Advanced Drug Delivery Reviews 65 (2013) 663–676

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Advanced Drug Delivery Reviews

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Therapeutic platforms based on gold nanoparticles and their covalent conjugateswith drug molecules☆

Leonid Vigderman, Eugene R. Zubarev ⁎Rice University, Department of Chemistry, 6100 Main Street, Houston, TX 77005, USA

☆ This review is part of the Advanced Drug Delivery Revnanoparticle platforms”.⁎ Corresponding author. Tel.: +1 713 348 3761; fax:

E-mail address: zubarev@rice.edu (E.R. Zubarev).

0169-409X/$ – see front matter © 2012 Elsevier B.V. Aldoi:10.1016/j.addr.2012.05.004

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 7 May 2012Available online 18 May 2012

Keywords:GoldNanoparticlesNucleic acidsDrug deliveryCovalent attachmentLigand exchangePhotothermal effect

This review will first look at the various covalent strategies that have been developed to attach drugs to goldnanoparticles as well as the strengths and limitations of such strategies. After examining general strategiesfor the synthesis of gold nanoparticles and their subsequent covalent functionalization, this review willfocus on nanoparticle conjugates for gene therapy, antibacterial, and anticancer applications including theuse of gold nanoparticles with intrinsically therapeutic properties. The effects of targeting and cellular uptakeof gold nanoparticles will also be discussed.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6632. Gold nanoparticle synthetic strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6643. General functionalization strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6644. Gold nanoparticles for gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665

4.1. Spherical nanoparticle conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6654.2. Delivery effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6664.3. Cellular response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6674.4. Photothermal release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

5. Gold nanoparticles for bactericidal applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6685.1. Amine-bound antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6685.2. Thiol-bound antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668

6. Gold nanoparticles for anticancer applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6696.1. Platinum-based conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6696.2. Conjugates with small organic molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6706.3. Gold nanoparticles as intrinsically therapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673

7. Towards clinical application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6748. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674

iews theme issue on “Inorganic

+1 713 348 5155.

l rights reserved.

1. Introduction

Gold nanoparticles have long been considered as possible drug deliv-ery vehicles applicable for therapy of a range of conditions. Among all ofthese systems, one of the most important elements is developing meth-odologies to properly functionalize the nanoparticles to have the desiredeffects. Countless reports exist of gold nanoparticles functionalized

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through the well-known gold–thiol covalent bond [1]. In addition totheir excellent surface functionalization chemistry, gold nanoparticlesare attractive for a number of reasons. They can be easily synthesizedin a variety of shapes including spherical, rod-like, core–shell, andmany others with sizes ranging from 1 nm to more than 100 nm [2–7].The optical and electronic properties of gold nanoparticles are highlyshape- and size-dependent [5,8], which has led to many biomedical ap-plications [9–17]. For instance, nanorods, nanoshells, and other goldnanocrystals are able to absorb light in the near infrared (NIR) region,which makes them particularly useful for the biological purposes giventhe high transparency of tissue to light of that wavelength [18]. In addi-tion, gold nanoparticles have generally shown themselves to be nontoxic,although there is some indication that non-covalently functionalizedparticles are not completely benign [19]. There are various strategiesfor using gold nanoparticles as a drug delivery vehicle, including sys-tems based on covalent binding, drug encapsulation [2,20–23], electro-static adsorption [24], and other non-covalent assemblies [25].However, this review will focus only on the direct covalent attachmentof several types of drugs to gold nanoparticles for use in gene therapy,bactericidal and anticancer applications (Fig. 1).

2. Gold nanoparticle synthetic strategies

It is first important to look at the general nanoparticle synthesisand functionalization strategies that could be useful for designing acovalent drug delivery system. The first choice that must be made iswhat shape and size of particle are most desired for the drug deliveryapplication (Fig. 1c). Detailed reviews covering the synthetic strategies forgold nanoparticles have been written, so only a brief reviewwill be givenhere [4,5,8]. Spherical nanoparticles are often chosen due to their facilesynthesis and have been thoroughly studied. Spherical nanoparticlescan be made with diameters ranging from 2 to 100 nm. The smallestdiameter nanoparticles are typically made by reduction of gold witha strong reducing agent in the presence of thiol molecules [26]. Particlesproduced by this method are immediately covalently functionalized asthey are synthesized. If a functional thiol is used in the initial step, itcan be further reacted using standard organic chemistry techniques[27–33]. Gibson et al. have used this technique successfully to synthe-size paclitaxel-functionalized gold nanoparticles [34] (Section 6.2).Alternatively, further covalent functionalization can be carried outwith a thiol place-exchange reaction with a separate functional thiol[20,35,36]. Though this technique is limited by the necessity for anexcess of thiol and does not completely replace the thiol monolayer, it

Fig. 1. a) Binding scheme in gold nanoparticle–drug conjugate

can be useful to install functional thiols as a base for chemical attach-ment of the desired molecules [37,38].

Small gold nanoparticles make excellent scaffolds for drug attach-ment, but they generally do not display a strong light absorbance inthe visible range. Gold nanoparticles of ca. 5 nm in diameter displaylarge optical absorption due to surface plasmon resonance whichmakes them very interesting as imaging and detection platforms [4].Larger nanoparticles in the range of 10–100 nm are usually synthe-sized by the Turkevich method and are capped with citrate ions [3].Efficient functionalization of this surface is then carried out by addinga thiol-containing capping agent. This simple procedure has led to thecovalent functionalization of nanoparticles with a wide variety of li-gands. Larger gold spherical nanoparticles scatter excellently, but ab-sorb light only in the visible range (520–550 nm), which may not beideal for biological application because of the low transparency of thebiological tissue to visible light [18]. On the other hand, gold nanorodsand nanoshells, among other morphologies, can be synthesized tohave a strong surface plasmon resonance in the near IR region,where the body is much more transparent [18]. Nanoshells, whichcontain a spherical dielectric core with a thin gold shell, have a citratecoated surface and can be functionalized in a similar way describedabove [39]. Gold nanorods, on the other hand, are generally synthesizedwith a cetyltrimethylammoniumbromide (CTAB) capping agent, whichmakes nanorods functionalizationmuchmore complicated and it is im-portant to deal with residual CTAB due to its inherent cytotoxicity[40,41]. Several groups have recently developed techniques for covalentfunctionalization of gold nanorods by carefully controlling their surfacechemistry [41–43]. Asmentioned earlier, both nanoshells and nanorodscan absorb NIR light which can be applicable for imaging or treatmentmodalities such as photothermal therapy of cancer (Section 6.3).

