nanomaterials in combating cancer-therapeutic applications

CLINICALLY RELEVANT

Nanomedicine: Nanotechnology, Biology, and Medicine10 (2014) 19–34

Review

Nanomaterials in combating cancer: Therapeutic applicationsand developments

Samina Nazir, PhDa,⁎, Tajammul Hussain, PhDa, Attiya Ayub, MSa,b, Umer Rashid, PhDb,Alexander John MacRobert, PhDc

aNanosciences and Catalysis Division, National Centre for Physics, Shahdra Valley Road, Quaid-i-Azam University Campus, Islamabad, PakistanbDepartment of Chemistry, Hazara University, Mansehra, KPK, Pakistan

cUCL Division of Surgery and Interventional Science, and Institute of Biomedical Engineering, University College London, Charles Bell House, London, UK

Received 3 April 2013; accepted 4 July 2013

nanomedjournal.com

Abstract

The development of novel nanomaterials and their use in biomedicine has received much attention in recent years. Significant advanceshave been made in the synthesis of nanomaterials with controlled geometry, physicochemical properties, surface charge, and surface tailoringwith bioactive polymers. These successful efforts have resulted in improved biocompatibility and active targeting of tumour tissues,leading to the development of a diverse range of nanomaterials that can recognize cancers, deliver anticancer drugs and destroy tumours by avariety of therapeutic techniques. The focus of this review is to provide an overview of the nanomaterials that have been devised for thedetection and treatment of various types of cancer, as well as to underline the emerging possibilities of nanomaterials for applications inanticancer therapy.

From the Clinical Editor: In this comprehensive review, the current state-of-the art of nanomaterials for cancer diagnosis and treatment ispresented. Emerging possibilities and future concepts are discussed as well.© 2014 Elsevier Inc. All rights reserved.

Key words: Nanoparticles; Cancer; Polymeric nanoparticles; Qdots; AuNPs

Cancer is the uncontrolled growth of tissues and their rapidinvasion without proper development and differentiation. Sixbiological aptitudes are considered to be ‘hallmarks of cancer’:proliferative signalling, evasion of growth suppressors, resis-tance to cell death, replicative immortality, angiogenesis,invasion and metastasis.1 Cancer is a major cause ofdevastating health outcomes and economic constraints inhuman life. Globally, cancer rates are increasing at adistressing rate. As indicated in the Cancer Facts and Figures2013, it is estimated that 1,660,290 new cancer cases whereas580,350 cancer deaths are expected in the United States only.2

A huge amount of research has already been carried outin the field of cancer, resulting in a number of availablediagnostic and treatment options, including magnetic reso-nance imaging (MRI), computed tomography (CT), biosen-sing, radiotherapy, chemotherapy, gene therapy, andimmunotherapy. Radiotherapy, or radiation therapy, involvesthe treatment of cancer with ionizing radiation such as high-energy X-rays, gamma rays, or particle beam radiations that

Declaration of Interests: Authors declare no conflict of interest.⁎Corresponding author:E-mail address: [email protected] (S. Nazir).

1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2013.07.001

Please cite this article as: Nazir S, et al, Nanomaterials in combating can2014;10:19-34, http://dx.doi.org/10.1016/j.nano.2013.07.001

destroy the target tissue by damaging its DNA. Hyperther-mia, the use of heat, is also being studied for itseffectiveness in sensitizing tissue to radiation. Other recentresearch has focused on the use of radiolabeled antibodies todeliver doses of radiation directly to the cancer site (radio-immunotherapy). Radiotherapy may be used alone or incombination with chemotherapy or surgery. Although pain-less, it has some severe side effects such as permanent hairloss, fetal damage, skin problems, and secondary malignan-cies – the radiation itself is the source of mutations inhealthy genes and can cause cancer. Chemotherapy, bycontrast, involves a range of cytotoxic drugs such asvinblastine, doxorubicin, and taxol. The challenges here arebiodistribution (non-tumour selectivity), hypersensitivity, andacquisition of multidrug resistance (MDR). Furthermore,these drugs degrade to toxic moieties, resulting in nephro-toxicity and cardiotoxicity. To devise a successful treatmentregime, it is important to consider the major limitations ofseveral therapeutic agents such as poor solubility, rapiddeactivation, unfavourable pharmacokinetics, and limitedbiodistribution. A wide range of nanomaterials has beenintroduced in an effort to devise more comprehensive andversatile diagnostic and treatment solutions for malignancies.

cer: Therapeutic applications and developments. Nanomedicine: NBM

http://dx.doi.org/10.1016/j.nano.2013.07.001




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SEGUNDO

Second

Figure 1. Nanodrug carriers are designed to stay longer in the blood andaccumulate at tumour sites due to the enhanced permeability and retention(EPR) effect. The EPR effect in solid tumours is related to the anatomic andpathophysiological differences between tumour and normal tissues. Forexample, angiogenesis leads to high vascular density in tumours, large gapsexist between the endothelial cells in tumour blood vessels, and tumourtissues show selective extravasations and retention of macromolecular drugs.Developed nanodrugs deliver payloads based on various factors such as theincreased temperature and lower pH at the site of growing tumours.

20 S. Nazir et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 19–34

The range of diagnostic/therapeutic nanomaterial tools isextensive. Drug delivery and imaging with nanomaterialsbenefits from the anatomical changes coupled with thepathophysiological conditions at the diseased site.3 Nanomater-ials are usually accumulated at a higher concentration at thediseased site than conventional drugs.3 This enhanced drugtargeting leads to decreased systemic toxicity and successfuldelivery, even to hard-to-target disease sites such as brain.4

Inorganic nanomaterials include quantum dots (QDs),metallic nanostructures, metal oxides, superparamagnetic ironoxides (SPIONs), gold nanoparticles (AuNPs), and carbonnanotubes (CNTs); organic nanomaterials include liposomes,natural and synthetic polymers forming nanocapsules anddendrimers.5 Several of these are sufficiently small in size(10–100 nm) to penetrate the capillaries and be taken up by thetissues; others that are larger in size passively target and deliverat disease-specific anatomic sites.6 Many nanomaterials arebiocompatible, meaning that they do not alert the immunesystem, and biodegradable, breaking down to form harmlessmetabolic products. Moreover, a variety of materials such asQDs, AuNPs, and SPIONs exhibit unique optical, electrical, andmagnetic properties,7 which are helpful in imaging theintracellular localization and trafficking of these devices.Drugs can also be delivered at a specific site after beingattached, encapsulated, absorbed, entrapped, or dissolved in thenanomaterial matrix. An emerging methodology is the use ofmultifunctional nanodevices, such as polymeric micelles ordendrimers, to target cancers8 These devices contain not only thedrug payload but also targeting agents such as antibodies orligands to target specific receptors, as well as MRI contrastagents.3,8 Recent advances in the biomedical use of nanomater-ials also include gene delivery (gene therapy), delivery ofantigens (vaccination), and other therapeutic applications incardiac diseases, dental repair, and orthopaedics.3

This review describes a comprehensive overview of thediverse range of nanomaterials emerged for the detection andtreatment of cancers as well a brief picture of some diagnosticand treatment strategies using these materials. The first sectionfocuses on tumours as a drug delivery target. The pathology ofcancer is briefly described, followed by an overview of theunique properties of nanomaterials that make them promising fortumour-targeted diagnosis and therapy. The various nanomater-ials explored to date will be briefly described, including thesynthetic aspects and approaches used for their application inunimodal and multimodal diagnostic imaging and therapy. Adiverse range of emerging possibilities of nanomaterials incancer therapy will be described including those which arecommercialized.

How cancer differs from normal tissue

Cancer is an extensive group of diseases affecting differentparts of the body. It arises from the accumulation of geneticmutations that control cell cycles. The unlimited and self-sufficient growth of cells is responsible for the majorcharacteristics of cancers, including uncontrolled growth,invasion of adjacent tissue, metastasis, and cell immortality.1

Because of their particular anatomy and physiology, it is feasibleto target tumours with various nanoprobes, for diagnosis andtreatment. This possibility is due to the enhanced permeabilityand retention (EPR) effect,9 a characteristic pathological featureof tumour tissue in conjunction with low extracellular pH,hypoxia, angiogenesis, and abnormal lymphatics.

