Review

Multifunctional nanoparticles properties and prospects for their use in human medicine
Nuria Sanvicens and M. Pilar Marco
Applied Molecular Receptors Group (AMRg), CSIC Networking Research Center of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jorge Girona, 1826, 08034 Barcelona, Spain

A major aim of medicine has long been the early and accurate diagnosis of clinical conditions, providing an efcient treatment without secondary effects. With the emergence of nanotechnology, the achievement of this goal seems closer than ever. To this end, the development of novel materials and devices operating at the nanoscale range, such as nanoparticles, provides new and powerful tools for imaging, diagnosis and therapy. This review focuses on the signicant improvements in performance that nanoparticles offer compared with existing technologies relevant to medicine. Specically, we address the design of multifunctional nanoparticles as an alternative system for drug and gene delivery, which has great potential for therapy in areas, such as cancer and neuropathologies. Moreover, we discuss the controversy generated by the possible toxic health effects of nanoparticles. Introduction Nanotechnology is considered by many as the next big revolution. This technological leap in controlling materials at the nanoscale has, in the past decade, driven developments enabling the use of nanodevices, such as nanoparticles, that have found applications in elds ranging from electronics and communications, through to optics, chemistry, energy and of course biology. Nanomedicine, the application of nanotechnology to healthcare, holds great promise for revolutionising medical treatments and therapies in areas, such as imaging, faster diagnosis, drug delivery and tissue regeneration, as well as the development of new medical products. Indeed, materials and devices of nanometric dimensions (1100 nm) are already approved for clinical use and numerous products are being evaluated in clinical trials [1]. However, as discussed later, there are toxicological concerns and ethical issues that accompany nanomedicine that might cast a shadow over the promising future of this emerging eld. This article presents an overview of the current applications of nanoparticles in medicine. In particular, we focus on the development of novel multifunctional nanoparticles and illustrate their potential application in drug and gene delivery for cancer and neuropathological therapy. The current limitations of nanoparticle-based approaches are discussed, with special emphasis given
Corresponding author: Sanvicens, N. (nsdqob@iiqab.csic.es).

