Multifunctional nanoparticles –properties and prospects for their usein human medicineNuria Sanvicens and M. Pilar Marco

Applied Molecular Receptors Group (AMRg), CSIC Networking Research Center of Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN), Jorge Girona, 18–26, 08034 Barcelona, Spain

Review

A major aim of medicine has long been the early andaccurate diagnosis of clinical conditions, providing anefficient treatment without secondary effects. With theemergence of nanotechnology, the achievement of thisgoal seems closer than ever. To this end, the develop-ment of novel materials and devices operating at thenanoscale range, such as nanoparticles, provides newand powerful tools for imaging, diagnosis and therapy.This review focuses on the significant improvements inperformance that nanoparticles offer compared withexisting technologies relevant to medicine. Specifically,we address the design of multifunctional nanoparticlesas an alternative system for drug and gene delivery,which has great potential for therapy in areas, such ascancer and neuropathologies. Moreover, we discuss thecontroversy generated by the possible toxic healtheffects of nanoparticles.

IntroductionNanotechnology is considered by many as the next ‘bigrevolution’. This technological leap in controllingmaterialsat the nanoscale has, in the past decade, driven develop-ments enabling the use of nanodevices, such as nanopar-ticles, that have found applications in fields ranging fromelectronics and communications, through to optics, chem-istry, energy and of course biology. Nanomedicine, theapplication of nanotechnology to healthcare, holds greatpromise for revolutionising medical treatments and thera-pies in areas, such as imaging, faster diagnosis, drugdelivery and tissue regeneration, as well as the develop-ment of new medical products. Indeed, materials anddevices of nanometric dimensions (1–100 nm) are alreadyapproved for clinical use and numerous products are beingevaluated in clinical trials [1]. However, as discussed later,there are toxicological concerns and ethical issues thataccompany nanomedicine that might cast a shadow overthe promising future of this emerging field.

This article presents an overview of the current appli-cations of nanoparticles in medicine. In particular, wefocus on the development of novel multifunctional nano-particles and illustrate their potential application in drugand gene delivery for cancer and neuropathologicaltherapy. The current limitations of nanoparticle-basedapproaches are discussed, with special emphasis given

Corresponding author: Sanvicens, N. (nsdqob@iiqab.csic.es).

0167-7799/$ – see front matter � 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.20

to the lack of knowledge of the toxicological risks associatedwith the exposure to nanoparticles. Finally, we summarisethe future challenges that lie ahead.

Multifunctional nanoparticlesNanoparticles are constructs that possess unique physicaland chemical properties associated with their being of 1–100 nm in size. The general characteristics and compo-sition of nanoparticles are described in Box 1 and areillustrated in Figure 1. Nanometre-sized particles are inthe same range of dimension as antibodies, membranereceptors, nucleic acids and proteins, among other biomo-lecules. These biomimetic features, together with theirhigh surface:volume ratio and the possibility of modulatingtheir properties, make nanoparticles powerful tools forimaging, diagnosis and therapy [2,3]. Thus, nanoparticlesoffer significant improvements in performance comparedwith existing technologies (Box 2). As a result, commercia-lisation of nanoparticle-based therapeutics is gainingmomentum, with an increasing number of available pro-ducts on the market [4]. Table 1 gives examples of com-mercial nanoparticle-based products. Other issues relatedto nanoparticle technology, such as biomolecule–nanopar-ticle hybrid systems and their use towards medical diag-nostics and therapeutics, have been reviewed elsewhere[5–7].

Despite the benefits that nanoparticles have rendered tomedicine (Box 2), some applications remain challenging;for instance, in vivo real-timemonitoring of cellular events,specific targeting to the action site or efficient drug deliveryinside the target cell. In this context, the design of multi-functional nanoparticles could significantly improvealready existing nanoparticle characteristics and help tosurmount these challenges. Whereas monofunctionalnanoparticles provide a single function – a liposome cantransport drugs but does not have the inherent property todistinguish between healthy and unhealthy cells or tissues– multifunctional nanoparticles combine different func-tionalities in a single stable construct. For example, a coreparticle could be linked to a specific targeting function thatrecognises the unique surface signatures of their targetcells. Simultaneously, the same particle can be modifiedwith an imaging agent to monitor the drug transportprocess, a function to evaluate the therapeutic efficacy ofa drug, a specific cellular penetration moiety and a thera-peutic agent [8] (Figure 2). Table 2 summarises some of the

08.04.005 425

Box 1. Organic and inorganic nanoparticles

Nanoparticles are materials of two or more dimensions, with a size

in the range of 1–100 nm. Nanoparticles show unique size-

dependent 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 ele-

ments (inorganic nanoparticles). Liposomes, dendrimers, carbon

nanomaterials and polymeric micelles are examples of organic

nanoparticles.