Cancers demonstrate irregular cell growth, aided by thedevelopment of a network of new blood vessels (angiogenesis),and prompted by various signals from cancerous tissuesincluding hypoxia, low pH, hypoglycaemia, mechanical stress,immune or inflammatory responses, and genetic mutations.10

These blood vessels are highly porous, with large spacesbetween the endothelial cells.9 They are convoluted, dilated,and irregular in diameter, possess a chaotic architecture, andpresent a disorganized vasculature including large openings,excessive branching, fenestration.11 With extensive leakage ofblood plasma components into the tumour microenvironment,the macromolecules are not rapidly cleared from the interstitialspace of the tumour, causing the EPR effect.9 As a consequence,the passive transport of macromolecules leads to their accumu-lation in tumours at considerably higher concentrations than innormal tissues, mostly 10–100 times higher within 1–2 days(Figure 1).12

Another important property of growing tumours is their pH.Tumour tissues, with their rapid cell division, require highmetabolic rates to meet their energy demands. Aerobic andanaerobic respiration inside the tumour leads to respiratory by-products, such as lactic acid and carbonic acid, being released

Figure 2. Active transport includes the endocytosis of specific ligand-modified drugs designed to be recognized by the various tumour cellreceptors and able to recognize small ligands such as folic acid, sugars, RGDpeptides, or antibodies.

21S. Nazir et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 19–34

into the surrounding environment. The poor drainage ofextracellular fluid from tumours results in the accumulation ofH+ ions in the tissue, leading to a decreased pH than in thenormal tissues.13 The acidic pH at the site of a growing tumourhas also been used for targeted delivery of nanoparticlesincluding polymeric micelles and polymer–drug conjugates,which destabilize at mild acidic pH and deliver their payloads atthe tumour site.13

The delivery of antitumor drug is also achieved by targetingsome of the proteins or glycoprotein that are over expressed atthe tumour cell surface and are capable of recognizing certainpeptides, proteins, carbohydrates, or other ligands. The conceptof targeted drug delivery is attractive as it retains some of theadvantages of the topical application of drugs, including highlocal concentration and low systemic exposure. Tumour-bindingpeptides including the RGD (arginine–glycine–aspartic acid)and NGR (asparagine–glycine–arginine) have been identifiedand used in the development of targeting nano assemblies.14

Moreover, studies on the expression of known cell-surfacereceptors in tumour tissue have led to the recognition of anumber of overexpressed molecular markers, including the αvβ3and αvβ5 integrins in angiogenic vessels, interleukin-11receptorα and folate receptor (FR) on the tumour surface.14 The targetingof such receptors helps in the selective uptake of the drugpayloads to the tumour cells than to the neighbouring healthycells (Figure 2). The targeted delivery of various nanomaterialsfor tumour treatment is described in the following sections.

Application of nanomaterials for cancer imagingand therapy

Conventional cancer therapies face challenges such as poorbioavailability and intrinsic toxicity. The therapeutic efficiency

of many useful drugs is compromised by such toxicity issues.Nanomaterials, with their altered pharmacological and therapeu-tic efficiencies have overcome some of these conventionallimitations. In the last three decades, research efforts in this fieldhave resulted in innovative nanostructures, including polymericand non-polymeric nanoparticles, QDs, SPIONS, AuNPs,nanowires, whiskers, tubes, pores, and chips.15 These recentlyintroduced nanomaterials and devices have demonstratedefficacy for use in applications such as in-vitro and in-vivodiagnosis, therapeutics, drug delivery and targeting. Many ofthese nanomaterials have unique properties that make them idealfor certain types of imaging or treatment modalities. Acomprehensive overview of such nanomaterials is given in thefollowing sections.

Polymeric nanoparticles

A wide range of natural and synthetic polymers constitute aplatform for synthesis of a variety of nanoparticles. Naturalpolymers that are widely used in nanoparticle synthesis includechitosan, dextran, albumin, heparin, gelatin, and collagen.5,6 Inaddition, a variety of synthetic polymers are employed fornanomaterial fabrications and drug encapsulation: for example,polyethylene glycol (PEG), polyglutamic acid (PGA), poly-D,L-lactide-co-glycolide (PLGA), polycarprolactone (PCL), poly-lactic acid (PLA), and N-(2-hydroxypropyl)-methacrylamidecopolymer (HPMA) have been widely used to preparenanoparticles and encapsulate drugs for cancer therapy.5 Thesepolymeric forms include dendrimers, liposomes, nanospheres,and micelles (Figure 3).15

DendrimersDendrimers are a unique class of polymeric macromolecules

inspired by nature. They are complex spherical structurescontaining hyperbranched subunits and end groups stemmingfrom a central core. The first report on dendritic structures waspublished almost two decades ago when Tomalia and co-workersmanipulated such structures through a cascade process.13 Thebranches of such polymeric molecules can be decorated with awide variety of functionalities, all of which are well-oriented inspace and highly accessible, thus providing a repertoire oftuneable chemistry. Furthermore, there are many techniques fordesigning dendrimers using either a divergent or a convergentapproach.16 In the divergent approach, the synthesis is startedfrom a multivalent core unit onto which the consecutive ‘layers’of branching units are added.16 The layers of arms are extendedby adding building blocks in a stepwise exhaustive manner toreach the exterior surface. In the convergent approach, on theother hand, the exterior part of the molecule is designed first withthe successive synthesis of different-sized branches starting frombuilding blocks of surface groups.17 This approach helps tominimize structural defects during synthesis17 and also helps inadding versatile functionalities or morphologies in suchassemblies.

The highly compact monodispersed dendritic polymers havea well-organized treelike molecular structure inhabiting a rangeof inner and outer reactive sites (molecular functionalities).13

The high structural diversity of dendritic structures makes themversatile nanodevices for drug delivery purposes.13 The great

image of Figure�2

Figure 3. Organic nanoparticles. Different polymeric assemblies used for various types of tumor imaging and therapies including dendrimer (A), liposomes (B),micelle (C), polymer–drug conjugate (D), polymeric nanosphere (E) and polymer core-shell nanoparticle (F).


variations in their basic morphology and peripheral functional-ities can be used to tailor them for a wide range of applications.Small changes in surface properties, such as the substitution of asingle amino acid for another or the use of PEG, glycosylation,and acetylation help in avoiding undesired immune responses.18

A variety of therapeutic candidates, ranging from small drugs toviral DNA, can be attached or encapsulated using the interiorcore or the extended branching surface.13 These highlymonodispersed nanoparticles can carry a high-density payloadof drugs, and are able to selectively release a drug in its activeform at the desired site.18,19 Thus, the development of morebiocompatible and targeted delivery systems, high-resolutionnovel contrast agents, and non-viral vaccines is currently inprogress.

Dendrimers are attractive devices for the imaging andtreatment of various types of cancers as well as promising drugdelivery candidates. Drugs are encapsulated in the interior cavityor are attached covalently to the branching ends at the surface,thus enhancing the pharmacological properties of the drug.19

Before discussing their efficacy as drug carriers or imagingdevices, it is important first to figure out how the dendrimers willreach the target site and accumulate there to provide a targetedresponse.13 The EPR effect described earlier improves thedelivery of dendritic structures to tumour tissues, and the uniquepathological features of solid tumours–hypervascularization,inadequate lymphatic drainage, and increased permeability tomacromolecules – help to ensure specific accumulation of

dendritic structures there.13,18,19 The EPR response is largely afunction of particle size and does not exclusively depend on thebiophysical or chemical nature of the dendrimer.18 Passivedelivery can therefore be improved by fine tuning of size,surface, and functional properties to exploit the EPR effect, andmuch research has already been undertaken to developdendrimers with promising properties for the passive targetingof tumours.19 Despite the advances that have been made duringthe last two decades in using dendrimers as imaging or drugdelivery devices, this research is still in its infancy.13 A widerange of targeting ligands including oligosaccharides, polysac-charides, oligopeptides, and polyunsaturated fatty acids as wellas folate and tumour associated antigens (TAAs) have beendeveloped for the specific targeting and destruction of tumoursusing dendrimer-like devices.20 Folate is a useful targetingchoice since FR is overexpressed in the cancer cell membrane ina wide variety of human cancers such as those of the lung, breast,kidney, ovary, and brain. Dendrimer formulations using folateshowed enhanced selectivity and success compared to their non-targeted counterparts.21