to the lack of knowledge of the toxicological risks associated with the exposure to nanoparticles. Finally, we summarise the future challenges that lie ahead. Multifunctional nanoparticles Nanoparticles are constructs that possess unique physical and chemical properties associated with their being of 1 100 nm in size. The general characteristics and composition of nanoparticles are described in Box 1 and are illustrated in Figure 1. Nanometre-sized particles are in the same range of dimension as antibodies, membrane receptors, nucleic acids and proteins, among other biomolecules. These biomimetic features, together with their high surface:volume ratio and the possibility of modulating their properties, make nanoparticles powerful tools for imaging, diagnosis and therapy [2,3]. Thus, nanoparticles offer signicant improvements in performance compared with existing technologies (Box 2). As a result, commercialisation of nanoparticle-based therapeutics is gaining momentum, with an increasing number of available products on the market [4]. Table 1 gives examples of commercial nanoparticle-based products. Other issues related to nanoparticle technology, such as biomoleculenanoparticle hybrid systems and their use towards medical diagnostics and therapeutics, have been reviewed elsewhere [57]. Despite the benets that nanoparticles have rendered to medicine (Box 2), some applications remain challenging; for instance, in vivo real-time monitoring of cellular events, specic targeting to the action site or efcient drug delivery inside the target cell. In this context, the design of multifunctional nanoparticles could signicantly improve already existing nanoparticle characteristics and help to surmount these challenges. Whereas monofunctional nanoparticles provide a single function a liposome can transport drugs but does not have the inherent property to distinguish between healthy and unhealthy cells or tissues multifunctional nanoparticles combine different functionalities in a single stable construct. For example, a core particle could be linked to a specic targeting function that recognises the unique surface signatures of their target cells. Simultaneously, the same particle can be modied with an imaging agent to monitor the drug transport process, a function to evaluate the therapeutic efcacy of a drug, a specic cellular penetration moiety and a therapeutic agent [8] (Figure 2). Table 2 summarises some of the
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Box 1. Organic and inorganic nanoparticles
Nanoparticles are materials of two or more dimensions, with a size in the range of 1100 nm. Nanoparticles show unique sizedependent physical and chemical properties, for example, optical, magnetic, catalytic, thermodynamic and electrochemical. The chemical composition and the shape of a nanoparticle also influence its specific properties. Nanoparticles are prepared with organic polymers (organic nanoparticles) and/or inorganic elements (inorganic nanoparticles). Liposomes, dendrimers, carbon nanomaterials and polymeric micelles are examples of organic nanoparticles. Liposomes Liposomes are phospholipid vesicles (50100 nm) that have a bilayer membrane structure similar to that of biological membranes and an internal aqueous phase. Liposomes are classified according to size and number of layers into multi-, oligo- or uni-lamellar. Their amphiphilic nature enables liposomes to transport hydrophilic drugs entrapped within their aqueous interior and hydrophobic drugs dissolved into the membrane. Owing to their physicochemical characteristics, liposomes show excellent circulation, penetration and diffusion properties. Moreover, the liposome surface can be modified with ligands and/or polymers to increase drug delivery specificity [63]. Dendrimers Dendrimers are highly branched synthetic polymers (<15 nm) with layered architectures constituted of a central core, an internal region and numerous terminal groups that determine dendrimer characteristics. A dendrimer can be prepared using multiple types of chemistry, the nature of which defines the dendrimer solubility and biological activity. Dendrimers show intrinsic drug properties and are used as tissue-repair scaffolds. Moreover, dendrimers are excellent drug and imaging diagnosis-agent carriers through chemical modification of their multiple terminal groups [71]. Carbon nanotubes Carbon nanotubes belong to the family of fullerenes and are formed of coaxial graphite sheets (<100 nm) rolled up into cylinders. These structures can be obtained either as single- (one graphite sheet) or multi-walled nanotubes (several concentric graphite sheets). They exhibit excellent strength and electrical properties and are efficient heat conductors. Owing to their metallic or semiconductor nature, nanotubes are often used as biosensors. Carbon nanotubes can be rendered water soluble by surface functionalisation. Therefore, they are also used as drug carriers and tissue-repair scaffolds [72]. Inorganic nanoparticles, such as quantum dots, polystyrene, magnetic, ceramic and metallic nanoparticles, have a central core composed of inorganic materials that define their fluorescent, magnetic, electronic and optical properties. Quantum dots Quantum dots are colloidal fluorescent semiconductor nanocrystals (210 nm). The central core of quantum dots consists of combinations of elements from groups IIVI of the periodic system (CdSe, CdTe, CdS, PbSe, ZnS and ZnSe) or IIIV (GaAs, GaN, InP and InAs), which are overcoated with a layer of ZnS. Quantum dots are photostable. They show size- and composition-tuneable emission spectra and high quantum yield. They are resistant to photobleaching and show exceptional resistance to photo and chemical degradation. All these characteristics make quantum dots excellent contrast agents for imaging and labels for bioassays [73]. Magnetic nanoparticles Magnetic nanoparticles are spherical nanocrystals of 1020 nm of size with a Fe2+ and Fe3+ core surrounded by dextran or PEG molecules. Their magnetic properties make them excellent agents to label biomolecules in bioassays, as well as MRI contrast agents. They are also amenable to surface functionalisation for active targeting in vivo or for in vitro diagnostics [74].

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Gold nanoparticles Gold nanoparticles are one type of metallic nanoparticle; others are Au, Ni, Pt and TiO2 nanoparticles. Gold nanoparticles (<50 nm) can be prepared with different geometries, such as nanospheres, nanoshells, nanorods or nanocages. These particles show localised surface plasmon resonant properties, i.e. under the irradiation of light, the conduction electrons are driven by the associated electric field to a collective oscillation at a resonant frequency, thereby absorbing light and emitting photons with the same frequency in all directions. Gold nanoparticles are excellent labels for biosensors because they can be detected by numerous techniques, such as optic absorption, fluorescence and electric conductivity [75].