Liposomes

Liposomes are phospholipid vesicles (50–100 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 nanocrys-

tals (2–10 nm). The central core of quantum dots consists of

combinations of elements from groups II–VI of the periodic system

(CdSe, CdTe, CdS, PbSe, ZnS and ZnSe) or III–V (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 10–20 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].

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 biolumi-

nescence system (i.e. luciferin/luciferase) applications are limited

because they cannot be optimised in multicolour assays. Nanopar-

ticles 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 concentra-

tions 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 technol-

ogies 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 [78–80].

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].

Review Trends in Biotechnology Vol.26 No.8

426

common approaches that are in use currently to developmultifunctional nanocarriers.

Design of intelligent multifunctional nanoparticles

The design of nanoparticles that combine several proper-ties is an elaborate process that requires different steps [9],such as the depositing of metal layers onto a supportingnanoparticle core [10,11], modifying the biocompatible

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.

Review Trends in Biotechnology Vol.26 No.8

polymer used to stabilise the nanoparticle [12] and the useof different linkers [13,14]. In an attempt to overcome this,recently, Gao et al. reported a simple ‘one-pot’ syntheticprocedure to prepare fluorescent magnetic nanocrystals[15]. Furthermore, these different properties of multifunc-tional nanoparticles have to be coordinated so that theyoperate in an orchestrated way and indeed provide thedesired functionalities. An elegant example for this is thework by Sawant and coworkers. In this study, the nano-particles had specific targeting and cell-internalisationfunctions. Under normal conditions (i.e. when not boundspecifically to the target), the specific target function isexposed, providing a long circulation life and specific deliv-ery, whereas the cell-internalisation function remains hid-den. In this manner, the cell-internalisation function doesnot interfere with the nanoparticle circulation. Whenspecific binding was achieved – and owing to the low pHof the pathological environment – the penetration functionwas exposed, facilitating nanoparticle penetration withinthe cell [12].

Multifunctional nanoparticles for drug and gene delivery

Multifunctional delivery nanosystems are just emergingbut there are already in the literature several examples of

Table 1. Examples of commercial applications of nanoparticles

Nanoparticle component Application Indication

Liposomes Drug delivery Cancer

Drug delivery Vaccines: influenza, h

Drug delivery Fungal infection

Dendrimers Therapeutics HIV, cancer, ophthalm

Carbon nanotubes In vitro diagnostics Respiratory function

Imaging Atomic-force microsc

Quantum dots In vitro diagnostics,

imaging

Labelling reagents: W

cytometry, biodetecti

Magnetic

nanoparticles

In vitro diagnostics Cancer

Imaging, therapeutics Liver tumours, cardio

anaemia

Therapeutics Cancer

Gold nanoparticles In vitro diagnostics HIV

In vitro diagnostics,

imaging

Labelling reagents (P

blotting), angiograph

in vivo studies with multifunctional nanoparticles, whichserve to highlight the promising future of these novelnanomaterials. An excellent example of the suitability ofmultifunctional nanoparticles for simultaneous in vivoimaging and delivery of therapeutic products for cancertreatment is the work by Yang and coworkers. Theseauthors developed a multifunctional nanosystem combin-ing magnetic nanocrystals (for MRI), with therapeuticantibodies (for specific delivery) and the chemotherapeuticdrug doxorubicin (for synergistic therapy) [16]. In a similarapproach, Farokhzad and coworkers developed biocompa-tible nanoparticles for the specific delivery of docetaxel tolocalised tumours. Targeted delivery was achieved onthis occasion using aptamers that recognised a prostate-specific membrane antigen [17]. Multifunctional nanopar-ticles have also been used for in vivo imaging and siRNAdelivery and silencing in tumours. In a work by Medarovaand coworkers, synthetic siRNA, which targeted a geneof interest, was conjugated to magnetic nanoparticles(for MRI) conjugated to the fluorescent probe Cy5.5 (foroptical imaging). Moreover, nanoparticle translocation tothe cytosol was facilitated and, in turn, RNAi initiated bycoupling a membrane-translocation module to the nano-particle [13]. This study exemplifies that multifunctional

Company

Liplasome Pharma (Lyngby, Denmark),

Schering-Plough Corp (Kenilworth, NJ, USA)

epatitis A Berna Biotech AG (Basel, Switzerland)