Immunotherapy or radio-immunotherapy is quite promisingin delivery of drugs, toxins, or radio-isotopes to tumour cellswhile avoiding their healthy counterparts. A variety ofmonoclonal antibodies have been employed to recognize andbind selectively to TAAs delivering conjugated drugs, prodrugs,and radio-isotope successively to tumour sites.22 The decorationof an antibody with toxins, small molecules, or multiple radio-

image of Figure�3


isotopes renders it non-specific for the antigens it is designedfor.13 An attractive solution to this problem is the deployment ofhighly internally conjugated dendrimers containing only onesurface-linked antibody.13 Such dendrimers have successfullybeen used for the immunotherapy and radio-immunotherapy ofvarious types of tumours. Dendrimers conjugated with radio-isotopes, including 3H, 14C, 88Y, 111In, and125I, have allowedthe detailed study of the biodistribution of such molecules inanimal models.13 Fluorescently labelled dendritic structureshave also been extensively used to characterize the targeting,cell-surface binding, uptake, internalization, and even sub-cellular localization of such devices in tumour cells.13

Dendrimers have also found applications in the diagnosticimaging of cancer cells, such as MRI. Gadolinium (153Gd) isgenerally considered as the best magnetic resonance contrastagent, but attempts to conjugate gadolinium to conventionalpolymers as well as proteins have met with limited success.However, gadolinium-conjugated dendrimers have allowed theselective comprehensive targeting and imaging of tumours.23

Boron neutron capture therapy (BNCT) has also attractedattention for the effective removal of cancer cells. BNCTemploys alpha-particles produced from the stable boron isotope10B. When activated with low energy (0.025eV) or thermalneutrons, 10B produces lithium (7Li) nuclei and alpha-particles,leading to impressive degradation of tumour cells within theirmembranes.24 Boronated antibody-targeted dendrimers havebeen designed for the effective BNCT of gliomas in the rat.24

LiposomesLiposomes are closed bi-layer vesicles made up of phospho-

lipids containing an aqueous core. They can carry hydrophilic orhydrophobic payloads embedded either in the interior core or inthe lipid bi-layer respectively.25 They are biocompatible drugdelivery agents and have been successfully employed fordelivery of a variety of drugs including anticancer agents.Liposomes are, however, vulnerable to rapid clearance throughphagocytosis by macrophages derived from the reticuloendo-thelial system (RES).25,26 Nonetheless, their half-life in theblood circulation can be greatly increased through surfacePEGylation or lipid cross-linking.25 Liposomes accumulate attumours through passive targeting via the EPR effect.25

Different types of pH-sensitive liposomes have also beendeveloped and described in literature. Some of these release theircargo after a pH-provoked change in the structural order of theirlipids; others contains pH-responsive lipids that are hydrolysedupon the change in pH.26 Proteins and fusogenic peptides arealso used to trigger drug release after fusion with the endosomalmembrane.26 The incorporation of pH-sensitive polymers intothe lipid bi-layer of the liposomal structure also makes themresponsive to the drop in pH.26,27 pH-responsive polymersinclude copolymers of polyalkylacrylic acid, polyphosphazene,polyglycidols, and polymalic acids.27 The mechanism of drugefflux from the liposome to the target cell depends upon the typeof polymer used: it may be a simple destabilization of the lipidbi-layer, or fusion between the liposome and the endosomalmembrane.26,27

Several liposomal formulations have met with success overthe years in a number of animal tumour models. The usual lipid

prodrug-based liposomes have shown promise in drug and genedelivery.27 Currently several liposomal formulation are in theclinical practice containing different chemotherapeutics such asdoxorubicin (Doxil1/Caelyx1), doxorubicin (Myocet1), dauno-rubicin (DaunoXome1) and cytarabine (DepoCyte1) for treatingthe ovarian cancer, AIDS-related Kaposi’s sarcoma, multiplemyeloma, lymphomas or leukaemia with meningeal spread.28

Several other liposomal chemotherapeutic drugs containingdoxorubicin, annamycin, mitoxantrone, cisplatin, oxaliplatin,camptothecine, 9-nitro-20 (S)-camptothecin, irinotecan, lurtote-can, topotecan, paclitaxel, vincristine, vinorelbine and floxur-idine are at the various stages of cilical trials.28 Moreover,advances with cationic liposomes have led to the successfuldelivery of small interfering RNA (siRNA).29 Targetedliposomal delivery has been explored through the use of low-density lipoprotein (LDL) particles as well as haloperidol-associated ‘stealth’ liposomes for genetic therapy of breastcancer cells.30 Drug delivery and imaging has been combined insome studies of a murine tumour model. Thermally sensitiveliposomes contained doxorubicin and MnSO4, and the paramag-netic properties of manganese, similar to those of gadolinium,were used as a probe for in-vivo monitoring of the drug byMRI.31 The temperature-responsive particles entered the tumour,shattered, and released the MnSO4; this could be observedthrough the relaxivity of manganese nuclei under the appliedmagnetic field.31

Polymerosomes, polymeric micelles, and nanospheresPolymerosomes consist of a polymeric casing surrounding an

aqueous internal core. The casing generally contains onehydrophobic layer with two hydrophilic polymer faces.26 Theyare often considered to be more stable than liposomes, and aretherefore the subject ofmuch attention for drug delivery purposes.32

Polymerosomes that assemble and disassemble with changes in pHare particularly attractive for the delivery of antitumor drugs.Polyanion-based pH-sensitive polymerosomes are less studied thanthose consisting of cationic non-pH-responsive polymers.26 Thespontaneous self-assembly of polyanionic block copolymers inwater can be exploited to deliver sensitive drugs such as proteinsand nucleic acids by simply adjusting the pH to an optimum valuewhere vesicle formation ensues.33 Biodegradable polymerosomesprepared from poly(trimethylcarbonate)-b-poly(L-glutamic acid)were able to deliver doxorubicinwith an increased release ratewhenthe pH was lowered from 7.4 to 5.5.34 They have recently beentested for in-vivo doxorubicin delivery to a murine tumour modeland were found to be more effective than free doxorubicin.35

Polymerosomes usually have a smaller vesicle size and a thinnermembrane.

Polymeric micelles (PMs) are nanoscale core–shell structuresobtained after the self-assembly of ampiphilic block copolymersin aqueous media.26 The structures organize only at the criticalmicelle concentration of the copolymer.26 They can beformulated from a variety of biocompatible polymers such aschitosan, PEG, methacrylic acid (MAA), methyl methacrylate(MMA), PMAA, poly(amido amine), (PAMAM) poly(L-asparticacid) (PAsp), N-isopropylacrylamide (NIPAM), dimethylacryla-mide (DMAA), 10-undecenoic acid (UA), poly(10-undecenoicacid) (PUA), 2-hydroxyethyl methacrylate (HEMA),


ethylacrylate (EA), ethyl methacrylate (EMA), butyl methacry-late (BMA), N-(2-hydroxypropyl)-methacrylamide (HPMA)octadecyl acrylate (ODA), N-vinyl-2-pyrrolidone (VP), poly(N,N-dimethylaminoethyl methacrylate) (DMAEMA), polygluta-mic acid (PGA), poly(D,L-lactide) (PLA), poly(ε-caprolactone)(PCL) and poly-D,L-lactide-co-glycolide (PLGA).26 The coreconsists of a hydrophobic domain that shelters the drug payloadwhereas the shell contains the hydrophilic functionalities andaugments the aqueous solubility and steric stability of theconstructs.26 PMs are biodegradable and are quite useful forlocalized drug and gene delivery.36 Drugs are encapsulated intothe PMs during the emulsification process and are releasedduring the degeneration process in the body/target cells.36 Thechange in pH and temperature at the delivery site are usuallyresponsible for the disintegration of these drug carriers,26 whichin turn could be controlled by adjusting the composition andmolecular weights. Another subclass of nanoparticles, polyioncomplex micelles (PICMs), originates from oppositely chargedpolymers making up the micelle structure.26 The self-associationof polymers in such micelles is due to electrostatic interactionsbetween the oppositely charged polymeric chains.