3. General functionalization strategies

Itmay be assumed that simply attaching a thiolmoiety (Fig. 1a) to theligand of choice is enough to functionalize any gold surface. However, asingle thiol group bound to a gold surface has some limitations, whichcan actually be useful under some circumstances. As mentioned earlier,thiols bound to a gold surface are subject to exchangewith those in solu-tion. This dynamic chemical composition can be utilized as a methodof drug release, as was demonstrated by Rotello et al., due to the highlevels of intracellular glutathione present in many types of cells [36]. Inthis case, glutathione partially displaces the thiolmonolayer on the nano-particle surface, thus releasing therapeutic cargo which is covalentlybound to themonolayer. This approach demonstrates thatmore complex

s. b) Types of drugs used. c) Types of nanoparticles used.

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systems may be desired if higher stability is required in the system(Fig. 1a). For instance, multiple thiols can be grafted to a singlemolecule,providing more chemical stability [44,45]. Similarly, multiple bindingpoints can be provided by utilizing a cyclical disulfide which may bindin a multidentate fashion with gold surface to increase the bindingstrength. Examples of this approach include utilizing a 1,2-dithiane endgroup, a technique demonstrated to improve DNA-gold nanoparticleconjugate stability [46]. Similarly, thioctic acid is a convenient anchorgroup due to the presence of the free carboxyl group which can easilybe coupled to free amines or alcohols and has been utilized by variousgroups to create highly stable covalent conjugates of gold nanoparticles[21,47–49]. Finally, in situ dithiocarbamate formation was shown to bea convenient method of grafting to a gold surface [50,51]. In this tech-nique a molecule with a free amino group is mixed directly with goldnanoparticles and is converted to a dithiocarbamate ligand by additionof carbon disulfide. The dithiocarbamate has a similar bidentate motifwith lower propensity for degradation compared to a single thiol. All ofthese binding motifs represent stronger binding than the traditionalgold–thiol bond and are clearly useful in situations where this addedstrength is desirable. However, theremay be timeswhenweaker bindingmay be required especially if this may function as part of a drug releasemechanism. This has been achieved often through the use of non-covalent interactions based on electrostatics, van der Waals forces,hydrophobic effect, hydrogen bonding, etc. which have been usedto attach or trap molecules in a gold nanoparticle conjugate, andshould be considered as a viable option which is outside the scopeof this review. However, if one is looking in the realm of covalent in-teractions, the bonding between gold and amines can fulfill such arole, although the bond strength is significantly lower, ~6 kcal/molcompared to 47 kcal/mol for thiols, and so it is not a truly covalentbond [52]. Overcoming this weaker binding to effect drug releasecould be substantially easier than in the case of thiol-bound ligands[53].

4. Gold nanoparticles for gene therapy

Gold nanoparticle-mediated delivery of DNA and RNA has beenone of the major research thrusts over the past several years. Becauseof the centrality of nucleic acids in biological systems, learning tomodulate the transcription and translation of DNA and RNA isexpected to make gene therapy an important treatment methodologyin the near future [54–56]. Thus, being able to deliver a wide variety ofoligonucleotides such as plasmids, double stranded DNA (dsDNA), singlestranded DNA (ssDNA), and single stranded RNA (ssRNA) could havelarge therapeutic benefits. However, due to their highly negative charge,these molecules cannot enter the cell without some sort of deliveryvehicle, a multitude of which are currently in use or in development[57–61]. Gold nanoparticles of a variety of morphologies have beenused for this purpose, including nanospheres, nanorods, and nanoshells.

Fig. 2. Preparation of various types of gold nanoparticle conjugates with DNA, LNA,

Many of these systems employ non-covalent interactions to attach DNAto the nanoparticles [62–65], but there are several examples whereDNA, generally modified with a thiol moiety, is directly attached tothe nanoparticle surface.

4.1. Spherical nanoparticle conjugates

One of the first examples of this strategy was demonstrated byMirkin et al. who were able to efficiently coat citrate-stabilized spher-ical nanoparticles with a dense layer of ssDNA molecules (Fig. 2)functionalized with either single or multiple thiol groups and usethem in gene silencing applications [45]. This conjugate was internal-ized efficiently by cells, notwithstanding the high negative chargedensity imparted by the DNA, and the structures were resistant todegradation by proteases and high levels of endogenous glutathionelevels which had been shown previously to be able to displace thiolsfrom the surface of gold nanoparticles. Binding of complementaryDNA to the grafted ssDNA was strengthened due to the dense packingof DNA around the nanoparticles which led to increased efficacy ofthe drugs in gene silencing applications. This system is an examplewhere the gold nanoparticle is used not only to facilitate drug delivery,but also function as a critical part of the drug action itself.More recently,further extensions and optimizations of these gold nanoparticle–DNAconjugates were performed. Chemically modified nucleic acids with in-creased stability and binding properties known as locked nucleic acids(LNA) were used instead of regular DNA (Fig. 2) [66]. This change ledto increased melting temperature of the DNA and greatly increasedeffectiveness as measured by a 50% decrease in the targeted proteinlevels as compared to a 30% decrease observed with regular DNA atthe highest dosage levels.

It is important to understand and predict the structures of nano-particle conjugates in order to tailor the structure to its particularfunction, something that is generally very difficult to do. Further studieson oligonucleotide-functionalized gold nanoparticles were conductedthat addressed someof these concerns, namely looking at the various fac-tors that affect the structure of the DNA shell around gold nanoparticlesas it relates to a variety of factors such as salt concentration and surfacecurvature of the nanoparticle [67]. The packing density of ssDNA wasfound to decrease as the size of the particles increases up to about60 nm, after which it reaches a plateau close to the packing density ofthe ssDNA on a flat substrate. Moreover, based on the model developedfrom spherical nanoparticles, it was possible to predict the amount ofDNA which could be loaded onto rod-shaped particles with a highdegree of accuracy. The covalent attachment of oligonucleotides togold nanoparticles was found to be extendable to the covalent attach-ment of RNA to the gold surface as well. Specifically, Mirkin et al. wereable to conjugate anti-firefly luciferase siRNA to gold nanoparticles(Fig. 2) and studied their gene knockdown potential compared to stan-dard cationic lipid transfection agents [68]. Because RNA is highly

siRNA, and combined peptide and DNA. Adapted from [45], [66], [68], and [71].