Box 2. Applications for nanoparticles in medicine

Imaging Optical imaging techniques, such as fluorescence labelling, are used extensively in clinical diagnosis. However, the organic fluorophores used currently are not photostable and have low intensity. Likewise, fluorescence proteins (i.e. green-fluorescence protein) or bioluminescence system (i.e. luciferin/luciferase) applications are limited because they cannot be optimised in multicolour assays. Nanoparticles have helped to overcome these limitations. For example, quantum dots are resistant to photobleaching and photo, chemical and metabolic degradation. They exhibit high quantum yield and enable the simultaneous identification of multiple markers [76]. MRI is another important example of a technique that is used commonly in medicine for the 3D examination of biological events. However, MRI applications are limited by their insensitivity to low concentrations of imaging agent. For these reasons, intensive research efforts aim to develop new MRI contrast agents to enhance imaging. In this regard, superparamagnetic iron oxide nanoparticles have already proven effective in increasing contrast in magnetic imaging [40]. Diagnosis The accurate targeting and quantification of molecules indicative of cellular disorders at the single-molecule level is a demanding task for current high-throughput analysis systems. The combination of nanoparticles with other nanotechnology-based materials has the potential to address this emerging challenge and provide technologies that enable diagnoses at the level of single cells and single molecules [77]. There are already several nanoparticle-based commercialised systems for medical diagnostics [4] (Table 1). Nevertheless, the possibilities for nanoparticle applications in diagnostics are almost unlimited because nanoparticles enable the selective tagging of a wide range of medically important targets, including bacteria, biomarkers and individual molecules, such as proteins and DNA [7880]. Drug delivery Nanoparticle-based drug delivery provides many advantages, such as enhancing drug-therapeutic efficiency and pharmacological characteristics. For example, nanoparticles improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, increase specificity towards the target cell or tissue (therefore reducing side effects), improve bioavailability, diminish drug metabolism and enable a more controllable release of therapeutic compounds and the delivery of two or more drugs simultaneously for combination therapy [81,82].

common approaches that are in use currently to develop multifunctional nanocarriers. Design of intelligent multifunctional nanoparticles The design of nanoparticles that combine several properties is an elaborate process that requires different steps [9], such as the depositing of metal layers onto a supporting nanoparticle core [10,11], modifying the biocompatible

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Figure 1. Examples of nanoparticles. (a) Organic nanoparticles. From left to right: liposomes, dendrimers and carbon nanotubes. (b) Inorganic nanoparticles. From left to right: quantum dots, magnetic nanoparticles and gold nanoparticles. Box 1 provides further information and examples of other types of nanoparticles.

polymer used to stabilise the nanoparticle [12] and the use of different linkers [13,14]. In an attempt to overcome this, recently, Gao et al. reported a simple one-pot synthetic procedure to prepare uorescent magnetic nanocrystals [15]. Furthermore, these different properties of multifunctional nanoparticles have to be coordinated so that they operate in an orchestrated way and indeed provide the desired functionalities. An elegant example for this is the work by Sawant and coworkers. In this study, the nanoparticles had specic targeting and cell-internalisation functions. Under normal conditions (i.e. when not bound specically to the target), the specic target function is exposed, providing a long circulation life and specic delivery, whereas the cell-internalisation function remains hidden. In this manner, the cell-internalisation function does not interfere with the nanoparticle circulation. When specic binding was achieved and owing to the low pH of the pathological environment the penetration function was exposed, facilitating nanoparticle penetration within the cell [12]. Multifunctional nanoparticles for drug and gene delivery Multifunctional delivery nanosystems are just emerging but there are already in the literature several examples of
Table 1. Examples of commercial applications of nanoparticles
Nanoparticle component Liposomes Application Drug delivery Drug delivery Drug delivery Dendrimers Carbon nanotubes Quantum dots Therapeutics In vitro diagnostics Imaging In vitro diagnostics, imaging In vitro diagnostics Imaging, therapeutics Therapeutics Gold nanoparticles In vitro diagnostics In vitro diagnostics, imaging Indication Cancer