Enzon (Bridgewater, NJ,USA), Gilead Science

(Foster City, CA, USA)

ology, inflammation Starpharma (Melbourne, Australia)

monitoring Nanomix (Emerryville, CA, USA)

opy probe tip Carbon Nanoprobes Inc (Seattle, WA, USA)

estern blotting, flow

on

Evident Technologies (New York, NY, USA),

Quantum Dot Corp. (Hayward, CA, USA),

Nanoco Technologies Ltd (Manchester, UK)

Immunicon (Huntingdon Valley, PA, USA)

vascular disease, Advanced Magnetics (Cambridge, MA, USA)

Nanospectra Biosciences Inc (Houston, TX,

USA)

Amersham/GE (Little Chalfont, UK)

CR, RNA, Western

y and kidney

Nanoprobes Inc. (Yaphank, NY,USA)

427

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.

Review Trends in Biotechnology Vol.26 No.8

nanoparticles also constitute a promising non-viral gene-delivery system.

After two decades of intensive research and numerousclinical trials, a successful outcome for gene therapies totreat human diseases remains elusive. Viral vectors toexpress genes for therapeutic purposes still show cytotox-icity and immunogenicity problems, whereas the in vivotransfection efficiency of non-viral systems is low andtransient. Transfection efficiency is restricted owing to

Table 2. A summary of strategies for constructing multifunctional

Properties Benefits

Stability, biocompatibility Maintain drug levels in the blood, th

improving specificity

Specific targeting Increase efficiency, reduce toxicity

Intracellular penetration Modify nanoparticle pharmacokinetic

biodistribution, increasing drug effica

Imaging Report real-time nanoparticle biodist

Stimulus-sensitive drug release Control bioavailability, reduces toxici

aTo build multifunctional nanoparticles that include all the above listed properties st

Bifunctional nanoparticles are the simplest approach and there are already numerou

combination of multiple functions on a single particle is the surface chemistry requi

hydrophobic adsorptions, do not enable control over the composition, size and mult

guaranties more control over the different functionalities. Nevertheless, new chemical st

better-controlled and coordinated properties.

428

the major barriers present in the body. For example, theblood–brain and blood–retinal barriers limit drug trans-port to the brain and the eye, respectively. Because viralvectors do not cross these barriers and, as a result, do notreach the eye and the brain, and non-viral vectors lacksufficient efficiency, one of the current challenges in genetherapy is the design of more sophisticated non-viral sys-tems that are able to circumvent these barriers. Nanopar-ticles have already demonstrated their potential to pass

nanoparticlesa

Function Refs

erefore Polyethylene glycol [22]

Modified acrylic acid polymers [50]

Phospholipid micelles [51]

Polypeptides [52]

Antibodies [53]

Peptides [41]

Aptamers [17]

Carbohydrate [54]

Folic acid [55]

s and

cy

Peptides

Trans-activating transcriptional activator (TAT) [12]

Ligands

Transferrin [56]

Positively charged moieties

Cationic lipids [57]

Cationic polymers [58]

ribution Quantum dots [14]

Magnetic nanoparticles [13]

ty pH-labile [12]

Photosensitive [59]

Thermosensitive [60]

Magnetic sensitive [61]

Photothermal sensitive [10]

Redox sensitive [62]

ill represents a challenge but it is a goal that is becoming increasingly realistic.

s examples of them in the literature [63]. One of the present limitations for the

red. Non-covalent strategies, such as electrostatic and biospecific interactions or

ifunctionality of the nanoparticulate system. Attachment through chemical bond

rategies will afford multifunctional nanoparticles with improved reproducibility and

Review Trends in Biotechnology Vol.26 No.8

through biological barriers. Anticancer drugs, such asloperamide and doxorubicin, when bound to nanomater-ials, cross the intact blood–brain barrier and could then bereleased at therapeutic concentrations in the brain [18,19].Recently, Novartis commercialised Visudyne, a liposomalpreparation of the drug verteporfin for the treatment ofage-related macular degeneration. Administration of thisdrug is intravenous and there is some evidence that Visu-dyne crosses the blood–retinal barrier [4], although analternative explanation involving leakage to the retinais yet to be eliminated. In view of these precedents, multi-functional nanoparticles that combine gene delivery withthe ability to cross tissue and membrane barriers couldconstitute ideal non-viral vectors for gene therapy. One ofthe first indications of the potential of multifunctionalplatforms for gene delivery was the work performed byZhang and coworkers. With the aim of transferring genesto the retina, liposomes administered systemically intorhesus monkeys were targeted across the blood–retinalbarrier together with a monoclonal antibody against thehuman insulin receptor, resulting in specific gene expres-sion in the eye by using the opsin promoter [20]. Multi-functional vehicles based on nanorods, dendritic polymersand quantum dots constitute other examples of nanosys-tems designed for gene delivery [9,14,21]. Nevertheless,despite the promising future that multifunctional nano-particles show as drug and gene delivery systems, the fieldis still in a preliminary state, with only some preliminaryin vivo studies carried out. Moreover, issues, such as lack ofspecificity, metabolic stability, bioavailability and nano-particle toxicology, remain to be overcome.