Fig. 3. a) Innate immune response of RAW 264.7 cells treated with DNA conjugated to gold nanoparticle and cationic lipid reagent as measured by interferon levels in terms ofi) relative abundance of IFN-β, and amounts of ii) IFN-β, iii) IL-1β, and iv) IL-6. Adapted from [73]. b) Increased cellular response of human PBMCs to gold nanoparticle–DNA conjugatesin a variety of different pathways. Adapted from [75].

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susceptible to nucleases it was necessary to develop a nuclease-freesynthesis of gold nanoparticles which was achieved by treating withdiethylpyrocarbonate and autoclaving. This citrate gold nanoparticleswere able to withstand this treatment, although this procedure mightnot be transferrable to other nanoparticle systems, especially goldnanorods which have a tendency to reshape into spheres at elevatedtemperatures. The tight packing of the siRNA due to its covalent couplingto the nanoparticles surfacewas found to increase its stability in serumasmeasured by a nearly eightfold increase in half-life as compared tomolecular RNA. These particles were also found to be more thantwice as effective as traditionally transfected RNA after a period offour days as measured by luciferase fluorescence, again presumably dueto the increased resistance to degradation of the siRNA-nanoparticleconjugates. It was similarly possible to regulatemRNA levels by utilizingsingle stranded DNA–LNA chimera functionalized gold nanoparticles[69]. By binding a small, reporter DNA sequence containing a fluores-cent group to the nanoparticle-bound oligonucleotides, the particlesgained the ability to detect mRNA when it is bound to the surface. Thefluorescence is strongly quenched when the reporter is in proximityto the gold nanoparticle and binding of complementary mRNA leadsto desorption of the reporter into solution with concurrent return of

the fullfluorescent signal. Itwas theprecise nature of the covalent bindingto the gold nanoparticle surface along with the well-studied nucleotidebinding chemistry that allowed for the development of multifunctional“theranostic” systems. However, it is necessary to mention that this sys-tem combines covalent and non‐covalent attachment of nucleotides togold nanoparticles as the first ssDNA strand is covalently attached to thenanoparticle surface while the reporter strand uses well-known DNA hy-bridization chemistry to build further functionality onto the nanoparticle[69].

4.2. Delivery effects

As mentioned previously, the high negative charge, which differssignificantly from most positively charged transfection agents, andthe fact that the free DNA does not enter cells by itself, naturallyleads to the question of how such particles are able to be internalizedby cells. It was found that significant binding of serum proteins uponexposure of the nanoparticles to cell culture medium was occurring[70]. By varying the amount of bound DNA molecules per particle, itwas determined that increased levels of bound DNA led to increasedrecruitment of proteins to the surface of the nanoparticles. Increased

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serum protein recruitment levels related directly to increased uptakeof the nanoparticles into cells as compared to octaethylene glycolcoated nanoparticles. Clearly, for the applications in gene therapy,increased delivery of oligonucleotides into the cell should lead to an in-creased response. Beyond simply having the capability to deliver theDNA or RNA into cells, increased gene regulation ability would beexpected if delivery to the nucleus could be achieved. Mirkin et al.further demonstrated that by co-binding nuclear targeting peptidessuch as TAT or NLS (Fig. 2), they could increase the perinuclear deliveryof the nanoparticle conjugates, although they were still unable to showintranuclear localization of their nanoparticles [71]. This led to an almost50% increase in gene silencing ability compared to the standard anti-sense DNA gold nanoparticles given the same degree of nanoparticleuptake.

4.3. Cellular response

When designing drug delivery systems, it is important to under-stand the cellular response to the delivery vehicle in order to determineunintended effects of the treatment [72]. In particular, modulating theimmune response to a delivery vehicle could have important conse-quence for the effectiveness of the system, especially in the case ofnucleotide delivery, in which case foreign DNA is known to elicit particu-lar types of immune response. Mirkin et al. demonstrated that the innateimmune response as measured by quantifying levels of interferon-β(IFN-β) produced in vitro in macrophages is modulated by the structureof the DNA nanoparticle conjugate [73]. Specifically, a greater surfacedensity of bound DNA leads to a lesser immune response in an approxi-mately linear fashion (double DNA density led to half the IFN-β levels).There was an approximately 25-fold difference in IFN-β levels ascompared to DNA transfected with standard cationic lipids whilemaintaining similar gene silencing ability (Fig. 3a). Thus, the tightpacking of oligonucleotides around the gold core due to the covalentbonding has clear advantages in modulating the cellular response tothe gold nanoparticles. This was shown to be more generally true inHeLa cells with the examination of genome-wide expression profilingstudies of cells treated with several types of gold nanoparticles [74]. Thecellular response proved to be minimal in the case of oligonucleotide-functionalized gold nanoparticles (ssDNA, dsNA, and dsRNA) in termsof either gene expression, cell-cycle progression, or apoptosis induction.However, uptake of unmodified citrate-capped gold nanoparticles wasfound to have a significantly negative cellular response including the

Fig. 4. a) Release of covalently bound EGFP plasmid DNA from gold nanorods with NIR laserAdapted from [80]. c) Selective release of DNA from two differently sized nanorods with N

beginnings of apoptosis, which was attributed to the weakly attachedcitrate capping agent as opposed to the strongly bound, densely func-tionalized oligonucleotide-nanoparticles. This finding demonstratesthe importance of having good control over surface functionality, afact which naturally lends itself to the use of covalent attachmentmethods. However, recent studies have shown that cellular responseof immortalized lineage-restricted cell lines such as HeLa cells or 293Tcells can differ from the response of more biologically relevant systemssuch as peripheral blood mononuclear cells (PBMC) (Fig. 3b) [75]. Fur-ther research needs to be done on the biological effects of nanoparticle–oligonucleotide complexes in in vitro as well as in vivo experimentsbefore they can be used for real-world gene delivery applications.