in vivo studies with multifunctional nanoparticles, which serve to highlight the promising future of these novel nanomaterials. An excellent example of the suitability of multifunctional nanoparticles for simultaneous in vivo imaging and delivery of therapeutic products for cancer treatment is the work by Yang and coworkers. These authors developed a multifunctional nanosystem combining magnetic nanocrystals (for MRI), with therapeutic antibodies (for specic delivery) and the chemotherapeutic drug doxorubicin (for synergistic therapy) [16]. In a similar approach, Farokhzad and coworkers developed biocompatible nanoparticles for the specic delivery of docetaxel to localised tumours. Targeted delivery was achieved on this occasion using aptamers that recognised a prostatespecic membrane antigen [17]. Multifunctional nanoparticles have also been used for in vivo imaging and siRNA delivery and silencing in tumours. In a work by Medarova and coworkers, synthetic siRNA, which targeted a gene of interest, was conjugated to magnetic nanoparticles (for MRI) conjugated to the uorescent probe Cy5.5 (for optical imaging). Moreover, nanoparticle translocation to the cytosol was facilitated and, in turn, RNAi initiated by coupling a membrane-translocation module to the nanoparticle [13]. This study exemplies that multifunctional

Vaccines: inuenza, hepatitis A Fungal infection HIV, cancer, ophthalmology, inammation Respiratory function monitoring Atomic-force microscopy probe tip Labelling reagents: Western blotting, ow cytometry, biodetection Cancer Liver tumours, cardiovascular disease, anaemia Cancer HIV Labelling reagents (PCR, RNA, Western blotting), angiography and kidney

Magnetic nanoparticles

Company Liplasome Pharma (Lyngby, Denmark), Schering-Plough Corp (Kenilworth, NJ, USA) Berna Biotech AG (Basel, Switzerland) Enzon (Bridgewater, NJ,USA), Gilead Science (Foster City, CA, USA) Starpharma (Melbourne, Australia) Nanomix (Emerryville, CA, USA) Carbon Nanoprobes Inc (Seattle, WA, USA) Evident Technologies (New York, NY, USA), Quantum Dot Corp. (Hayward, CA, USA), Nanoco Technologies Ltd (Manchester, UK) Immunicon (Huntingdon Valley, PA, USA) Advanced Magnetics (Cambridge, MA, USA) Nanospectra Biosciences Inc (Houston, TX, USA) Amersham/GE (Little Chalfont, UK) Nanoprobes Inc. (Yaphank, NY,USA)

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Figure 2. Multifunctional nanoparticles for drug delivery. Multifunctional nanocarriers can combine a specific targeting agent (usually an antibody or peptide) with nanoparticles for imaging (such as quantum dots or magnetic nanoparticles), a cell-penetrating agent (e.g. the polyArg peptide TAT), a stimulus-sensitive element for drug release (Table 2), a stabilising polymer to ensure biocompatibility (polyethylene glycol most frequently) and the therapeutic compound. Development of novel strategies for controlled released of drugs will provide nanoparticles with the capability to deliver two or more therapeutic agents.

nanoparticles also constitute a promising non-viral genedelivery system. After two decades of intensive research and numerous clinical trials, a successful outcome for gene therapies to treat human diseases remains elusive. Viral vectors to express genes for therapeutic purposes still show cytotoxicity and immunogenicity problems, whereas the in vivo transfection efciency of non-viral systems is low and transient. Transfection efciency is restricted owing to

the major barriers present in the body. For example, the bloodbrain and bloodretinal barriers limit drug transport to the brain and the eye, respectively. Because viral vectors do not cross these barriers and, as a result, do not reach the eye and the brain, and non-viral vectors lack sufcient efciency, one of the current challenges in gene therapy is the design of more sophisticated non-viral systems that are able to circumvent these barriers. Nanoparticles have already demonstrated their potential to pass

