In vivo tumor targeting and spectroscopic detectionwith surface-enhanced Raman nanoparticle tagsXimei Qian1, Xiang-Hong Peng2, Dominic O Ansari1, Qiqin Yin-Goen3, Georgia Z Chen2, Dong M Shin2,Lily Yang2,4, Andrew N Young3, May D Wang5 & Shuming Nie1,2

We describe biocompatible and nontoxic nanoparticles for in vivo tumor targeting and detection based on pegylated gold

nanoparticles and surface-enhanced Raman scattering (SERS). Colloidal gold has been safely used to treat rheumatoid arthritis

for 50 years, and has recently been found to amplify the efficiency of Raman scattering by 14–15 orders of magnitude. Here

we show that large optical enhancements can be achieved under in vivo conditions for tumor detection in live animals. An

important finding is that small-molecule Raman reporters such as organic dyes were not displaced but were stabilized by thiol-

modified polyethylene glycols. These pegylated SERS nanoparticles were considerably brighter than semiconductor quantum

dots with light emission in the near-infrared window. When conjugated to tumor-targeting ligands such as single-chain variable

fragment (ScFv) antibodies, the conjugated nanoparticles were able to target tumor biomarkers such as epidermal growth factor

receptors on human cancer cells and in xenograft tumor models.

The development of biocompatible nanoparticles for in vivo molecularimaging and targeted therapy is an area of considerable currentinterest across a number of science, engineering and biomedicaldisciplines1–9. The basic rationale is that nanometer-sized particleshave functional and structural properties that are not available fromeither discrete molecules or bulk materials1–3. When conjugatedwith biomolecular targeting ligands such as monoclonal antibodies,peptides or small molecules, these nanoparticles can be used to targetmalignant tumors with high specificity and affinity10–13. In the‘mesoscopic’ size range of 10- to 100-nm diameter, nanoparticlesalso have large surface areas for conjugating to multiple diagnostic(e.g., optical, radioisotopic or magnetic) and therapeutic (e.g., anti-cancer) agents. Recent advances have led to the development ofbiodegradable nanostructures for drug delivery14–18, iron oxide nano-crystals for magnetic resonance imaging19,20, quantum dots for multi-plexed molecular diagnosis and in vivo imaging21–24, and nanoscalecarriers for short interfering RNA (siRNA) delivery25,26.

We report a class of nontoxic nanoparticles for in vivo tumortargeting and spectroscopic detection based on the use of pegylatedcolloidal gold and surface-enhanced Raman scattering (SERS). Colloi-dal gold has been safely used to treat rheumatoid arthritis for half acentury27,28, and recent work indicates the pegylated gold nanoparticles(colloidal gold coated with a protective layer of polyethylene glycol orPEG) exhibit excellent in vivo biodistribution and pharmacokineticproperties upon systemic injection29–31. In contrast to cadmium-containing quantum dots and other toxic or immunogenic nanopar-ticles, gold colloids have little or no long-term toxicity or other adverseeffects in vivo32,33. Equally attractive are the optical properties of

colloidal gold nanoparticles, which are able to amplify the Ramanscattering efficiencies of adsorbed molecules by as much as 1014- to1015-fold, allowing spectroscopic detection and identification of singlemolecules under ambient conditions34,35. Since its initial discovery in1997 (refs. 36,37), single-molecule and single-nanoparticle SERS hasbeen studied extensively, both for examining its enhancement mechan-isms and for applications in ultrasensitive optical spectroscopy38–42.In this work, we show that this enhancement effect can be realizedunder in vivo conditions for tumor detection in live animal models. Aserendipitous finding is that various Raman reporters (that is, spectro-scopic encoding chromophores adsorbed on the surface of colloidalgold) are not displaced by thiol-modified PEG, a nontoxic and hydro-philic polymer that is commonly used to improve drug biocompat-ibility and systemic circulation14–17. In fact, the thiol-PEG–coated goldparticles become so stable that their SERS signals do not change undervery harsh conditions including strong acids (0.1 M HCl), strong bases(0.1–1 M NaOH), concentrated salts (1–2 M NaCl) and organicsolvents (methanol, ethanol and dimethylsulfoxide). In comparisonwith near-infrared–emitting quantum dots22,23, our pegylated SERSnanoparticles were 4200 times brighter (on a particle-to-particle basis)under identical experimental conditions, whereas their hydrodynamicdiameters were about 4 times larger (64 times by volume). The PEGcoating layer also allows efficient conjugation to tumor-targetingligands; for example, we conjugated SERS nanoparticles with an ScFvantibody (MW ¼ 25 kDa) for in vitro and in vivo tumor targeting. Thissmall antibody fragment recognizes the epidermal growth factorreceptor (EGFR)43,44 that is overexpressed in many types of humanmalignant tumors. The results demonstrate highly specific recognition