4.4. Photothermal release

In some cases, release of the DNA from the delivery vehicle may bedesired. A variety of examples exist where non-covalent interactionsare used to bind DNA to the nanoparticle allowing for later release ofthe DNA, such as functionalizing gold nanoparticles to carry a positivecharge to which DNA can be electrostatically adsorbed [62–65,76].Another strategy is to covalently attach a base layer of DNA to thenanoparticle surface and then use the self-assembling properties ofDNA to further build up the system [77,78]. One popularway of releasingthe DNA is by using light-responsive nanoparticles such as nanorods,nanoshells, or nanocages. For these nanoparticles, tuning of their sizescan lead to intense absorbance in the near-IR transparency window.Laser irradiation at the appropriate frequency for these structures cancause photothermal effects as well as the generation of “hot” electronsthat can trigger a release of DNA covalently attached to a nanoparticle[79]. Chen et al. employed this strategy by inducing the release of athiolated Enhanced Green Fluorescent Protein (EGFP) plasmid DNAattached to gold nanorods by femtosecond laser irradiation at thelongitudinal plasmon wavelength of the rods (Fig. 4a) [80]. This ap-proach led to melting of the rods with concurrent release of DNA,which was shown to be efficacious for the release of up to 80% ofthe DNA molecules conjugated covalently to the nanorods surface.EGFP fluorescence in cells indicated successful plasmid transfection(Fig. 4b). Wijaya et al. developed a different method for the attachmentof thiolated DNA [81] to gold nanorods and were able to demonstratethe nanorods' use for gene delivery purposes with a similar strategyas Chen et al. [80,82]. They extended this work by synthesizing rods oftwo distinctly different aspect ratios, and thus different longitudinal

irradiation. b) Cells exhibiting EFGP fluorescence after nanorod-mediated transfection.IR laser irradiation matched to their plasmon absorption. Adapted from [82].

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peaks, which were functionalized with different oligonucleotidestrands. When mixed together, irradiation at the specific longitudinalplasmon of one type of rods led to their selective melting and thus therelease of the corresponding DNA sequence (Fig. 4c). In both cases, afemtosecond pulsed laser is required to melt the gold nanorods and re-lease the DNA, presumably by breaking or oxidizing the gold–thiolbond, which represents a significant limitation of this technique. Cyto-toxicity due to either residual surfactant on the NR surface [41] or dueto photothermal heating are also potential issues with this tech-nique [79,80]. Still, Chen et al. showed that cytotoxicity could be miti-gated by controlling the laser fluence. Clearly the high strength of thegold–thiol covalent bond, while lending itself to applications wherethat stability is important, may create some limitations when it comesto the release of DNA from nanoparticles. Other systems for DNA releaseutilize the same covalent technique, but depend on breaking weaker,non-covalent interactions such as those between complementarystrands of DNA to effect the release of theDNA [83] or even drugs loadedinto DNA [84]. In these systems, gold nanoshells can be excited at lowerlaser powers to produce the necessary photothermal effect to releaseDNA hybridized to covalently attached strands. However, Braun et al.demonstrated that while low laser power release of oligonucleotidesmay be possible, somewhat higher power might be necessary to effectendosomal escape such that the therapeutic effects would be observed[85]. Thus, it is important to understand the desired properties of thegold nanoparticle–drug conjugates before choosing a design strategy.

5. Gold nanoparticles for bactericidal applications

Another important class of gold nanoparticle–drug conjugates hasbeen designed for antibacterial purposes. Many different antibioticcompounds have been developed and are used extensively, thereforeit is necessary to understand how gold nanoparticles can improve theefficacy of these molecules (Table 1). It is also important to examinehow the covalent attachment chemistry of the antibiotics to goldnanoparticles can affect the properties of the resulting conjugates.

5.1. Amine-bound antibiotics

Several reports exist on gold nanoparticles non-covalently boundwith antibiotics and exhibiting enhanced bactericidal activity, includingthose that involve simple mixing of citrate-capped gold nanoparticleswith an antiobiotic drug [86–89] and/or reduction of gold chloride inthe presence of antibiotics [90]. These specific antibiotics contain freeamino groups, which have a strong affinity to gold surface and generallylead to aggregation as evidenced by a color change from red to blue/purple. In this case the gold–amine binding is not truly covalent as inthe case of gold–thiol binding. Although these studies demonstratedan increased activity against some types of bacteria, another reportwas also published that suggested that such nanoparticle aggregatesdo not show a measurable enhancement of bactericidal activity [91]. Itwas suggested that in order for the nanoparticle conjugates to be useful,

Table 1Summary of gold nanoparticle–drug conjugates for bactericidal applications showing thedrugs which have been studied, any extra treatment which is required, the attachmentchemistry to the gold surface, the type of bacteria tested, and the corresponding references.

Drug/treatment Attachment Tested against Reference

Vancomycin Thiol VRE, gram-negative [93]Vancomycin/photothermal

Thiol Gram-positive, gram-negative,VRE, MRSA, PDRAB

[97]

Ampicillin,streptomycin,kanamycin

Amine(putative)

Gram-positive, gram-negative [90]

Cefaclor Amine Gram-positive, gram-negative [92]Toluidine blueO/photosensizitization

Thiol Gram-positive [98]

they should be functionalized in a way that prevents aggregation andleads to a high surface coverage on the surface of nanoparticles. Whilethe gold–amine interaction is weaker than a gold–thiol bond, it is stillproblematic to mix gold nanoparticles with molecules containing mul-tiple amino groups, i.e. aminoglycoside antibiotics, as it can result inphysical cross-linking and uncontrolled agglomeration of particles. Itis expected that by moving to a system with only a single aminogroup, the degree of aggregation could be reduced, although this maynot be the only factor affecting the stability of the system. Indeed, Raiet al. used cefaclor, a second-generation cephalosporin, containing asingle amino group as the capping agent as well as reducing agent inthe synthesis of the gold nanoparticle conjugates [92]. These hybridstructures showed no signs of aggregation, were stable enough to bepurified via dialysis, and thus could be properly characterized, indicatingthe usefulness of an amine–gold bindingmotif. The conjugates displayedan increased bactericidal activity compared to free cefaclor in both gram-positive and gram-negative bacteria, and they showed lower degrada-tion over time than the free antibiotic drug. Althoughprevious reports in-dicate that the gold nanoparticles are important only as scaffolds todensely bind antibiotics, Rai et al. suggest that the particle structureacts to amplify the cell membrane damage initiated by the antibiotic, in-creases the cell penetration into gram-negative bacteria, and disruptsbacterial DNA [92].