Table 2. A summary of strategies for constructing multifunctional nanoparticlesa

Properties Stability, biocompatibility Benets Maintain drug levels in the blood, therefore improving specicity Function Polyethylene glycol Modied acrylic acid polymers Phospholipid micelles Polypeptides Antibodies Peptides Aptamers Carbohydrate Folic acid Peptides Trans-activating transcriptional activator (TAT) Ligands Transferrin Positively charged moieties Cationic lipids Cationic polymers Quantum dots Magnetic nanoparticles pH-labile Photosensitive Thermosensitive Magnetic sensitive Photothermal sensitive Redox sensitive Refs [22] [50] [51] [52] [53] [41] [17] [54] [55] [12] [56] [57] [58] [14] [13] [12] [59] [60] [61] [10] [62]

Specic targeting

Increase efciency, reduce toxicity

Intracellular penetration

Modify nanoparticle pharmacokinetics and biodistribution, increasing drug efcacy

Imaging Stimulus-sensitive drug release

Report real-time nanoparticle biodistribution Control bioavailability, reduces toxicity

a To build multifunctional nanoparticles that include all the above listed properties still represents a challenge but it is a goal that is becoming increasingly realistic. Bifunctional nanoparticles are the simplest approach and there are already numerous examples of them in the literature [63]. One of the present limitations for the combination of multiple functions on a single particle is the surface chemistry required. Non-covalent strategies, such as electrostatic and biospecic interactions or hydrophobic adsorptions, do not enable control over the composition, size and multifunctionality of the nanoparticulate system. Attachment through chemical bond guaranties more control over the different functionalities. Nevertheless, new chemical strategies will afford multifunctional nanoparticles with improved reproducibility and better-controlled and coordinated properties.

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through biological barriers. Anticancer drugs, such as loperamide and doxorubicin, when bound to nanomaterials, cross the intact bloodbrain barrier and could then be released at therapeutic concentrations in the brain [18,19]. Recently, Novartis commercialised Visudyne, a liposomal preparation of the drug verteporn for the treatment of age-related macular degeneration. Administration of this drug is intravenous and there is some evidence that Visudyne crosses the bloodretinal barrier [4], although an alternative explanation involving leakage to the retina is yet to be eliminated. In view of these precedents, multifunctional nanoparticles that combine gene delivery with the ability to cross tissue and membrane barriers could constitute ideal non-viral vectors for gene therapy. One of the rst indications of the potential of multifunctional platforms for gene delivery was the work performed by Zhang and coworkers. With the aim of transferring genes to the retina, liposomes administered systemically into rhesus monkeys were targeted across the bloodretinal barrier together with a monoclonal antibody against the human insulin receptor, resulting in specic gene expression in the eye by using the opsin promoter [20]. Multifunctional vehicles based on nanorods, dendritic polymers and quantum dots constitute other examples of nanosystems designed for gene delivery [9,14,21]. Nevertheless, despite the promising future that multifunctional nanoparticles show as drug and gene delivery systems, the eld is still in a preliminary state, with only some preliminary in vivo studies carried out. Moreover, issues, such as lack of specicity, metabolic stability, bioavailability and nanoparticle toxicology, remain to be overcome. Current limitations to the efcacy of nanoparticles Extensive in vivo application of nanoparticles will require a more exhaustive exploration of the physicochemical and physiological processes occurring in biological environments. For example, it is not yet possible to predict nanoparticle biodistribution according to their physicochemical properties. Moreover, nanoparticle biodistribution can be affected by undesirable interactions with biological systems and molecules, such as proteins, by a process known as opsonisation, or by the mononuclear phagocyte system, which consists of monocytes and macrophages that take up and metabolise foreign molecules and particulates. In this context, polymer polyethylene glycol (PEG) coatings minimise unwanted recognition and increase nanoparticle circulation half life [22,23]. Once nanoparticles reach their target site, and despite their small size, they do not enter into biological systems, such as cells or organelles, easily. Therefore, it is essential to design strategies that, rst, enable nanoparticles to recognise the unique surface signatures of their target cells and, second, enable nanoparticles to enter the cells and then access specic organelles. There are already several examples in the literature that illustrate different strategies for intracellular uptake and the efcient delivery of nanoparticles into target organelles, such as endoand lyso-somes, mitochondria and the nucleus [24]. In this context, recent innovative imaging studies aimed to elucidate the processes involved in the transport and distribution of nanoparticles in the body and their detailed