Received 16 July; accepted 3 December; published online 23 December 2007; doi:10.1038/nbt1377

1Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, 101 Woodruff Circle, Suite 2001, Atlanta, Georgia 30322,USA. 2Winship Cancer Institute, 3Department of Pathology and Laboratory Medicine, 4Department of Surgery, Emory University School of Medicine, 1365 Clifton Road,Atlanta, Georgia 30322, USA. 5Departments of Biomedical Engineering and Electrical and Computer Engineering, Georgia Institute of Technology, 313 Ferst Drive, UAWhitaker Building 4106, Atlanta, Georgia 30332, USA. Correspondence should be addressed to S.N. (snie@emory.edu).

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and Raman detection of human cancer cells, as well as active targetingof EGFR-positive tumor xenografts in animal models.

RESULTS

Design and characterization of pegylated SERS nanotags

Figure 1a shows the design and preparation of pegylated goldnanoparticles with embedded spectroscopic tags and their schematic

structures. Also shown are their opticalabsorption spectra (Fig. 1b), transmissionelectron microscopy (TEM) structures(Fig. 1c), and hydrodynamic size data(Fig. 1d). The original gold particles (60-nmdiameter) were encoded with a Raman repor-ter and stabilized with a layer of thiol-PEG.Previous research has shown that gold nano-particles with a core size of B60–80 nm aremost efficient for SERS at red (630–650 nm)and near-infrared (785 nm) excitations41.This spectral region is known as a ‘clearwindow’ for optical imaging because thehemoglobin (blood) and water absorptionspectra are minimal45. Beyond the SERSeffect, we also achieved resonance Raman

enhancement by using reporter molecules with electronic transitionsat 633 nm or 785 nm. The gold plasmonic resonance spectra remainedessentially unchanged (o1-nm red shifts), even when the goldparticles were coated with a large number of molecules (about1.4–1.5 � 104) and stabilized with a layer of PEG molecules(Fig. 1b). We note that single-molecule SERS occurs only at specialactive sites or junctions36–39, and it is not required for tumor

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Figure 2 Comparison of pegylated SERS nanoparticles and

near-infrared-emitting quantum dots in the spectral region

of 650–750 nm. (a,b) Optical absorption (red curves) and

emission spectra (blue curves) of SERS nanoparticles (a)

and QD705 (b) under identical experimental conditions.

(c,d) SERS and fluorescence images of single goldnanoparticles (c) and single quantum dots (d) dispersed on

glass slides and acquired under the same conditions

(EM-CCD camera, 633 ± 3 nm excitation, and 655 nm

long-pass emission). The speckles (d) are optical

interference fringes, which become visible at low light levels.

(e,f) Line plots (e) and statistical analysis (f) of the brightness differences between SERS nanoparticles and quantum dots. The corresponding signals are

highlighted by red and blue lines in the images c and d. S.d. in the Raman and quantum dot signals are indicated by error bars.

Figure 1 Design, preparation and properties of

pegylated gold nanoparticles for in vivo tumor

targeting and spectroscopic detection.