5.2. Thiol-bound antibiotics

Using the standard gold–thiol binding approach is also an excellentstrategy for binding antibiotics to gold nanoparticles. Early work by Guet al. reported on coating small 5 nm gold nanoparticles with vancomy-cin through a phase transfer mechanism [93]. They mixed an aqueoussolution of bis(vancomycin) cystamide, with a toluene solution of qua-ternary ammoniumbromide-coated gold nanoparticles, leading to theirphase transfer into aqueous solution (Fig. 5a). These nanoparticles weretested against a variety of vancomycin-resistant enterococci (VRE) aswell as some gram-negative bacteria like E. coli and showed excellentactivity against all bacterial strains even when vancomycin itself wasnot effective. The activity of these particles is explained by the effect ofmultivalency in which multiple vancomycin moieties present enhancedbinding to the terminal D-Ala-D-Ala moieties (Fig. 5b) [93–95]. It isalso important to note that the exact orientation of the vancomycin ishighly important to maximize the binding to bacteria [96], whichmakes covalent binding an ideal method to attach vancomycin to thenanoparticle systems. Similarly, bis(vancomycin)cystamide was used tofunctionalize polygonal particles with absorption in the near IR [97].Such particleswere shown to bind to a variety of bacteria types includingVRE, MRSA, and PDRAM and once bound, they can be used forphotothermal destruction of these bacterial types with high efficiency.This treatmentwas also found to be nontoxic to non-bacterial cells as de-termined by MTT assays with human epidermoid carcinoma epithelialcells. As an alternative to antibiotics, photosensitizers can also be cova-lently conjugated to gold nanoparticles and used to kill bacteria. Thelight-activated antimicrobial agent (LAAA) toluidine blue O (TBO) wascovalently coupled to the carboxyl group on tiopronin-functionalizedgold nanoparticles and found to be highly stable [98]. The exact amountof TBO and tiopronin per each gold nanoparticlewas determined by TGA,NMR, and other techniques, facilitated by the discrete structures formedvia covalent functionalization. These conjugates demonstrated four-folddecrease in minimum bactericidal concentration under white light orlaser illumination as compared to free TBO, which was attributed to anenhanced light absorbance of nanoparticles functionalized by TBO. It isconceivable that a direct covalent link is not required for the enhance-ment of photodynamic therapy as it is possible that gold nanoparticlesmay increase the levels of reactive oxygen species [99]. It must benoted that reports have also been made of gold nanorods that havebeen coated electrostaticallywith polyelectrolytes and further covalentlyfunctionalized for use in photothermal treatment [100] or combined

Fig. 5. a) Synthetic scheme for covalent gold nanoparticle–vancomycin conjugates. b) Illustration of multivalent binding of a nanoparticle to the terminal D-Ala-D-Ala moieties on abacterial membrane. Adapted from [93].

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photodynamic and photothermal treatment of bacteria [101]. For goldnanorods, direct covalent functionalization can be more challenging, asmentioned before, so a wider variety of functionalization schemes hasbeen developed.

6. Gold nanoparticles for anticancer applications

Gold nanoparticles carrying anticancer payloads represent anotherimportant class of covalent conjugates. A variety of therapeutic ap-proaches have been developed which take advantage of the well-researched covalent chemistry of gold nanoparticles (Fig. 6). Cancer isa major target of new therapeutics because of the drawbacks associatedwith current treatment strategies which include large systemic side-effects due to the non-specificity of many cancer drugs such as cisplatin[102], paclitaxel [103], tamoxifen [104], and doxorubicin [105]. Goldnanoparticles can address some of these issues in several ways.First, attachment to gold nanoparticles can lead to increased toxicitycompared to free drug. This is useful, but must ultimately be combinedwith increased specificity of the drug conjugate to cancer cells. This canbe mediated by passive targeting through enhanced permeability andretention effect (EPR) based on the size of gold nanoparticle conjugates[106] or by active targeting using antibodies or other targetingmoieties.In addition, the gold nanostructures themselves can have the ability tokill cells by virtue of intrinsic therapeutic effects such as photothermaleffect.

6.1. Platinum-based conjugates

The first type of drug-nanoparticle conjugates that will be discussedis based on platinum compounds. Several examples exist in which aplatinum(IV)-nanoparticle complex has been used as a drug delivery

vehicle and a prodrug for intracellular release of platinum(II) ions[107–109]. This strategy is beneficial because of the reduced reactivityof Pt(IV) compared to Pt(II) with biological agents and thus higher bio-availability and lower systemic toxicity [110]. Thus far, there exist two re-ports of platinum complexes covalently attached to gold nanoparticles,which utilize a Pt(IV) prodrug strategy. In the first report, Lippard et al.synthesized oligonucleotide-functionalized gold nanoparticles with aterminal dodecyl amine moiety and attached a carboxyl-containingPt(IV) complex by using standard carbodiimide coupling chemistrywith EDC and NHS (Fig. 7a) [110]. The platinum complex was designedto generate Pt(II) species by means of intracellular reduction of Pt(IV)by glutathione molecules. Subsequent loss of axial ligands would leadto the release of free cisplatin. It was confirmed that the conjugatewas taken into cells as was shown for regular DNA-functionalizednanoparticles. Significantly, the anticancer activity of the complexes toseveral cancer cell types was shown to be equal or greater than theactivity of cisplatin, although most of IC50 values were of similarmagnitude (Fig. 7b). It is important to note that for nanoparticledrug delivery carriers in general, even having the same activity asthe unconjugated drug would be an excellent result if the drug canbe delivered more efficiently to cancer site with fewer systemicside effects. A similar strategy was adopted byMin et al. for attachingPt(IV) prodrugs to gold nanorods [111]. Gold nanorods were first cova-lently functionalized with a diamino(polyethylene glycol) by using insitu dithiocarbamate formation on one terminus [112] and using the sec-ond amino group for coupling with a carboxyl-containing platinum(IV)compound. Cytotoxicity measurements based on the MTT assay forthree different cancer lines showed a much higher toxicity comparedto free cisplatin, with IC50 values ranging from 9 to 65 times lower. Thiswas correlated to increased intracellular levels of platinum ions in nano-rod-treated cells, indicating that the nanoparticle delivery vehicle can be