Though the PMs and PICMS have a narrow size distribution(10 to 100 nm) and proved as effective drug delivery agents.However, they become relatively unstable after dilution in the bodyfluids.26 The principal drawback of such constructs is theircomparatively unstable structure, which leads to the leakage ofdrug payload at increased ionic strength, as well as after dilution inthe body fluids.26 As a result, there has been an attempt toformulate stable ionic gels by cross-linking the micelle core withdivalent metal ions such as Ca2+.37 The resulting PM effectivelyencapsulated chemotherapeutic candidates such as cisplatin anddoxorubicin.38 The chemically cross-linked micelle of PEG-b-PAsp was also able to capture and stabilize lysosomes.39 PH-sensitive polyanion PMs are used for the delivery of variousnucleic acids including siRNAand antisense oligonucleotides.36,40

Such polyanion PMs involve bridging with polycation moleculesfor stability of the core structure.40 To obtain a non-toxic,endosomolytic, and efficiently networking cationic polymer, thePEG copolymer of PEG-b-poly(aminoethyl methacrylate) or PEG-b-poly(propylmethacrylate-co-methacrylic acid) (PEG-b-P(PrMA-co-MAA)) has been used with methacrylic acid (MAA)or poly(amido amine) (PAMAM) dendrimers.40–42 These micelleconstructs are fairly stable in serum and effectively protect theirnucleic acid cargo against enzymatic degradation.42,43 In order toachieve the receptor-mediated uptake of PICMs, the micelles wereadornedwith the transferrin receptor (CD71) targeting antibody viaeither a thioether or a disulfide linkage.42,43 It was found that thetargeted PICMS effectively stabilized 2′-F-modified siRNA andeffectively down regulated the oncoprotein Bcl-2 in humanprostate adenocarcinoma cells in-vitro.42

Polymeric nanospheres (PNS) are insoluble colloidal nano- ormicro particulates possessing a polymeric core with sizesranging from about 10 to 1000 nm. They are mostly designedas pH-sensitive drug delivery systems intended for oral deliveryin order to survive the strongly acidic environment of thestomach.44 They are used for oral delivery of labile peptidic andpeptidomemetic drugs such as the HIV protease inhibitors,44

HIV reverse transcriptase inhibitors,45 cyclosporin,46 and

insulin. PNS are mostly produced from MAA copolymers ofthe Euragit family, such as L100, S100, and L100-55, marketedas gastro-resistant covering agents. Oral delivery of variousEuragit-based polymer formations has been tested and foundeffective for improving the bioavailability of drug payloads ascompared to the micro-emulsion system.46 The delivery systemprotected the drug against degradation and additionally ensured ashorter transit time in the stomach and better bio-adhesiveness tothe gastrointestinal mucosa.46 PNS have also been obtained fromchitosan and pH-sensitive polyanions. Chitosan and γ-PGA-based PNS cross-linked with sodium tripolyphosphate andmagnesium sulphate resulted in efficient insulin loading andprotection for oral delivery.47,48 In a more highly acidicenvironment (pH 1.2–2.0), most of the carboxylic acids of theγ-PGA were protonated and the PNS became unstable, whichcould lead to drug leakage in the stomach under fastingconditions.48 However, enclosing the freeze-dried PNS in anenteric-coated capsule resulted in enhanced protection in thehighly acidic environment, as compared to free insulinencapsulated in coated capsules, and demonstrated increasedplasma drug levels after oral delivery in rats.48

FullerenesFullerenes are the spheroidal carbon nanostructures, with

exceptional physical, photochemical, and electrochemical prop-erties. They have been remodelled to carry gadolinium atoms forMRI of tumours and have been surface functionalized withreceptor agonists and antagonists for tumour targeting.49 Thewater-soluble gadolinium metallofullerenes had a prolongedblood circulation time, up to 48 h, and delayed clearance by theexcretory system.50 Further advances has resulted in enhancedproton relaxivities obtained by manipulating the pH effect.51

Fullerene formulations now need to be surface tailored to achieveactive tumour targeting, and investigated for their in-vivo safetyaspects; in addition, all of the findings need to be tested atclinically applicable MRI field strengths.

Inorganic nanoparticles

Various form of inorganic nanoparticles are used for thediagnosis and treatment of various forms of cancers includingQD, SPIONS, AuNPs and other metallic and non-metallicnanoparticles or nanoclusters (Figure 4). A detailed overview ofthese inorganic particles is given in the following sections.

Quantum dotsFluorescent QDs are inorganic semiconductor nanocrystals 2–

10 nm in size, exhibiting a broad absorption band but a symmetric,narrow emission band, typically in the visible to near infrared(NIR) spectral range.52 Their excitation by single-wavelength laserlight leads to absorption with a broad spectrum of colours. Theabsorption probability of QDs increases as they decrease in size,and is shifted towards the blue end of the spectrum.53 The quantumconfinement effect comes into play when the size of thesemiconductor crystal becomes less than the Bohr exciton radius,leading to unique electronic and optical features.52

Earlier QD nanocrystals usually were composed of hundredsto thousands of atoms of groups II–IV (such as zinc, cadmium,

Figure 4. Some inorganic nanoparticles used for the imaging and treatment of tumours: superparamagnetic iron oxide nanoparticle (SPION), (A) inorganiccore–shell nanoparticle such as QD (B), AuNP (C) and inorganic nanoclusters (D).


selenium, and tellurium) and groups III–V (such as indium,arsenic, and phosphorus).53 The quantum yield of suchsemiconductor cores was greatly improved by constructing ananoshell of comparatively higher band gap material around thecentral core. Recently, ultra-small nanocrystals of any metalexhibiting the quantum confinement effect have been catego-rized as QDs. The optical properties of QDs make them suitablefor highly sensitive, long-term, and multi-target bio-imagingapplications.7 The surface properties of the QD crystals aretailored to achieve better solubility, biocompatibility, and targetselectivity. Thiol groups are usually anchored on the surface,providing terminal carboxyl groups.7 These surface functional-ities make it possible to add targeting proteins, includingtransferrin or antibodies, so that the nanocrystals can bind tospecific receptors on the cell surface.7,54 Such receptor-basedinternalization enables the cell and nuclear localization of QDs asthey have highly sensitive fluorescent imaging properties withmolar extinction coefficient as high as 0.5–5 × 106 M-1cm-1.52

This efficient photon absorption leads to nanomaterials that are10–50 times brighter and several thousand times more photo-stable than conventional imaging dyes.52,7

The synthesis and development of QDs as multifunctionalimaging probes is a multistep process. Hot solution-phase QDsynthesis is the most popular methodology. The QDs aresynthesized at elevated temperatures in a non-polar organicliquid mixture with high boiling point containing usuallytrioctylphosphene (TOP) and trioctylphosphene oxide (TOPO)as surfactants.54 The hydrophilic medium coordinates with theunsaturated metal, facilitates the kinetics, and defines the particlesizes in the nanometre range.54 The reverse-micelle (water-in-oil) method is another option, using a surfactant such as sodiumbis-2-ethylhexyl-sulfosuccinate (AOT) with oil and water as athree-component stable emulsion yielding highly photo-stableQDs at room temperature, as reported for Mn-doped CdS/ZnScore–shell QDs using AOT/heptane/water as the reverse-micelle

medium.55 A silica coating on the QD surface ensuresbiocompatibility and an increased shelf life even at low pH.Further outer coatings with multifunctional silane reagentsprovide the opportunity for surface modifications such as theaddition of targeting agents and drug payloads.56 Surfacetailoring and bio-conjugation with peptides, antibodies, proteins,and DNA has greatly enhanced the in-vivo applications ofQDs.57–60 Such bio-conjugation can take the form of eithercovalent bonding or electrostatic interactions. Proteins contain-ing cysteine or histidine can be directly attached to the ZnSsurface through in-situ disulfide linkages between the sulphuratoms of ZnS and the respective sulphur-containing amino acidresidues.