Current limitations to the efficacy of nanoparticlesExtensive in vivo application of nanoparticles will requirea more exhaustive exploration of the physicochemical andphysiological processes occurring in biological environ-ments. For example, it is not yet possible to predict nano-particle biodistribution according to their physicochemicalproperties. Moreover, nanoparticle biodistribution can beaffected by undesirable interactions with biological sys-tems and molecules, such as proteins, by a process knownas opsonisation, or by the mononuclear phagocyte system,which consists of monocytes andmacrophages that take upand metabolise foreign molecules and particulates. In thiscontext, polymer polyethylene glycol (PEG) coatings mini-mise unwanted recognition and increase nanoparticle cir-culation half life [22,23].

Once nanoparticles reach their target site, and despitetheir small size, they do not enter into biological systems,such as cells or organelles, easily. Therefore, it is essentialto design strategies that, first, enable nanoparticles torecognise the unique surface signatures of their targetcells and, second, enable nanoparticles to enter the cellsand then access specific organelles. There are alreadyseveral examples in the literature that illustrate differentstrategies for intracellular uptake and the efficient deliv-ery of nanoparticles into target organelles, such as endo-and lyso-somes, mitochondria and the nucleus [24]. In thiscontext, recent innovative imaging studies aimed to eluci-date the processes involved in the transport and distri-bution of nanoparticles in the body and their detailed

interactions with cellular components once the nanoparti-cles have entered the cell. Tada and coworkers analysedthe movement of quantum dots functionalised withtumour-targeting antibodies injected into mice from capil-lary vessels to the perinuclear region of cancer cells [25].

Inside the cell, nanoparticles can remain structurallyunaltered, can be modified or can be metabolised. Studiesaimed at investigating how nanoparticles are processedmetabolically are still lacking. Ideally, once they haveexerted their function, it would be desirable if nanoparti-cles could be secreted or degraded without any toxic sideeffects. Approaches to this end are to coat nanoparticleswith biodegradable polymeric materials that are already inuse in biomedicine [26] or to design novel nanoparticulatesystems with biodegradable polymers.

When evaluating the potential of nanoparticles for invivo applications, toxicity is a crucial factor to consider.Surprisingly, despite the great potential nanoparticlesshow for medical applications, our knowledge of the healtheffects of nanoparticle exposure is still limited. Whethernanoparticles are a hazard for humans remains unclear.Furthermore, to date, no studies have been performed thataddress the possible toxic effects of multifunctional nano-particles.

Nanotoxicology

Studies have revealed that the same properties that makenanoparticles so unique – that is, primarily, their smallsize, large surface area, chemical composition, solubilityand geometry – could also be responsible for their potentialhazard to human health. For example, size and concen-tration affect the cytotoxicity of quantum dots. Severalstudies have reported that there is an inverse relationshipbetween quantum dot size and concentration and theiradverse effects, with smaller sizes and higher concen-trations being more cytotoxic [27,28]. In this context, Lov-ric and coworkers found that quantum dot-induced celldeath was more pronounced in small green-emitting quan-tum dots than large red-emitting quantum dots [29].Regarding nanoparticle dose-dependent cytotoxicity,higher concentrations (even at levels used for studies) ofnano-60 fullerene, gold and iron oxide nanoparticlesresulted in higher levels of cell death [30–32]. Geometryalso influences nanoparticle toxicity. Carbon nanomater-ials with different geometry, such as single-walled ormulti-walled nanotubes and nano-60 fullerenes, exhibitdifferential cytotoxicities in vitro [33], with single-wallednanotubes being the most toxic and nano-60 fullerenes theleast toxic. Some nanoparticles contain toxic elements,such as cadmium and selenium in quantum dots, that,without a protective coating preventing premature break-down, could lead to unpredictable collateral damage [34].Unfortunately, surface modifications also seem to have arole in cytotoxic effects. Studies with quantum dots demon-strate that their toxicity could be attributable to the sur-face molecules surrounding the quantum dot rather thanthe metallic core itself [35]. Similarly, gold nanoparticletoxicity varies depending on the nature of the surfacecoating applied to the nanoparticle [36]. Therefore, withinthe same class of nanoparticle, properties change in linewith size, geometry, concentration and surface compo-