Fig. 6. Overviewof anticancer drugs covalently conjugated to gold nanoparticles. Dashed lines represent an omitted portion of a linker. Helical segment represents a DNA oligonucleotide.Wavy line indicates an oligo- or polyethylene glycol containing segment.

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quite important if it can enhance the cellular uptake of the drug viaendocytic pathways (Fig. 7c).

6.2. Conjugates with small organic molecules

Another type of common anticancer therapeutics is based on smallorganic molecules such as paclitaxel, docetaxel, tamoxifen, etc. Suchsmall molecules either already have reactive functional groups orcan be modified in a way which can be used to couple them to goldnanoparticles. As mentioned earlier, several basic strategies can beapplied to build the conjugate systems. First, one can synthesize a

Fig. 7. a) Coupling of Pt(IV) prodrug to oligonucleotide-functionalized gold nanoparticles. b) Ccell uptake and cisplatin release from gold nanorods conjugated with Pt(IV) prodrug [111].

functionalized nanoparticle to which the drug will be attachedthrough subsequent covalent chemistry. Gibson et al. used such anapproach to synthesize well-characterized paclitaxel-functionalizedgold nanoparticles and demonstrated the utility of covalent chemistryto accurately characterize the resulting conjugate (Fig. 8) [34]. In thiscase, the mercaptophenol-functionalized gold nanoparticles weresynthesized using the one-phase synthesis first developed by Brustet al. [113]. A carbodiimide-based esterification was used to attachpaclitaxel with oligoethylene glycol spacer. It was important to usethis flexible spacer, whichwas attached at the C-7 position of paclitaxel,to allow for proper NMR characterization. Using 1H NMR, along with

ytotoxicity of these nanoparticles to various cell lines. Adapted from [110]. c) Schematic of

Fig. 8. Covalent attachment of paclitaxel to mercaptophenol-functionalized gold nanoparticles. Adapted from [34].

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other characterization methods, it was possible to quantify the numberof paclitaxel molecules per nanoparticle, something that could bedifficult with non-covalent systems. Hwu et al. synthesized severalsystems based on paclitaxel functionalized at the C-2 position usinga phosphodiester linkage [114]. This compound was attached to aPEG linker containing a thiol group for attachment to citrate-capped gold nanoparticles or maleimide-coated magnetitenanoparticles. The phosphodiester bond was designed to be prefer-entially cleaved by phosphodiesterases in cancer cells and did notshow paclitaxel release in serum alone. Interestingly, while both thegold and magnetite nanoparticles demonstrated high water solubility,only magnetite nanoparticles showed any appreciable release of pacli-taxel in the presence of phosphodiesterase, which occurred only after4 days of treatment. However, this is not surprising given that the goldnanoparticles aggregated at the high salt concentration, unlike theirmagnetite counterparts. The authors claimed an IC50 value of 0.6 pMfor the magnetite nanoparticles, which would be a spectacular break-through considering that the cytotoxicity of regular paclitaxel rangesfrom 2 to7 nM [115], although the conditions for the MTT assay werenot described in detail. Still, it is clear that the solubility of the nanopar-ticle–drug complex as well as the free drug itself is highly important foranticancer applications. This is particularly true for paclitaxel, whichmustbe encapsulated in micelles of Cremophor El solvent that can have itsown harmful side effects.

Recently, Mirkin et al. have synthesized oligonucleotide–goldnanoparticle conjugates with paclitaxel in order to increase its solubilityin aqueousmedia [116]. Paclitaxelwas esterified through the C-2positionand coupled to thiolated, fluorescent DNA, which was immobilized ontocitrate-capped gold nanoparticles as described before. Paclitaxel, whichis highly insoluble in water, was solubilized by attaching to the goldnanoparticles, including in high-salt buffers. The gold nanoparticle–DNA–paclitaxel conjugates were shown to be internalized by thecells and showed increased cytotoxicity compared to free paclitaxel inMTT assays, and even showed toxicity to paclitaxel-resistant cell lines,suggesting that nanoparticle-mediated cell uptake could fight drug-efflux in drug-resistant cancers. However, comparisons were not madeto the standard delivery method (solution in Cremophor El), whichtypically displays IC50 values of 2–7 nM [115], compared to thevalues of 4–100 nM for the nanoparticle conjugates in pure water[116]. Still, the possibility for better targeting, increased effectivenessagainst the drug-resistant cells, and high solubility without CremophorEl could make this conjugate a promising paclitaxel-delivery platform.

El-Sayed and coworkers reported the first example of covalent cou-pling of tamoxifen to gold nanoparticles by attaching it to a thiolatedPEG linker [117]. This procedure was somewhat more involved thanthe attachment of paclitaxel because of the lack of a simple chemicalfunctionality, such as the free hydroxyl group in paclitaxel. Instead,the tertiary amino group was converted to a secondary amine, andthen alkylated with a PEG compound that was subsequently thiolatedthrough several steps. This tamoxifen–PEG–thiol was added to citrate-capped particles, which showed 2.7 times higher potency than that of

free tamoxifen. This was shown to be a consequence of the increasedcellular uptake rate of the nanoparticle–drug complex compared tofree tamoxifen and was not due to any multivalency effects, such asthose discussed for antibiotic–gold nanoparticle complexes [93]. Parti-cle uptake was mediated by binding to estrogen receptors, which areoverexpressed in 75–80% of breast cancers, suggesting that this strategycould be used to selectively target breast cancers compared to regularcells [117]. Another type of anticancer drug that has been successfullyconjugated to gold nanoparticles was a polypeptide drug, Kahalilide F[118]. This thirteen residue peptide drug was synthesized with a cyste-ine modification that could covalently bind to citrate-capped goldnanoparticles, although it was demonstrated that the amount of pep-tide loaded onto the nanoparticles was much higher than expectedfrom a purely covalent binding. It was suggested that a multilayer coat-ing of the peptide molecules onto the nanoparticles was responsiblefor the observed increased loading of the drug. The increased cellularuptake of the nanoparticle system led to a higher anticancer activitywhen compared to the free peptide. However, it is important to notethat one of the benefits of covalent structures is that they are easilypurified and then can be analyzed directly. In this case, it is not clearif the free peptide was still present in the system, which could make itdifficult to accurately determine the actual bioactivity of the conjugate.