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interactions with cellular components once the nanoparticles have entered the cell. Tada and coworkers analysed the movement of quantum dots functionalised with tumour-targeting antibodies injected into mice from capillary vessels to the perinuclear region of cancer cells [25]. Inside the cell, nanoparticles can remain structurally unaltered, can be modied or can be metabolised. Studies aimed at investigating how nanoparticles are processed metabolically are still lacking. Ideally, once they have exerted their function, it would be desirable if nanoparticles could be secreted or degraded without any toxic side effects. Approaches to this end are to coat nanoparticles with biodegradable polymeric materials that are already in use in biomedicine [26] or to design novel nanoparticulate systems with biodegradable polymers. When evaluating the potential of nanoparticles for in vivo applications, toxicity is a crucial factor to consider. Surprisingly, despite the great potential nanoparticles show for medical applications, our knowledge of the health effects of nanoparticle exposure is still limited. Whether nanoparticles are a hazard for humans remains unclear. Furthermore, to date, no studies have been performed that address the possible toxic effects of multifunctional nanoparticles. Nanotoxicology Studies have revealed that the same properties that make nanoparticles so unique that is, primarily, their small size, large surface area, chemical composition, solubility and geometry could also be responsible for their potential hazard to human health. For example, size and concentration affect the cytotoxicity of quantum dots. Several studies have reported that there is an inverse relationship between quantum dot size and concentration and their adverse effects, with smaller sizes and higher concentrations being more cytotoxic [27,28]. In this context, Lovric and coworkers found that quantum dot-induced cell death was more pronounced in small green-emitting quantum dots than large red-emitting quantum dots [29]. Regarding nanoparticle dose-dependent cytotoxicity, higher concentrations (even at levels used for studies) of nano-60 fullerene, gold and iron oxide nanoparticles resulted in higher levels of cell death [3032]. Geometry also inuences nanoparticle toxicity. Carbon nanomaterials with different geometry, such as single-walled or multi-walled nanotubes and nano-60 fullerenes, exhibit differential cytotoxicities in vitro [33], with single-walled nanotubes being the most toxic and nano-60 fullerenes the least toxic. Some nanoparticles contain toxic elements, such as cadmium and selenium in quantum dots, that, without a protective coating preventing premature breakdown, could lead to unpredictable collateral damage [34]. Unfortunately, surface modications also seem to have a role in cytotoxic effects. Studies with quantum dots demonstrate that their toxicity could be attributable to the surface molecules surrounding the quantum dot rather than the metallic core itself [35]. Similarly, gold nanoparticle toxicity varies depending on the nature of the surface coating applied to the nanoparticle [36]. Therefore, within the same class of nanoparticle, properties change in line with size, geometry, concentration and surface compo429