(a) Preparation and schematic structures of the

original gold colloid, a particle encoded with a

Raman reporter, and a particle stabilized with

a layer of thiol-polyethyleneglycol (thiol-PEG).

Approximately 1.4–1.5 � 104 reporter molecules(e.g., malachite green) are adsorbed on each

60-nm gold particle, which is further stabilized

with 3.0 � 104 thiol-PEG molecules. (b) Optical

absorption, (c) transmission electron microscopy

(TEM); and (d) dynamic light scattering size data

obtained from the original, Raman-encoded, and

PEG-stabilized gold nanoparticles as shown in a.

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Control nanotag(with nonspecific lgG)

Control nanotag(without ScFv)

EGFR-negativecell

EGFR-positivecell

Pure tag(solution)

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EGF receptor

ScFv EGFR antibody

HS-PEG-CONH-ScFv

PEG-SH shell

a

Figure 3 Cancer cell targeting and spectroscopic detection by using

antibody-conjugated SERS nanoparticles. (a) Preparation of targeted SERS

nanoparticles by using a mixture of SH-PEG and a hetero-functional PEG

(SH-PEG-COOH). Covalent conjugation of an EGFR-antibody fragment

occurs at the exposed terminal of the hetero-functional PEG. (b) SERS

spectra obtained from EGFR-positive cancer cells (Tu686) and from EGFR-

negative cancer cells (human non-small cell lung carcinoma NCI-H520),

together with control data and the standard tag spectrum. All spectra were

taken in cell suspension with 785-nm laser excitation and were corrected

by subtracting the spectra of nanotag-stained cells by the spectra of

unprocessed cells. The Raman reporter molecule is diethylthiatri-

carbocyanine (DTTC), and its distinct spectral signatures are indicated

by wave numbers (cm–1).

detection. In fact, with a large number of reporter molecules adsorbedon the particle surface, the achieved total signal intensities exceededthat of single-molecule SERS. The PEG coating was clearly observed asa thin white layer of B5 nm by TEM negative staining, whereas theparticle’s ‘wet’ hydrodynamic diameter increased by 20 nm afterpegylation, as measured by hydrodynamic light scattering (DLS) inbuffered saline. At a core particle size of 60 nm, a minimum of 30,000thiol-PEG molecules (MW ¼ 5 kDa) per gold nanoparticle wasnecessary to achieve complete protection against salt-induced colloidaggregation. This surface coverage corresponded to a footprint area ofB0.35 nm2 per PEG molecule, in agreement with that reported byanother group46 for thiol-PEG adsorbed on colloidal gold in a brushconformation. After this shielding layer was completed, the use ofadditional thiol-PEG up to 10- to 20-fold excess had little effect on thecoating thickness, as measured by both TEM and DLS.

We investigated the stability of pegylated gold nanoparticles bymeasuring their SERS signals (both frequency and intensity) under awide range of conditions including concentrated salts (1–2 M), strongacids (0.1 M HCl), strong bases (1 M NaOH) and organic solvents(methanol, ethanol and dimethyl sulfoxide or DMSO) (Supplemen-tary Figs. 1 and 2 online). In the absence of PEG protection, the goldnanoparticles rapidly ‘crash’ (that is, aggregate and precipitate) underthese harsh conditions. With PEG protection, the gold particles andtheir SERS spectra are completely stable, with only minor relativeintensity changes at pH 1–2 (due to protonation and relative orienta-tion changes of the reporter molecule on the gold surface)47.