There are several other reports on antibody-conjugated QDsfor the specific targeting of specific tumours in mice61,62 Onesuch report describes QD conjugation with prostate specificmembrane antigen (PSMA). Tumour cells were grafted into anude mouse and after the tumour developed, the antibody-conjugated QD–PSMA was injected through the tail vein.63 Theexperiment resulted in an intense QD signal in the targetedtumour for the antibody-conjugated particles, whereas little or nosignal was displayed after passive targeting of the tumourtissues.63 QDs conjugated to human epidermal growth factorreceptor 2 (HER2) and epithelial growth factor receptor (EGFR)antibodies were demonstrated to accumulate in human breastcancer and pancreatic tumour tissues in mice respectively64,65

Another in-vivo study showed that QD conjugated with folicacid targeted the FR and accumulated in mouse lymphoma cellsbut were not found in normal body tissues.66 QDs conjugatedwith RGD peptide were also found to be promising. One studyreported on QD–RGD conjugates injected into xenograft humanglioblastoma in mice.67 The NIR-specific CdTe QDs conjugatedwith DNA and siRNA showed different behaviours. DNA–QDsconjugated with polyethyleneimine (quantocomplexes), whenused for in-vivo gene therapy using a plasmid DNA,

image of Figure�4


accumulated in the liver, lungs, spleen within 5 minutes afterinjection.68 Moreover, using anti-vascular endothelial growthfactor (VEGF) specific siRNA suppressed VEGF production andreduced tumour size.69

With all the advances already made in QDot nanotechnology,biocompatibility, distribution, metabolism/excretion, and safetyissues are still major concerns. New approaches such as theaddition of a silica coating or a biocompatible polymer coatinghave increased the biocompatibility and minimized the toxicityof these ultra-small particles, resulting in more water-soluble andsafer formulations.70 Similarly several reports contained thehighly luminescent, cadmium free QDots for a better biocom-patibility and lesser toxicity in cellular environment.71,72

Nevertheless, the safe breakdown and elimination of nanode-vices require more comprehensive study.

Superparamagnetic iron oxide nanoparticlesSPIONs have unique magnetic properties that make them

attractive materials for advanced biomedical applications. Theycan help in medical imaging, serving as contrast agents inMRI; are capable of killing malignant and infected cellsthrough thermal therapy, acting as miniaturized heaters; andcan be employed for targeted drug delivery to cancer cells andother disease sites.7 SPIONs acquire a large magnetic momentin an externally applied magnetic field, thus attaining super-paramagnetic properties.7 They are capable of producing highcontrast per unit of particles, so that smaller quantities ofSPIONs are sufficient for imaging therapy, thereby reducingthe toxicity issues.7,73 Additionally, their surface can beengineered with a variety of functionalities, enhancing theirbiocompatibility and biodegradability for widespread biomed-ical applications.73 SPIONs degrade by leakage of the free ironions, which are added to the body’s native iron pool,incorporated into haemoglobin, and degraded through thenormal iron recycling pathway.7

SPIONS are usually synthesized by co-precipitation ofFe(OH)2 and Fe(OH)3 suspensions. The synthesized SPIONscan then be functionalized with a variety of biodegradable andbiocompatible polymers such as PEG, polyethyleneoxide (PEO),dextran, and polysaccharides to achieve better biocompatibilityand stability in blood plasma.7

In-vivo MRI. MRI is one of the most important applications ofSPIONs. The largemagnetic moment of SPIONs distorts the localmagnetic moment of water molecules in tissues, resulting inenhanced contrast between tumour and normal tissues. SPIONsare readily taken up by the RES, resulting in the successfuldetection of smaller tumours.74 Similarly, the macrophage-mediated uptake of SPIONs leads to their accumulation in thelymphatic system and the subsequent detection of lymph nodemetastasis.75 A comparative study of SPIONs and gadoliniumchelates as contrast agents forMRI observed a prolonged imagingresponse from SPIONs due to their increased uptake followed byslow diffusion from the tumour site after internalization.74

SPIONs have been extensively studied for the detection anddiagnosis of various solid tumours.76,77 Specific targeting ofdiseased tissues may allow the more precise diagnosis of cancersat several stages of malignancy. Clinicians are now greatly

interested in the active targeting of malignant areas for theprecise marking of tumour boundaries within healthy tissue.77

As a result, the active targeting of SPIONs through functiona-lization with antibodies, nanoparticles, short peptides, and smalltargeting ligands is attracting increasing interest from thescientific community. A recent finding involving SPIONs coatedwith β-cyclodextrin and pluronic polymer has demonstrated veryeffective curcumin loading efficiency.78 The drug-loadedformulation exhibited haemocompatibility as well as greatlyimproved imaging contrast properties. Another group hasreported the effective targeting of glioma tumours over-expressing MMP-2 through SPIONs functionalized with chlor-otoxin (a targeting peptide).79 Similarly, SPIONs functionalizedwith RGD peptide have been successfully used to target cancercells.80 The RGD peptide has high affinity for αvβ3 integrin,which is a well-known angiogenesis marker.

Thermal therapy. SPIONs can convert the electromagneticenergy supplied by an externally applied alternating magneticfield into heat.81 This generated heat can be used for the selectivedestruction of tumour cells, which are more vulnerable thannormal body cells to this heating. SPIONs have been successfullyused as hyperthermia agents in the initial clinical setting.81

On-going research is focusing on the selective targeting oftumour tissue and its ablation by controlled heating ofSPIONs.82 The dual role of SPIONs, as the imaging probe inMRI and as agents in the subsequent hyper-thermal treatment fordestruction of tumour tissue, is attracting increasing interest inthe attempt to devises after imaging and treatment tools forcombating various cancers.82,83

In-vivo drug delivery. In addition, polymers and cappingagents can be attached to the SPION surface for increasedbiocompatibility and bioavailability, using biodegradable mate-rials such as cellulose, dextran, PEG, or PLGA.73 Standardchemotherapeutic drugs can be incorporated into the polymershell around the SPION core. PEG-coated doxorubicin-contain-ing SPIONs have successfully delivered the sufficient amount ofdrug to the malignant Lewis lung carcinoma in-vivo.73 The drugwas released more rapidly in the mildly acidic tumourenvironment than at the neutral pH of the vasculature, leadingto a lower toxicity to the normal tissues.73 Recently, paclitaxel(PTX) loaded superparamagnetic iron platinum nanoparticles(SIPPs) were encapsulated in the PEG–functionalized biotincontaining phospholipid micelle and were studied for theirtargeting efficiency, PTX release and in-vivo MRI contrastenhancement efficiency.84 The resulting SIPP-PTX multifunc-tional, stealth micelles (SPMs) were conjugated to an antibodyagainst prostate-specific membrane antigen (PSMA). PSMAconjugated SPMS resulted in the significant reduction in thegrowth of C4-2 prostate cancer xenografts in nude mice alongwith a successful MRI contrast enhancement in vivo.84

Multifunctional water-soluble SPIONs have also beendeveloped for the positron emission tomography (PET)/MRI.85

The targeted delivery was achieved using the tumour targetingpeptide cyclo(Arg-Gly-Asp-D-Phe-Cys) (c(RGDfC)). A 64Cuchelator, macrocyclic 1,4,7-triazacyclononane-N,N′,N″-triaceticacid (NOTA), was conjugated onto the distal ends of the


PEGylated SPIONs for PET imaging while doxorubicin wasconjugated onto PEG linkers via pH-sensitive bonds.85 Thesedual imaging/treatment probes combined a much higher levelof tumour accumulation and non-invasive and quantitative PETimaging in vivo with an effective cytotoxicity to tumour cellsin-vitro.

Gold nanoparticlesThe use of gold in medicines has been known since ancient

times in cultures such as those of China, Egypt, and India.Eastern civilizations used gold-based herbal formulations tomaintain health.86 In the West, gold-based medicines havehistorically been used for nervous conditions, epilepsy, leprosy,syphilis, plague, and diarrhea.87 The bacteriostatic effect of goldwas first explored in early 20th century, when gold was found tobe active against Bacillus tuberculosis, leading to gold-basedtuberculosis therapy.86 This laid the foundations of modern gold-based drug therapy. Later, gold-based formulations came intoclinical practice for the treatment of various diseases such aspsoriasis, arthritis, and rheumatism.86 The first generation ofgold-based pharmaceuticals included sodium aurothiomalate andsodium aurothioglucose, which were principally used forrheumatoid arthritis.13 These drugs were water soluble, distrib-uted and cleared from kidneys, liver, and spleen.13 However,several side effects were reported, such as nephrotoxicity, liver-toxicity, skin reactions, and mouth ulcers.13 A second-generationgold-based drug, aurofin, contains aphosphine ligand and istherefore more lipophilic, displaying a better retention time andreduced nephrotoxicity.13 More recently, gold has been used inmany modern medical devices such as gold-plated stents,pacemakers, middle ear implants, and dental prostheses.3

Whereas, several nanogold complexes have emerged withpromising antimicrobial, antitumor, antimalarial, antirheumatic,and anti-HIV activities.3 The following sections describe thesynthesis and various applications of AuNPs for the diagnosisand treatment of cancers.