Another interesting example of anticancer drug that has beenconjugated to gold nanoparticles is 5-fluorouracil, a drug which in-hibits DNA and RNA synthesis [119]. Agasti et al. synthesizedfluorouracil-functionalized gold nanoparticles of 2 nm size [35].The drug was incorporated through a terminal UV-photocleavableortho-nitrobenzyl (ONB) group. These particles were synthesized bythe place exchange reaction of pentanethiol-capped gold nanoparticleswith the fluorouracil-containing thiol as well as a zwitterionic thiol forincreased solubility. Irradiation of nanoparticles with 365 nm UV lightresulted in controllable release of the drug, which exerted its cytotoxiceffect only when it was released from the particle surface. The lack oftoxicity of the conjugates before the photo-induced release of the ligandcould be very beneficial for targeted treatment of cancer in vivo in coop-eration with the passive targetingmechanism (EPR effect). Interestingly,unlikemost of the other systems, the zwitterionic thiols reduce the cellu-lar uptake [20], indicating that high uptake is not always necessary in thedesign of gold nanoparticle–drug conjugates. This was also shown to bethe case for delivery of a phthalocyanine (Ptc) drug for photodynamiccancer therapy, wherein release of the drug outside of the cell led tomore efficient photodynamic therapy compared to internalized covalentnanoparticle–Ptc conjugates [120]. However, this may have been due topoor uptake of the Ptc particles as a result of their PEG coating [41] be-cause the release of drug can bemediated by cell uptake as demonstratedby Rotello and coworkers [36]. In this report, gold particles were func-tionalized by a place-exchange reaction of octanethiol-capped goldnanoparticles with a cationic thiol and a dye-containing thiol (Bodipy).The presence of the cationic thiols leads to higher cellular uptake of thenanoparticle conjugate as opposed to the zwitterionic systemmentionedearlier. It was proven that intracellular glutathione caused the release of

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nanoparticle-bound thiols as confirmed by large fluorescence due to freeBodipy dye, which is quenched when attached to the gold nanoparticlessurface.

Recently, gold nanoparticles have been covalently grafted withdoxorubicin for treatment of drug-resistant breast cancer [48]. Doxoru-bicin was attached through a thioctic acid-PEG linker to the surface of30 nm citrate-capped gold nanorods through a hydrazone group(Fig. 9a). The hydrazone is stable at normal physiological pH, but ishydrolyzed at the reduced pH levels found in endosomes [121].These nanoparticles inhibited the growth of drug resistant cancercells by allowing a large uptake of the particles, followed by acid-sensitive release from endosomes which led to a rapid increase indoxorubicin concentration inside the cancer cell (Fig. 9b). This overcomesdrug efflux from the cells which is responsible for drug-resistance [122],

Fig. 9. a) Structure of gold nanoparticles covalently bound to doxorubicin through an acidendosomes. c) Cell viability of drug-resistant breast cancer cells after 24 h. d) Viability afte

and increases the cytotoxicity compared to free drug (Fig. 9). Similar ef-fects were observed in the gold nanoparticle–DNA–paclitaxel conjugatedescribed earlier [116]. A hydrazone-linkeddoxorubicin thiolwas also at-tached to small gold nanoparticles (ca. 2 nm diameter) and tested intumor cylindroids, a three-dimensional in vitro cancermodel. This systemdiffered in that nanoparticle surface was engineered to carry a densenegative or positive charge, which was found to have a large effect onnanoparticle uptake in the tumor cylindroids [123] with positivelycharged particles having the highest degree of uptake, as has been seenbefore [124]. Doxorubicin releasewas visualized through itsfluorescence,though cytotoxicity was not determined. Given that differences betweenthe studies include nanoparticle size, synthesis, and functionalizationmethod it is difficult to draw conclusions and more detailed studies inthis area would be useful.

-labile hydrazone linkage. b) Uptake of nanoparticles and release of doxorubicin fromr 48 h. Adapted from [48].

Fig. 10. a) Bright field optical microscopy image of cancer cell treated with gold rods functionalized by cationic thiol. b) TEM image of a section of a cell treated with cationic goldnanorods. Adapted from [41].

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6.3. Gold nanoparticles as intrinsically therapeutic agents

In the synthesis of covalently functionalized conjugates designedto destroy cells, gold nanoparticles can act as more than just drug de-livery vehicles. The unique properties of these systems mean that at-tachment of a drug may not be necessary. One area of research thathas received a large amount of attention is the use of near-IR activegold nanostructures including nanorods, nanoshells, and nanocages.Several reviews have already been published on this topic and thuswill not be discussed in great detail here [2,18,39,40,125]. Briefly, alaser irradiation of anisotropic gold nanocrystals results in a dissipa-tion of energy in the form of heat, which may result in cell death. Im-portantly, for application of such nanostructures in vivo, it is generallynecessary to properly functionalize the nanoparticle surface to mitigatethe cytotoxic effects and to target the particles. Vigderman et al. recent-ly reported a cationic thiol exchange for the cytotoxic CTAB on the sur-face of as-synthesized gold nanorods [41,126]. This surfactant exchangeallowed for the loading of more than 2 million gold nanorods per cell,which were visualized by optical (Fig. 10a) and transmission electronmicroscopy (Fig. 10b). This number is at least 10 times higher thanthat reported for CTAB-coated rods and therefore such high loadingmay lead to more efficient photothermal therapy of cancer cells.