The observation of intense SERS signals with a thiol-PEG coating iscounterintuitive because the reporter molecules on the particle surfaceare expected to be displaced by thiol compounds (which are known tospontaneously form a monolayer on gold)46. Also surprising is that arange of Raman reporters such as crystal violet, Nile blue, basicfuchsin and cresyl violet were not displaced by thiol-PEG, evenwithout an anchoring isothiocyanate (-N ¼ C ¼ S) group. In fact,the SERS signals of crystal violet and other dyes were stronglyprotected by thiol-PEG, and were stable for 411 months at 25 1C.A common feature for these reporter dyes is that they are positivelycharged and contain delocalized pi-electrons. In contrast, organic dyeswith negative charges such as sodium fluorescein gave only weak andunstable SERS signals on the citrate-stabilized gold particles (alsonegatively charged) used in this work. Thus, we believe that bothelectrostatic interactions and delocalized pi-electrons are importantfor strong dye adsorption35, likely at gold surface sites that do notcompete with thiol-PEG adsorption. It is also possible that thethiol-PEG layer protected and stabilized the adsorbed reporter dyesby steric shielding and electronic interactions.

For cellular and in vivo imaging applications, we comparedthe excitation and emission spectral properties of pegylated gold

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nanoparticles and near-infrared quantum dots. The gold nanoparticlesprovided much richer spectroscopic information, and their emissionpeaks (full width at half maximum FWHM ¼ 1–2 nm) were20–30 times narrower than those of quantum dots (FWHM ¼40–60 nm) (Fig. 2a,b). Under identical experimental conditions, thepegylated gold particles were 4200 times brighter (on a particle-to-particle basis) than near-infrared–emitting quantum dots in thespectral range of 650–750 nm (see single particle images inFig. 2c,d, and statistical data in Fig. 2e,f). The pegylated goldnanoparticles had hydrodynamic sizes of B80 nm (diameter) andwere completely nontoxic to cultured cells when tested over 3–6 d. Inthe absence of surface-enhanced Raman signals, near-infrared goldnanoshells have recently been used as a contrast enhancement agentfor optical coherence tomography as well as for photothermal tumorablation48, but this approach does not provide molecular signaturesfor spectral encoding or multiplexing.

Spectroscopic detection of cancer cells

For cancer cell detection, targeted gold nanoparticles were prepared byusing a mixture of thiol-PEG (B85%) and a heterofunctional PEG(SH-PEG-COOH) (B15%). The heterofunctional PEG was covalentlyconjugated to an ScFv antibody (MW ¼ 25 kDa), a ligand that bindsto the EGFR with high specificity and affinity49 (Fig. 3a). UV-Visabsorption and fluorescence data indicated that B600 copies of theScFv ligand were conjugated to each gold nanoparticle. Figure 3bshows cellular binding and SERS spectra obtained by incubating theScFv-conjugated gold nanoparticles with human carcinoma cells. Thehuman head-and-neck carcinoma cells (Tu686) were EGFR positive(104–105 receptors per cell)50, and were detected by strong SERSsignals. In contrast, the human non-small cell lung carcinoma(NCI-H520) did not express EGFR, showing little or no SERS signals.To confirm targeting specificity, we preincubated Tu686 cancer cells ina tenfold excess of free ScFv EGFR antibody, and then added EGFR-labeled SERS nanoparticles for competitive binding studies. Afterthree rounds of washing, the cells showed only negligible SERS signals.We also tested and confirmed the binding specificity of SERSnanoparticles conjugated to secondary antibodies in a two-sitesandwich format. For control cancer cells (EGFR negative) and controlnanoparticles (plain PEG-coated nanotags and PEG-nanotagsfunctionalized with a nonspecific IgG antibody), the spectra showed

a weak but reproducible background (Fig. 3b). This low backgroundwas probably caused by residual SERS nanoparticles in the mixingsolution that were not completely removed during cell isolation,but there could also have been contributions from nonspecificbinding or nanoparticle internalization. We used an infrared dye(diethylthiatricarbocyanine or DTTC) as a spectroscopic reporter,and achieved surface-enhanced resonance Raman scattering (SERRS)at 785-nm excitation. This resonance condition did not lead tophotobleaching because the adsorbed dyes were protected fromphoto-degradation by efficient energy transfer to the metal particle.The resonance effect can further increase the surface-enhanced Ramansignals by 10- to 100-fold36, sensitive enough for Raman molecularprofiling studies of single cancer cells (Supplementary Fig. 3 online).This sensitivity is important for investigating the heterogeneousnature of cancer tissue specimens removed by surgery, and circulatingtumor cells captured from peripheral blood samples. Single-cellprofiling studies are of great clinical significance because EGFR is avalidated protein target for monoclonal antibody and protein-kinase–based therapies43,44.