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sition. However, not only do nanoparticle characteristics vary, the experimental conditions also appear to have an effect on the results. Thus, although some authors indicate that quantum dots can induce apoptosis through activation of members of the caspase family of proteases [37], others show that quantum-dot-mediated cell death occurs in a caspase-independent manner [38]. In summary, the effects of exposing cells to nanoscale materials might differ considerably from those elicited by contact with bulk (i.e. nonnanoscale) materials. Only a few studies have evaluated the risks associated with exposure to nanoparticles and the results have been inconclusive. For example, it has been suggested that nanoparticles affect biological behaviour at the cellular, subcellular, protein and gene levels. Lovric and coworkers showed that naked CdTe quantum dots (i.e. without a ZnS coating) induced cell death through damage to the plasma membrane, mitochondrion and nucleus [38]. In accordance with this, Sayes et al. found that nano-C60 particles disrupted the integrity of the plasma membrane, although these authors did not observe associated changes in mitochondrial activity or DNA content [31]. By contrast, other authors have indicated that nanoparticles are biologically inert materials and therefore suitable for in vivo applications. In support of this hypothesis, several studies have demonstrated that nanoparticles injected into live animals produce no detectable toxicity [3941]. Examples of these contradictory results are shown in Table 3, which highlight the inuence of the experimental conditions and the physicochemical characteristics of the nanoparticles in the results. Amidst this controversy, nanotoxicology has emerged as the discipline that aims to investigate the safety of nanotechnologies. Specically, nanotoxicology aims to assess the risks associated with exposure to nanomaterials, to explore the routes of entry of nanoparticles into the organism and to study the molecular mechanisms of nanoparticle toxicity [42,43]. Today, many questions await resolution in the nanotoxicology eld. Studies undertaken to identify the molecular basis for nanoparticle-induced toxicity have shown that reactive oxygen species (ROS)

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have a key role [34]. Nevertheless, the precise mechanisms responsible for generating the oxidative damage and the identity of the molecules involved are still unclear. Figure 3 illustrates some of the possible ROS-mediated mechanisms that are associated with nanoparticle toxicity in the cell. Currently, we know that the small size of nanoparticles implies a high surface area. However, this does not necessarily mean that they should possess more reactivity in the cellular environment or have increased potential for toxicity. In fact, nanoparticle-associated toxicity should depend more on whether accumulation in specic organs occurs because such a deposition of nanoparticles could provoke intracellular changes that might affect cell integrity and hence organ function. In this context, animal studies have shown that nanoparticles target the blood stream and the central nervous system and can induce inammatory reactions in the lungs [44]. Nanoparticle accumulation has also been observed in the liver, spleen, lymph node and bone marrow [41,45,46]. Unfortunately, at present, the overall behaviour of a nanoparticle regarding nonspecic adsorptions, organ distribution and residence time once it enters the body is still unpredictable. To attempt to control and minimise the undesirable effects of nanoparticles, chemical approaches, such as surface treatment, the addition of functional groups and composite formation, are applied to nanoparticle design. PEG coatings are used to minimise unwanted recognition by endogenous proteins and increase circulation half life [22]. PEG has also been used to avoid nanoparticle uptake by macrophages of the mononuclear phagocytic system, another important barrier to controlled drug delivery [23]. Similarly, by means of adjusting the composition of the coating material, nanoparticle clearance from the body has been facilitated [26]. Recent years have seen substantial efforts towards understanding nanoparticle behaviour and providing a basis for assessing their toxic responses. Nevertheless, further pharmacological studies, such as blood circulation and clearance half life, organ biodistribution and accumulation, are needed before the promise of nanoparticle technologies can translate into clinical applications. Crucially, there is still a potential gulf between the

Table 3. Examples of cytotoxicity studies on nanoparticles

Nanoparticle Pegylated dendrimers Cationic dendrimers Carbon nanotubes Single-wall carbon nanotubes CdSe quantum dots CdTe quantum dots Cell or animal Male CH3 mice Male CH3 mice Male Dunkin Hartley guinea pigs Male Crl:CD(SD)IGS BR rats B16F10 melanoma cells C57BL/6 mice Human hepatoma HepG2 cells Sprague-Dawley rats COS-7 cells Rat pheochromocytoma cell line PC12M Nude mouse BALBc mice Toxicity No toxicity was observed at doses up to 2.56 g/kg i.p. injection 100% mortality at 160 mg/kg i.p. injection Do not induce any abnormalities of pulmonary function or measurable inammation High-dose induced mortality within 24 h post-instillation Pulmonary inammation (granuloma formation) No detectable toxicity No detectable toxicity Cytotoxicity in a concentration- and size-dependent manner Few signs of functional toxicity Transient reduction in motor activity Excellent biocompatibility No toxicity observed Dose-dependent diminishing viability Reduced ability to respond to nerve growth factors No toxicity or physiological complications Dose-dependent toxic effect Refs [64] [65] [66] [67] [39] [39] [27] [27] [68] [30] [69] [70]