El-Sayed et al. demonstrated that covalent attachment of a nucleartargeting peptide to gold nanorods leads to their rapid uptake and sub-sequent localization in both the cytoplasm and the nucleus [127]. It waslater shown that spherical gold nanoparticles conjugated with nucleartargeting peptides cause damage to DNA and lead to cytokinesis arrestresulting in apoptosis of cancer cells [128]. The mechanism of thisprocess was not fully understood, however, and the process wasnot evident at lower nanoparticle concentrations [129]. More recently,

Fig. 11. Bombesin-conjugated gold nanorod uptake into GRP-receptor positive cells through

it was demonstrated that masking the cationic charge of nanoparticlesby supramolecular assembly of a cucurbit [7] uril host over thediaminohexane guest covalently attached to 2 nm gold nanoparticlesleads to a loss of their cytotoxic effect [130]. After the uptake of thesenanoparticles, cells can be treated with 1-adamantylamine, whichmore strongly binds the cucurbit [7] uril hosts, thereby releasing freecationic gold nanoparticles into the cytosol where they exert their cyto-toxic effect.

Evidently, for such systems to be effective in cancer treatment,gold nanostructures must be targeted to cancer cells and avoid accumu-lation in healthy tissue. Asmentioned earlier, the enhanced permeabilityand retention properties of some tumors lead to the preferential accumu-lation of nanoparticles in cancer tissue due to the leaky vasculature pre-sent in these areas [106,131]. This is a passive targeting mechanismwhich could be applicable to most nanoparticle drug delivery systems.Active targeting involves covalent attachment of molecules which canbind specifically to cancer cells. Small-molecule based targeting systemscan be used, such as tamoxifen-coated particles discussed earlier, whichbind specifically to breast cancer cells. In addition, it is possible to usefolic acid [132] and polyunsaturated fatty acids [133]. Biopolymers canalso be utilized for that purpose as was exemplified by hyaluronic acid[133,134], various peptides [133], and oligonucleotides [133,135]. Inaddition, gold nanorods covalently functionalized by bombesin pep-tides, a gastrin-releasing peptide (GRP), were shown to bind withhigh affinity to breast cancer cells and be taken up by the cells via aGRP receptor-mediated endocytosis process (Fig. 11) [49,136]. Finally,gold nanoparticles can be conjugated to antibodies, which would beexpected to increase the specificity of their delivery to cancer in invitro and in vivo experiments. Antibodies are often targeted to over-expressed binding domains on cancer cells as compared to regular cells

receptor-mediated endocytosis and the lack of uptake into GRP-receptor negative cells.

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[137]. Such a strategy has often been achieved through covalent modifi-cation of gold nanoparticles and examples existwith various nanoparticletypes including nanorods [138,139], nanoshells [140,141], and nanocages[142].

7. Towards clinical application

Much of theworkwhich has been described up to this point is limitedto the in vitro testing of therapeutic gold nanoparticle systems. In order totransition these technologies towards clinical applications, it will benecessary to understand their activity in vivo in both animal modelsand eventually in humans. It is possible that some systems which maybe effective in vitro may not only lose their activity in vivo, but mayalso lead to unexpected side effects. While a detailed examination ofthese effects is outside the scope of this review, it is important tomention the general factors which must be considered. One of themain issues is the biodistribution of the gold nanoparticles withtime, which has been reviewed in detail [143,144]. The EPR effect isoften cited as a passive targeting mechanism leading to increased accu-mulation of nanoparticles inside of solid tumors. However, it is importantto address the extent of this effect as well as the possible buildup ofparticles in other organs. Accumulation is commonly seen in theliver, spleen, and kidneys, which are functioning to clear the goldnanoparticles from the body, but can also occur in other areas includ-ing the brain and lungs, which may or not be desirable [143]. Themethod of nanoparticle administration could also influence this bio-distribution [143]. Furthermore, the nanoparticle size and the surfacefunctionality can have a large effect. Active targeting through antibodies,peptides, or other methods is likely to be crucial, as mentioned earlier,butmight not preclude non-specific accumulation in the liver, spleen, etc.

The extent of nanoparticle targeting which is acceptable will de-pend on the application. For instance, laser-induced hyperthermictreatment of tumors will necessitate delivery of nanoparticles to thetumor site, but accumulation of nanoparticles in other areas maynot be overly problematic given highly localized laser-treatmentarea. On the other hand, imperfect delivery of particles functionalizedwith toxic anticancer drugs could have more side effects. Finally, thestudy of nanoparticle pharmacokinetics including the clearance ofnanoparticles from the body after treatment is still in its infancy[144]. Understanding this clearance and any long-term nanoparticletoxicity, will be necessary for their clinical application. Due to thelarge effects of size, shape, and surface functionalization on the invivo activity of therapeutic gold nanoparticles, it will be necessary tocarefully evaluate each new system before it can be applied clinically.

8. Conclusions

Covalently modified gold nanoparticles have attracted a great dealof interest as drug delivery vehicles. Their predictable and reliablesurface modification chemistry, usually through gold–thiol binding,makes the desired functionalization of nanoparticles quite possibleand accurate. A variety of therapeutic molecules have been attached inthismanner including various oligonucleotides for gene therapy, bacteri-cidal compounds, and anticancer drugs. Anisotropic gold nanoparticlesalso exhibit optical and chemical properties that can make them thera-peutic in their own right, and yet still require proper surface func-tionalization to make them useful. The ability to properly purify andaccurately analyze the structure of such hybrid inorganic–organic parti-cles shouldmake them an attractive system for continued drug develop-ment. Transitioning to testing these gold nanoparticle–drug conjugatesfrom in vitro to in vivomodels will be an important step for the future re-search, and has already been undertaken in some cases. It is expectedthat enhancing in vivo targeting of nanoparticle–drug conjugates and un-derstanding their biodistributionwill be highly important. Just as impor-tantly, understanding and learning how to control the intracellular fate ofnanoparticles will be necessary for further development in this field.

Acknowledgement

Financial support by the NSF (DMR-1105878) is gratefully acknowl-edged by the authors.

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