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Review Trends in Biotechnology Vol.26 No.8

sition. However, not only do nanoparticle characteristicsvary, the experimental conditions also appear to have aneffect on the results. Thus, although some authors indicatethat quantumdots can induce apoptosis through activationof members of the caspase family of proteases [37], othersshow that quantum-dot-mediated cell death occurs in acaspase-independent manner [38]. In summary, the effectsof exposing cells to nanoscale materials might differ con-siderably from those elicited by contact with bulk (i.e. non-nanoscale) materials.

Only a few studies have evaluated the risks associatedwith exposure to nanoparticles and the results have beeninconclusive. For example, it has been suggested thatnanoparticles affect biological behaviour at the cellular,subcellular, protein and gene levels. Lovric and coworkersshowed that ‘naked’ CdTe quantum dots (i.e. without a ZnScoating) induced cell death through damage to the plasmamembrane, mitochondrion and nucleus [38]. In accordancewith this, Sayes et al. found that nano-C60 particles dis-rupted the integrity of the plasma membrane, althoughthese authors did not observe associated changes in mito-chondrial activity or DNA content [31]. By contrast, otherauthors have indicated that nanoparticles are biologicallyinert materials and therefore suitable for in vivo appli-cations. In support of this hypothesis, several studies havedemonstrated that nanoparticles injected into live animalsproduce no detectable toxicity [39–41]. Examples of thesecontradictory results are shown in Table 3, which highlightthe influence of the experimental conditions and the phy-sicochemical characteristics of the nanoparticles in theresults.

Amidst this controversy, nanotoxicology has emerged asthe discipline that aims to investigate the safety of nano-technologies. Specifically, nanotoxicology aims to assessthe risks associated with exposure to nanomaterials, toexplore the routes of entry of nanoparticles into the organ-ism and to study the molecular mechanisms of nanopar-ticle toxicity [42,43]. Today, many questions awaitresolution in the nanotoxicology field. Studies undertakento identify the molecular basis for nanoparticle-inducedtoxicity have shown that reactive oxygen species (ROS)

Table 3. Examples of cytotoxicity studies on nanoparticles

Nanoparticle Cell or animal Toxicity

Pegylated dendrimers Male CH3 mice No toxicity

Cationic dendrimers Male CH3 mice 100% mor

Carbon nanotubes Male Dunkin Hartley guinea pigs Do not ind

inflammat

Single-wall carbon

nanotubes

Male Crl:CD(SD)IGS BR rats High-dose

inflammat

CdSe quantum dots B16F10 melanoma cells No detecta

C57BL/6 mice No detecta

CdTe quantum dots Human hepatoma HepG2 cells Cytotoxici

Few signs

Sprague-Dawley rats Transient

Fe3O4 nanoparticles COS-7 cells Excellent b

No toxicity

Fe2O3 nanoparticles Rat pheochromocytoma cell line

PC12M

Dose-depe

Reduced a

Pegylated gold

nanoparticles

Nude mouse No toxicity

Gold nanoshells BALBc mice Dose-depe

430

have a key role [34]. Nevertheless, the precise mechanismsresponsible for generating the oxidative damage and theidentity of themolecules involved are still unclear. Figure 3illustrates some of the possible ROS-mediated mechan-isms that are associated with nanoparticle toxicity in thecell. Currently, we know that the small size of nanoparti-cles implies a high surface area. However, this does notnecessarily mean that they should possess more reactivityin the cellular environment or have increased potential fortoxicity. In fact, nanoparticle-associated toxicity shoulddepend more on whether accumulation in specific organsoccurs because such a deposition of nanoparticles couldprovoke intracellular changes that might affect cell integ-rity and hence organ function. In this context, animalstudies have shown that nanoparticles target the bloodstream and the central nervous system and can induceinflammatory reactions in the lungs [44]. Nanoparticleaccumulation has also been observed in the liver, spleen,lymph node and bonemarrow [41,45,46]. Unfortunately, atpresent, the overall behaviour of a nanoparticle regardingnonspecific adsorptions, organ distribution and residencetime once it enters the body is still unpredictable. Toattempt to control and minimise the undesirable effectsof nanoparticles, chemical approaches, such as surfacetreatment, the addition of functional groups and compositeformation, are applied to nanoparticle design. PEG coat-ings are used to minimise unwanted recognition byendogenous proteins and increase circulation half life[22]. PEG has also been used to avoid nanoparticle uptakeby macrophages of the mononuclear phagocytic system,another important barrier to controlled drug delivery [23].Similarly, by means of adjusting the composition of thecoating material, nanoparticle clearance from the body hasbeen facilitated [26]. Recent years have seen substantialefforts towards understanding nanoparticle behaviour andproviding a basis for assessing their toxic responses.Nevertheless, further pharmacological studies, such asblood circulation and clearance half life, organ biodistribu-tion and accumulation, are needed before the promise ofnanoparticle technologies can translate into clinical appli-cations. Crucially, there is still a potential gulf between the

