adriamycin).[136] The particles were also coated with a folate-conjugated block copolymer for targeting purposes. The drug was released at acidic pH values due to the carboxylate protonation of the methacrylic moieties which lead to the break of the ionic bonds between drug and polymer. Moreover, PEG coating improves the particle biocompatibility, enhancing their cellular uptake. It should be noted that the thickness of the polymer layer is crucial, as it may not be capable of preventing oxidation of MNPs if the coating is too thin. A good method for controlling the thickness of the polymer layer is given by the atom transfer radical polymerization.[137–139] Moreover, amphiphilic polymers have been employed both to bestow NPs with aqueous dispersibility properties and to conjugate biomolecules onto the MNP’s surface. Typically, –COO− groups are widely exploited to improve water solubility. The use of amphiphilic polymers is straightforward and fast (i.e., ligand-exchange method), considering that it simply relies on hydrophobic interactions. For instance, a poly(maleic anhydride)-based polymer modified with organic molecules and hydrophobic side chains was synthesized and used to coat several types of nanoparticles (quantum dots [QDs], gold, and iron oxide NPs).[140] The advantage of such a polymer relies on the fact that the maleic anhydride portions react spontaneously with molecules bearing amino terminal groups, without the need of any additional compound for the coupling reaction and generating at the same time free carboxylic groups useful to enhance water dispersibility. Poly(maleic anhydride)-based polymers were successfully conjugated with PEG or glucose to confer biocompatibility to gold-coated MNPs.[141]
Adv. Healthcare Mater. 2014, 3, 160–175
Polysaccharides such as alginate, gum Arabic, starch chitosan and dextran, and polyamino acids have been successfully employed as coating agents.[131] Chitosan increases the particle biocompatibility and enhances particle adhesion to cells because the positively charged polymer coating interacts electrostatically with the negatively charged glycocaly present on the cellular surface. However, polysaccharides show low mechanical resistance and the microemulsion technique cannot be employed for the in situ particle coating as polysaccharides are difficult to solubilize in organic solvents. Furthermore, nonpolymeric stabilizers such as fatty acids have been successfully employed to improve MNPs stability in organics.[142] By means of ligand-exchange approach, the oleic acid moieties present onto the surface of such particles can be replaced with other capping agents bearing hydroxyl groups useful for further derivatization.[143.] MNPs can also be embedded in phospholipid bilayers (liposomes), allowing enhanced uptake in cells for drug and gene delivery.[144] Silica coating is another method for improving stability, biocompatibility, and surface functionalization of MNPs by exploiting silanol groups, which can easily react with silane coupling agents and alcohols.[114,145,146] (3-Aminopropyl)triethoxysilane, aminopropyl trimethoxysilane, and (3-mercaptopropyl) triethoxysilane are the most frequently employed agents used for introducing functional groups for further derivatization. The silica matrix can be used for drug loading, thus employing silica-coated MNPs both as diagnostic and therapeutic agents.[145,147,148] Silica coating is usually performed by hydrolysis and condensation of tetraethoxy silane (Stöber method), by microemulsion or sol–gel processes. Depending on the synthetic parameters (pH in particular), either silica NPs embedded with several MNPs or single coated MNPs can be obtained. The thickness of the silica layer can be controlled down to 2 nm.[149] Li et al. encapsulated RNA molecules within mesoporous silica MNPs, which showed in vivo tumourtumor inhibition.[115] The silica coating has also a protective role preventing interactions between magnetic particle and molecules linked on its surface. For instance, the attachment of a dye molecule to iron oxide NPs may lead to quenching of the fluorescence emission of such a dye, due to short-range energy transfer phenomena. This problem can be overcome by embedding the dye within a polymeric or silica matrix, thus increasing the distance between dyes and MNPs. Moreover, PEG-silanes can be used to make hydrophobic oleic acid-coated MNPs easily
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dispersible in aqueous media, by means of the ligand-exchange technique.[71] Unfortunately silica is not stable under basic conditions and precise control over silica deposition on MNPs is difficult to achieve. Gold is another coating agent used due to its capability to protect the iron core from oxidation and allow further functionalization. Furthermore, gold coating can be exploited in cancer therapy due to its ability to convert energy into heat by alternating magnetic field, radiowaves, or near-infrared light.[150,151] This targeted heat generation is useful against tumors resistant to chemotherapy, or for improved therapeutic efficacy when combined with radiotherapy or classic anticancer drugs. Usually, the deposition of gold layer is carried out by reduction of Au(III) on the surface of iron oxide NPs. Other authors employed sonochemical methods to produce iron/gold core/ shell NPs with narrow size distribution. For instance, Tamer and Gündogˇdu synthesized monodisperse gold-coated iron oxide NPs (12.5 ± 3 nm) first by coprecipitation of Fe (II) and Fe (III) mixture, then by sonication of the MNP dispersion obtained in the presence of HAuCl4 and NaBH4.[152] Recently, carbon has been used as coating agent due to its high thermal, chemical stability, and biocompatibility. MNPs coated with carbon show higher magnetic moment compared with their corresponding oxides because they are usually in the metallic state.[153] However, the synthetic methods for obtaining carbon-coated MNPs often produce agglomerated particles. Moreover, the carbon coating is often inhomogeneous and a fine control over the coating thickness still has to be achieved. 4.2. Biocompatibility and Biodistribution The particle size and shape affect particle toxicity and their internalization within cells. This latter process is also affected by the particle surface coating. In physiological conditions, any type of NPs is likely to undergo a covering process by the biomolecules (mainly proteins) present in the serum. Because such a protein corona is the outmost layer, it can affect the particle interactions with cells. This fact has been proven to be independent of the particle core, considering that both organic (polystyrene) and inorganic (QDs and gold NPs) nanosystems showed such a corona effect.[152] The presence of a polymeric layer surrounding the particle core can reduce such a protein corona effect. Recently, polymer -coated iron oxide NPs showed better stability in biological fluids compared with particles covered by low molecular compounds (citric acid).[153] In general, the MNP cytotoxicity is also related to the production of reactive oxygen species, probably via Fenton or Haber– Weiss reactions, which may lead to protein and lipid oxidation as well as DNA damage. Thus functional coating is required to minimize toxic effects and to aid intended application. The large surface-to-volume ratio of the NPs compared with bulk systems leads to higher potential cytotoxicity and agglomeration issues, which are also affected by the particle size and surface coating. A recent study conducted on PVA-coated iron oxide NPs demonstrated that the biocompatibility of the NPs can be enhanced by increasing the polymer/iron mass ratio (r value).[154] It has been proven that the best size for in vivo applications should be