Synthesis of gold nanoparticles. In 1994, Brust introduced atoluene/water-based two-phase method for the synthesis of thiol-capped AuNPs with a controlled size range, from 1.5 to 5 nm.88

AuCl4 was transferred to the oil phase using tetraoctylammoniumbromide and then thiolated through ligand replacement.88 Asubsequent reductionwith NaBH4 results in highly monodispersedair-stable AuNPs. Other reducing/stabilizing agents such ashydrazine, mesitylsulfonic acid (MSA), oxalate, maleate, andseveral synthetic and natural polymers such as PVP, PVA, PMAA,starch, cellulose, dendrimers and variety of functionalizedpolymers such as including those of PMAA, PEG, thiols havealso been reported for the size-controlled synthesis of AuNP.89,90

The production of larger nanoparticles through the chemicalreduction method meets with only limited success, yielding apolydispersed solution.90 However, a seeding approach isfruitful, using AuNP or AgNPs as the catalyst surface for thereduction of Au3 + in the presence or absence ofhydroxylamine.91 Consequently, this seed-mediated growthapproach has been adopted to control the shape and size ofAuNPs.92 A gold salt solution reduced in the presence ofNaBH4-reduced AuNP seeds (3–4 nm in diameter), using

ascorbic acid as the reducing agent, cetyltrimethylammoniumbromide (CTAB) as micellar template, and small amount ofsilver ions for shape induction, has been used for the productionof spheroid or rod-like AuNPs.91 This approach has beensuccessively improved to harvest monodispersed AuNPs ofmany different shapes in higher yields than any previousreport.93,94 Other methods for the synthesis of AuNPs includephotochemical reduction (using UV, NIR, microwave, orradiofrequency radiation),94 ultrasonics, thermolysis, physicalreduction,95 biological reduction,96 and solvent evaporationtechniques.97 Additionally, a simple and cost-effective micro-wave irradiation method has been introduced for the productionof shape-controlled AuNPs.98 In this method a gold salt solutioncontaining CTAB and alkaline 2,7-dihydroxy naphthalene (2,7-DHN) is readily reduced within just 90 seconds to AuNPs ofvarious shapes – spherical, rod-like, triangular, and polygonal.

Many bacteria, fungi, and plant extracts are reported to reducemetal salts to the respective nanoparticles. These microorgan-isms or plant extracts usually contain a specific class of enzymescalled reductases, or otherwise they have the highly function-alized phytochemicals responsible for the reduction of Au3+ to agood yield of pure AuNPs.96,99,100

Gold nanoparticles for cancer detection and diagnosis. Thebiomedical applications of colloidal AuNPs with size-dependentproperties, including colour, and dimensional similarities tobiomolecules have been the subject of much interest in cancerresearch. AuNPs has proved as very useful probes for themicroscopic imaging of tissues. The unique light absorption andlight scattering properties of AuNPs help in distinguishinghealthy cells from cancerous.101 Conventional exogenousimaging agents include organic fluorophores and lanthanidechelates. Organic fluorophores are readily photobleached,leading to low quantum yields, and have a broad emissionwindow, whereas lanthanide chelates are non-selectively local-ized in extravascular space.102 Quantum confinement andsurface plasmon resonance (SPR) in these nanoparticles havefurther increased interest in their use as contrast agents in typicalimaging modalities for the detection and management of varioustypes of cancers.101 A single 80 nm gold nanoparticle is foundwith a surface plasmon resonance optical scattering intensityequivalent to the efficiency of 200 of the most efficient QDotsand 500000 of the most efficient organic fluorophore (AlexaFluor) dyes.101 AuNPs give very flexible colorimetric contrastwhich can be easily tailored by controlling the shapes and sizesof AuNPs103 or through surface modifications. Furthermore,they are non-toxic, biocompatible (i.e. they do not elicit anyallergic or immune responses),104,105 and can be easily used foroptical imaging, helping to detect and manage various types ofcancers. Antibody-conjugated AuNPs have made cancer imag-ing much easier even using a confocal reflectancemicroscope.101 El-Sayed et al has demonstrated that simpler,less expensive dark-field optics could be used for the canceridentification, staging and treatment monitoring using immunelabelled AuNPs in vitro.101 Similarly, in-vivo cancer imaging isachieved by establishing a colorimetric contrast between normalbody cells and the tumour cells or tumour vasculature. Currently,


the method is widely used for both the imaging and spectroscopyof AuNPs in living cells and tissues.106,107

The optical imaging of cancers, including photoacousticimaging108 and two-photon luminescence,109 is facilitated bytuning the SPR of AuNPs in the visible or NIR regions. The SPRof AuNPs tuned to a NIR window results in light absorption/scattering upon excitation in the NIR region (600–1000 nm).108

This spectral region, called the ‘phototherapeutic window’, hasmaximum penetration power into most human tissues. At thesewavelengths here is low absorption by normal cell constituentsand relatively inefficient scattering of red light by cellorganelles.108,109 The penetration depth of light is therefore ata maximum in this region, permitting the deeper imaging ofcancerous cells. Photoacoustic imaging is reported using 15 nmAuNPs as the cell contrast agents when excited by a short pulselaser.108 An ultrasonic array was used in the experiment tocollect and recreate the initial heat distribution to image the targetcells. AuNPs have also been used to image prostate cancer(LnCAP) and skin cancer (A431) cells using two-photonluminescence imaging.109

CT is a non-destructive medical imaging method. Usually, anX-ray absorbing contrast agent is employed to enhance thecontrast between the different types of cells.110 AuNPs have ahigh X-ray absorption coefficient, and they are also versatile:their sizes and shapes can be controlled and their surface can befunctionalized with desired materials. Recently, coated AuNPshave been used for in-vivo CT studies for cancer detection.110–112 In one such studies, AuNPs with the layer-by-layer (LBL)assembly of poly(acrylic acid) (PAA) and poly(allylaminehydrochloride) (PAH) was used to detect the human hepatocel-lular carcinoma cell line (FOCUS) through spatial harmonicimaging (SHI).112 The modified AuNP were able to penetratethrough the FOCUS cell pellets several millimetres in diametersand were detected with the SHI technique.112

MRI is another non-invasive imaging technique thatgenerally makes use of gadolinium complexes as contrastagents, but AuNPs have found applications here too. Thesegadolinium chelates such as gadoliniumdiethyltriaminepentaa-cetic acid (Gd-DTPA) produce low magnetic fields and haveshown to exhibit kidney toxicity.113 DNA-templated Au-NPchains have shown an increased phagocytosis capability by the3D cancer cell sacffolds.114 Although the LBL encapsulatedAuNP experienced comparatively weaker local magnetic fields,the greater cell uptake of DNA-AuNP was able to producestatistically equivalent image contrast in T2-weighted MRIimages.114

Au radio-isotopes such as Au198 have been widely used totreat different types of cancers. Moreover, Au radio-colloidscontaining the different radio-active nuclei such as 125I, 64Cu,99mTc have been found very effective in the deep tissue andhighly mineralized near-bone imaging.115 AuNP c[RGDfk(C)]conjugates labelled with 99mTc resulted in significant tumoruptake (3.65% ID/g) just after 1h post-injection after whendelivered to an athymic mice bearing C6 human glioma.116 Non-radioactive AuNPs have also been found as effective radio-sensitizer in cancer radiation therapy. In a recent in vitro and in-vivo radiosensitization study, the effect of PEG-coated AuNPshave revealed the size-dependent radiosensitization effect, the

smaller being more effective in scattering of 5 Gy gammaradiation.117 The accompanied immune response, pathology andblood biochemistry studies indicated that the PEG-AuNPs didnot cause spleen and kidney damages, but they resulted goldaccumulation and damage in liver.117