Fe3O4 nanoparticles Fe2O3 nanoparticles Pegylated gold nanoparticles Gold nanoshells

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Figure 3. Possible reactive oxygen species (ROS)-mediated mechanisms associated with nanoparticle toxicity. Nanoparticles are able to target mitochondria directly, which can lead to mitochondrial disruption and, in turn, to ROS production. Oxidative stress owing to excess ROS generation induces over-expression of antioxidant enzymes in an attempt to control ROS levels. At high levels of oxidative stress, antioxidant defences are overwhelmed, which leads to inflammatory and cytotoxic responses. Oxidative stress might induce collateral damage, such as lipid peroxidation, protein denaturation, nuclear and DNA damage and immune reactivity. More detailed information regarding nanoparticle toxic effects in biological systems can be found elsewhere [34,42].

animal-based toxicity studies undertaken and their eventual translation into protocols that will be safe for humans. Concluding remarks Although nanomedicine is a relatively new area of biotechnology, the possibilities for new therapies to treat illness and disease seem endless. Nanoparticles are already appearing in commerce as novel tools for molecular imaging, diagnosis and drug delivery formulations [4]. Of note, some nanoparticles have intrinsic therapeutic properties themselves. For example, owing to the multivalent display of ligands on their surface, dendrimers have the ability to block the binding between cells, viruses, bacteria and proteins. [47]. Cerium oxide and platinum nanoparticles show antioxidant properties that herald a promising future for the treatment of oxidative-stress-related conditions, such as neurodegenerative disorders, including Alzheimers and Parkinsons diseases [48,49]. In this same context, the emerging development of novel multifunctional nanosystems, in which the combination of different functions in a single nanoparticle affords biocompatibility, biostability and biodistribution, provides new potential for therapeutic applications that, undoubtedly, will revolutionise the medical landscape. Nevertheless, one has to bear in mind that the biomedical applications of nanoparticles require their direct ingestion or injection into the body. Therefore, a better understanding of the effects that these new materials have on human health is imperative before clinical use can ensue. Nanoparticles must be evaluated on a particle-by-particle basis and a rational characterisation strategy must include absorption, distribution, metabolism and excretion (ADME) tests and physicochem-

ical and toxicological characterisation, involving both in vitro tests and in vivo animal studies. Actions have already been taken to develop research strategies to evaluate the specic risks associated with exposure to each particular nanoparticle. The Seventh Research Framework Programme of the European Union includes among its objectives the study of the impact of engineered nanoparticles on health and the environment and the validation, adaptation and/or development of risk assessment methodology for engineered nanoparticles (FP7 Cooperation Work Programme 2008). Recently, the US FDA has launched comprehensive recommendations for improving the scientic knowledge of nanotechnology (July 2007) and the American National Nanotechnology Initiative (NNI), comprising 26 federal departments and agencies, will invest over US$96 million in environmental, health and safety R&D to address the potential hazards linked to nanotechnology. Despite all these efforts, our knowledge of the health effects on exposure is still limited. Therefore, the development of nanoparticles must proceed in parallel with the assessment of the toxicological effects of these new materials. In this context, one has to bear in mind that each nanoparticle property (i.e. small size, large surface area, chemical composition, solubility and geometry) determines the biological response. Consequently, it is imperative to characterise the physicochemical properties of the nanoparticle under consideration to correlate them with the biological results.
Acknowledgements
We acknowledge the support of the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain.
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