Refs

was observed at doses up to 2.56 g/kg i.p. injection [64]

tality at 160 mg/kg i.p. injection [65]

uce any abnormalities of pulmonary function or measurable

ion

[66]

induced mortality within 24 h post-instillation Pulmonary

ion (granuloma formation)

[67]

ble toxicity [39]

ble toxicity [39]

ty in a concentration- and size-dependent manner [27]

of functional toxicity

reduction in motor activity [27]

iocompatibility [68]

observed

ndent diminishing viability [30]

bility to respond to nerve growth factors

or physiological complications [69]

ndent toxic effect [70]

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].

Review Trends in Biotechnology Vol.26 No.8

animal-based toxicity studies undertaken and their event-ual translation into protocols that will be safe for humans.

Concluding remarksAlthough nanomedicine is a relatively new area of biotech-nology, the possibilities for new therapies to treat illnessand disease seem endless. Nanoparticles are alreadyappearing in commerce as novel tools for molecular ima-ging, diagnosis and drug delivery formulations [4]. Of note,some nanoparticles have intrinsic therapeutic propertiesthemselves. For example, owing to the multivalent displayof ligands on their surface, dendrimers have the ability toblock the binding between cells, viruses, bacteria andproteins. [47]. Cerium oxide and platinum nanoparticlesshow antioxidant properties that herald a promising futurefor the treatment of oxidative-stress-related conditions,such as neurodegenerative disorders, including Alzhei-mer’s and Parkinson’s diseases [48,49]. In this same con-text, the emerging development of novel multifunctionalnanosystems, in which the combination of different func-tions in a single nanoparticle affords biocompatibility,biostability and biodistribution, provides new potentialfor therapeutic applications that, undoubtedly, will revo-lutionise the medical landscape. Nevertheless, one has tobear in mind that the biomedical applications of nanopar-ticles require their direct ingestion or injection into thebody. Therefore, a better understanding of the effects thatthese new materials have on human health is imperativebefore clinical use can ensue. Nanoparticles must be eval-uated on a particle-by-particle basis and a rational charac-terisation strategy must include absorption, distribution,metabolism and excretion (ADME) tests and physicochem-

ical and toxicological characterisation, involving both invitro tests and in vivo animal studies.

Actions have already been taken to develop researchstrategies to evaluate the specific risks associated withexposure to each particular nanoparticle. The SeventhResearch Framework Programme of the European Unionincludes among its objectives the study of the ‘impact ofengineered nanoparticles on health and the environment’and the ‘validation, adaptation and/or development of riskassessment methodology for engineered nanoparticles’(FP7 Cooperation Work Programme 2008). Recently, theUS FDA has launched comprehensive recommendationsfor improving the scientific knowledge of nanotechnology(July 2007) and the American National NanotechnologyInitiative (NNI), comprising 26 federal departments andagencies, will invest over US$96 million in environmental,health and safety R&D to address the potential hazardslinked to nanotechnology. Despite all these efforts, ourknowledge of the health effects on exposure is still limited.Therefore, the development of nanoparticles must proceedin parallel with the assessment of the toxicological effectsof these new materials. In this context, one has to bear inmind that each nanoparticle property (i.e. small size, largesurface area, chemical composition, solubility and geome-try) determines the biological response. Consequently, it isimperative to characterise the physicochemical propertiesof the nanoparticle under consideration to correlate themwith the biological results.

AcknowledgementsWe acknowledge the support of the Networking Research Center onBioengineering, Biomaterials and Nanomedicine (CIBER-BBN),Barcelona, Spain.

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