Photothermal therapy. Non-invasive photothermal therapyhas also shown promise in the treatment of cancers. Photo-thermal therapy employs a non-ionizing light source, usually inthe visible to NIR region, which penetrates deeply into thetissues with minimal scattering.118 The optically active AuNPscan transform light radiation to localized heat, killing the tumourtissues.118 Tailoring the surface plasmon properties of AuNPshas resulted in gold nanospheres, nanoshells, nanorods, andnanocages with effective photothermal properties that can killcancers, bacteria, viruses, and protozoans.118–120 The techniquemay prove potentially useful in clinical settings as SPR andsurface functionalities of AuNPs can be tuned for the precisemarking of tumour boundaries within normal tissues, followedby the heat treatment. Au nanorods irradiated with a wide rangeof NIR radiation has resulted in a longitudinal absorption band inthe NIR region leading to effective photothermal killing oftumours.120 In another study, Au nanocubes demonstrated veryhigh photoluminescence (PL) quantum yield, about 200 timeshigher than that of nanorods.121 As a result, these nanocubeswere successfully used for the photokilling as well asphotoimaging of human liver cancer cells and human embryokidney cells using a laser scanning confocal microscope.121 Au-silicon nanowires composite structures have also been employedfor the efficient capture and photothermal therapy of circulatingtumor cells.122

In an in-vivo study involving mice, Stern et al observed that93% of tumour was removed using a high dose of nanoshells (8.5μL/g).123 While other experiments with low doses resulted onlyin tumour arrest, only the high doses resulted in the completeremoval of tumours.123

As well as photothermal studies, AuNPs have also beenemployed for other tumour treatment modalities, such asphotodynamic therapy (PDT). PDT is the photo-oxidation ofcancer tissues through the production of reactive oxygen species(ROS) on the surface of photoactive agents (dyes such asphthalocyanine or semiconductor crystals) delivered to tumours.There are several literature reports of the successful delivery andinternalization of photoactive dyes by conjugation to AuNPs.The AuNP photosensitizer conjugates resulted in effectiveenergy or electron transfer between the photoactive dye andthe nanoparticle, leading to an effective photodynamic effect inin-vitro cancer models.124,125 Other reports describe the use ofsemiconductors such as TiO2, ZnO, and Fe3O4for the photody-namic destruction of cancers, through either necrosis orapoptosis.126,127

Although the EPR effect leads to the effective delivery ofnanoparticle-based therapeutics into tumours, selective thermo-lysis was not achieved in smaller tumours and single metastaticcells.3 Occasionally heat diffused from the host particles and,upon longer exposure time, the damaged tissue area increased,disturbing the normal tissue.3 Bearing in mind that AuNPs cancarry up to 150 antibodies conjugated through a bifunctional PEG

Table 1Approved/marketed nanomaterials in cancer therapy.

No Product(s)/device Nanoplatform/active agent Status Active mechanism Company/Reference

1 Doxil/Caelyx1 Doxorubicin HCl Liposome (PEGylated) Approved Chemotherapy Ortho Biotech(acquired by JNJ)144

2 DaunoXome Liposomal daunorubicin Approved Chemotherapy Galen Ltd.144

3 Myocet Liposomal doxorubicin (non-PEGylated) Approved Chemotherapy Sopherion Therapeuticsand Cephalon, Inc.144

4 Abraxane Paclitaxel albumin nanoparticle Approved Chemotherapy Celgene144

5 Feridex Dextra coated Iron oxide nanoparticles Approved MRI imaging Berlex Laboratories144

6 Endorem Carboxydextra coated Iron oxidenanoparticles

Approved MRI imaging Guerbet144

7 Resovist/supravist Dextra coated Iron oxide nanoparticles Approved MRI imaging Bayer Schering Pharma AG144

8 ThermoDox Heat-activated liposomal Encapsulationof doxorubicin

Approved phase III Chemotherapy Celsion144,28

9 Lumirem, Sinerem,FeraSpin

Iron Oxide NPs Approved/investigational

Enhanced MRIContrast

Guebert28

10 Ontak Protein NP (IL-2 Protein)for T-Cell Lymphoma

Approved (1999) Targeteddelivery

Seragen, Inc.145

11 Rexin-G Targeting protein taggedphospholipid/microRNA-122

Phase II/IIIapproved in Philippines

Targeted genetherapy

Epeius Biotechnologies Corp.144

12 NanoTherm Iron oxide nanoparticles Approved AC MagneticHeating

MagForce, Nanotechnologies AG145


linker, photothermal therapy experiments were designed toselectively target the highly metastatic tumour cells targetedthrough their upregulated tumour cell-surface receptors.128,129

Most of the targeted photothermal studies that have beenconducted involved either human EGFR or HER2, because ofthe easy availability of monoclocal antibodies for these tworeceptors.128 AuNPs conjugated with anti-EFGR antibodies haveresulted in selective targeting and photothermal killing of cancercells in in-vitro experiments.130 EFGR-responsive cells werekilled with only half of the energy required to kill the EFGR-negative cells.130 In another study, the treatment of a HER2-overexpressed breast cancer cell line by gold nanocages resultedin selective cell death under in-vitro conditions,131 and HER-2targeted gold nanoshells resulted in similar effects, as reported byseveral other groups.132,133 AuNP–anti-EFGR conjugates havealso been reported for non-invasive radiofrequency (RF)-basedtumour therapy.134 The AuNP–anti-EFGR conjugates weresuccessfully internalized through receptor-mediated endocytosis(RME). The AuNPs accumulated in cytoplasmic endolysomesand were activated by a non-invasive RF electric field, resultingin hyperthermia and consequent killing of tumour cells.134

Similarly, anti-metadherin (anti-MTDH)-conjugated fluorinatedAuNPs were reported, containing radioactive and optical labelsfor tumour treatment.135

Drug delivery using gold nanoparticles. There are manyreports on the utilization of AuNPs for the delivery of antitumordrugs. They have proved to be effective and relatively safe drugcarriers, especially for the delivery of highly toxic drugs. Severaltypes of drugs, including proteins and DNA as well as smallerdrug molecules such as chemotherapeutic agents, have beendelivered by tailoring the surface chemistry of AuNPs. Tumournecrosis factor alpha (TNF-α) is a cytokine that has excellentantitumor properties, but its high toxicity severely limits itstherapeutic application.136 However, PEG-coated stealth AuNPs

containing TNF-α have resulted in good tumour damage withreduced systemic toxicity.137 In-vivo experiments with theseconjugates resulted in little or no accumulation in liver, spleen,and other healthy organs.138 Such a construct (AurimuneTM,CYT-6091) is currently undergoing phase-I clinical trials for theassessment of its pharmacokinetics and properties.137 Dhar et alreported a platinum(IV) complex tethered DNA–AuNP conju-gate for in-vitro treatment of human carcinoma cell linesincluding lung carcinoma (A549), osteosarcoma (U2OS),prostate cancer (PC3), and cervical cancer (HeLa).139 Thisnanoparticle had promising antitumor properties, in some caseseven exceeding that of cisplatin.139 In a similar study of AuNP–methotrexate the drug–AuNP conjugate accumulated rapidly inLewis lung carcinoma (LL2) cells, leading to high tumourtoxicity, whereas a similar dose of free methotrexate had noantitumor effect.140

AuNPs have facilitated the targeted delivery of severalimportant small-molecule therapeutic agents to tumours. Thisdelivery is achieved through a combination of the EPR effect andRME. The drug is initially accumulated in the tumour vasculaturethrough the EPR effect followed by RME-mediated uptakefacilitated by the small molecules anchored to the AuNP surface.One such delivery agent is folic acid, which has been extensivelyexploited to successfully target the FR that is overexpressed inmany types of tumours including lung, ovary, breast, prostate,brain, and kidney tumours.13 AuNP–folate conjugates havepermitted selective accumulation in FR-responsive tumoursleading to tumour imaging and photothermal therapy.141 Detailedmechanistic studies in human epithelial carcinoma revealed anFR-mediated RME of AuNP–folate conjugates. The targetingand imaging of dendrimer-entrapped AuNP–folate was alsoexplored by Shi et al.142 The subsequent TEM analysis of KBcells (a human epithelial carcinoma cell line) revealed selectiveaccumulation of the conjugated drug in high FR-expressingtumours.143 Similarly, the peptide bombesin (BBN) was

Table 2The expected future nanomedicine products against cancers.

No Product(s)/device Nanoplatform/active agent Status Active mechanism Company/reference

1 Cyclosert Cyclodextrin nanoparticles(Cyclodextrin NP/SiRNA)

Phase I Gene therapy Insert Therapeutics(now Calando Pharmaceuticals)144

2 CRLX101 Cyclodextrin NPs/camptothecin Phase II Chemotherapy Cerulean Pharma144

3 CPX-351 Liposomal cytarabine and daunorubicin Phase I Chemotherapy Celator Pharmaceuticals144

4 LE-SN38 Liposomal SN38 Phase II chemotherapy Neopharm144

5 INGN-401 Liposomal/FUS1 Phase I Tumor suppressor gene therapy Introgen144

6 NC-6004 Polymeric nanoparticle (PEG-polyaspartate)formulation of cisplatin

Phase I Chemotherapy NanoCarrier Co.144

7 NK-105 Polymeric nanoparticle (PEG-polyaspartate)formulation of paclitaxel

Phase II Chemotherapy Nippon Kayaku Co. Ltd.144

8 NK-911 Polymeric nanoparticle(PEG-polyaspartate)formulation of doxorubicin

Phase II Chemotherapy Nippon Kayaku Co. Ltd.144

9 NK-012 Polymeric micelle of SN-38 Phase II Chemotherapy Nippon Kayaku Co. Ltd.144

10 SP1049C Glycoprotein of doxorubicin Phase II Chemotherapy Supratek Pharma Inc.144

11 SPI-077 PEGylated liposomal cisplatin Phase II Chemotherapy Alza Corporation144

12 ALN-VSP Lipid nanoparticle formulation of siRNA Phase I Gene therapy Alnylam Pharmaceuticals144

13 OSI-7904L Liposomal thymidylate synthase inhibitor Phase II Chemotherapy OSI Pharmaceuticals144

14 OSI-211 Liposomal lurtotecan Phase II Chemotherapy OSI Pharmaceuticals144

15 Combidex Iron oxide Phase III Tumor imaging Advanced Magnetics144

16 Aurimune Aurimune Colloidal gold/TNF Phase II Targeted Thermaltherapy

CytImmune Sciences144

17 BIND-014 Polymeric nanoparticle formulation of docetaxel Phase I Chemotherapy BIND Bioscience144

18 SGT53-01 Transferrin targeted liposome with p53 gene Phase I Gene therapy SynerGene Therapeutics144

19 PEG-IFNα2a PEG-asys Phase I/II Chemotherapy Genentech144

20 PEG-IFNα2b PEG-intron Phase I/II Chemotherapy Merck144

21 ADI-PEG20 PEG-arginine Phase I - Polaris144

22 JNS002 Doxorubicin Phase II Chemotherapy 28

23 Liposomalannamycin

Annamycin Phase II Chemotherapy 28

24 LEM Mitoxantrone Preclinical Chemotherapy 28

25 SPI-77 Cisplatin Phase II Chemotherapy 28

26 Lipoplatin Cisplatin Phase III Chemotherapy 28

27 LiPlaCis Cisplatin Phase I Chemotherapy 28

28 L-NDDP/auroplatin

Cisplatin analogue Phase II Chemotherapy 28

29 MBP426 Oxaliplatin Phase I Chemotherapy [28]30 NL CPT-11 Nanoliposomal camptothecine Trial Chemotherapy http://www.clinicaltrials.gov/31 L9NC 9-nitro-20 (S)-camptothecin Trial Chemotherapy http://www.clinicaltrials.gov/32 IHL-305 Irinotecan Phase I Chemotherapy http://www.clinicaltrials.gov/33 LE-SN38 SN38 (active metabolite of irinotecan) Trial Chemotherapy http://www.clinicaltrials.gov/34 PEP02 Irinotecan Phase I Chemotherapy 28

35 OSI211 Lurtotecan Phase II Chemotherapy 28

36 TLI Topotecan Trial Chemotherapy http://www.clinicaltrials.gov/37 PNU-93914 Paclitaxel Trial Chemotherapy http://www.clinicaltrials.gov/38 PNU-93914 Paclitaxel Trial Chemotherapy [http://www.clinicaltrials.gov/,28

39 LEP-ETU Paclitaxel Trial Chemotherapy [http://www.clinicaltrials.gov/,28]40 MarqiboW Vincristine Phase II Chemotherapy 28

41 VLI Vinorelbine Trial Chemotherapy [http://www.clinicaltrials.gov/,28]42 CPX-1 Fixed combination of irinotecan and floxuridine Phase I Chemotherapy 28

43 CPX-351 Fixed combination of cytarabineand daunorubicin

Phase I Chemotherapy 28

44 Clariscan Iron Oxide nanoparticle Phase III Targeted MRI contrast Nycomed28,144,145

45 AuroShell Gold Nanoshell for solid tumor treatment Phase I IR Laser Heating Nanospectra Biosciences145

46 NanoXray Proprietary NP Phase I X-Ray-Inducedelectron emission

Nanobiotix145

47 BIND-014 Polymeric nanoparticleformulation of docetaxel

Phase I Chemotherapy(various cancers)

BIND Bioscience144


exploited for its high affinity for gastrin-releasing peptide (GRP),which is overexpressed in a variety of cancers including breast,prostate, and small-cell lung carcinoma.143 In-vivo studies

involving AuNP–BBN constructs displayed selective uptake inGRP-receptor rich pancreatic acne as well as the prostate tumour,while a reduced RES organ uptake was observed.143

http://www.clinicaltrials.gov/

mailto:[email protected]









Conclusions

A variety of nanomaterials are being investigated for thediagnosis, imaging, and therapy of cancers. The nanometre size ofthe particles is responsible for a diverse range of promisingproperties, including the absorption, scattering, fluorescence,photoacoustic, Raman enhancement, and magnetic resonance,making inorganic nanoparticles immediately suitable for exploita-tion in different imaging modalities. Their capability for thermo-therapy and PDT is another benefit, in addition to drug delivery.

Inorganic and organic nanoparticles are available for prefer-ential uptake and residence at tumour sites due to the EPR effectand RES-mediated uptake into tumour cells. Self-assembledpolymeric nanoparticles such as liposomes, micelles, polymero-somes, and spheres can be used for the pH- and temperature-triggered release of drug payloads at target sites. The use ofdifferent biodegradable polymers in surface tailoring andfunctionalization to reduce toxicity profiles and increase bloodcirculation times of inorganic nanoparticles, metallic and non-metallic, is also of potential interest. Some of the successfullyapproved and commercialized nanomedicines for the treatmentand detection of cancers are listed in Table 1 whereas many othersat the various stages of clinical trials are listed in Table 2.

The nanomaterials described earlier have already been thesubject of intensive research for clinical solutions in the field ofnanomedicine. Other very promising materials have also beenexploited for the in-vitro diagnosis, imaging, and treatment oftumours by thermotherapy, PDT, radiotherapy, BNCT, and drugdelivery. Such materials include CNTs and metallic/non-metallicNPs other than those described above, such as SiO2, TiO2, andNiO2. The effective modification and functionalization of thesenanoprobes with well-established chemistries will providefurther control of the localization, biodistribution, biocompati-bility, and efficacy of nanomaterial systems in vivo.

Although nanomaterials have found promising applicationsin biomedicine, especially in the imaging and treatment ofcancer, the successful adoption of this technology in clinicalpractice requires issues regarding long-term toxicity andeffective removal from body fluids to be addressed. Intelligentnanomaterials that are stable in the body, with longercirculation times and improved localization to diseased areas,without compromise to their therapeutic efficiency, will have tobe designed. Nanomaterials with these improved propertiesmust meet the demands of short-term therapeutics whileavoiding any adverse effects of long-term exposure. Furtherresearch is needed to ensure confidence in translatingnanomaterials into clinical applications.

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