In vivo tumor targeting and detection

A major challenge in in vivo optical imaging and spectroscopy is thelimited penetration depth, due to light scattering and absorption inanimal tissues51. To determine whether SERS spectra can be acquiredfrom pegylated gold nanoparticles buried in animal tissues, weinjected small dosages of nanoparticles into subcutaneous and deepmuscular sites in live animals. Highly resolved SERS signals wereobtained from subcutaneous as well as muscular injections (Fig. 4).The in vivo SERS spectra were identical to that obtained in vitro (salinesolution), although the absolute intensities were attenuated by 1–2orders of magnitude. Based on the high signal-to-noise ratios, weestimated that the achievable penetration depth was about 1–2 cm forin vivo SERS tumor detection (also confirmed by deep tissueinjection studies).

For in vivo tumor targeting and spectroscopy, the gold nanoparti-cles conjugated with the ScFv antibody were injected systemically(through tail veins) into nude mice bearing a human head-and-necktumor (Tu686). Figure 5 shows SERS spectra obtained 5 h afternanoparticle injection by focusing a near-infrared, 785-nm laser beamon the tumor site or on other anatomical locations (e.g., the liver or aleg). Substantial differences were observed between the targeted andnontargeted nanoparticles in the tumor signal intensities, whereas theSERS signals from nonspecific liver uptake were similar. This resultindicates that the ScFv-conjugated gold nanoparticles were able totarget EGFR-positive tumors in vivo. Time-dependent SERS datafurther indicate that nanoparticles were gradually accumulated in

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Figure 4 In vivo SERS spectra obtained from pegylated gold nanoparticles

injected into subcutaneous and deep muscular sites in live animals. The

injection sites and laser beam positions are indicated by circles on the

animal. A healthy nude mouse received 50 ml of the SERS nanoparticles

tags (1 nM) by subcutaneous (1–2 mm under the skin) or muscular (B1 cm

under the skin) injection. The subcutaneous spectrum was obtained in 3 s,

the muscular spectrum in 21 s, and the control spectrum (obtained in an

area away from the injection site) also in 21 s. The reference spectrum (red)was obtained from the SERS nanoparticles in PBS solution in 0.1 s. The

spectral intensities are adjusted for comparison by a factor (�1, �30 or

�210) as indicated. The Raman reporter molecule is malachite green, with

spectral signatures at 427, 525, 727, 798, 913, 1,169, 1,362, 1,581

and 1,613 cm–1. These features are distinct from the animal skin Raman

signals (see the skin spectrum). Excitation wavelength, 785 nm; laser

power, 20 mW.

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the tumor for 4–6 h, and that most of theaccumulated particles stayed in the tumor for424–48 h.

In vivo nanoparticle distribution and intracellular localization

Quantitative biodistribution studies using inductively coupledplasma–mass spectrometry (ICP-MS) revealed that the targeted goldnanoparticles were accumulated in the tumor 10 times more efficientlythan the nontargeted particles (Fig. 6 and Supplementary Fig. 4online). The ICP-MS data also confirmed nonspecific particle uptakeby the liver and the spleen, but little or no accumulation in the brain,muscle or other major organs, similar to the biodistribution datareported for gold nanoshells injected into healthy mice31. Ultrastruc-tural TEM studies further revealed that the SERS nanoparticles weretaken up by the EGFR-positive tumor cells, and were localized inintracellular organelles such as endosomes and lysosomes (Supple-mentary Figs. 5 and 6 online). The in vivo endocytosed nanoparticleshad crystalline and faceted structures, in agreement with the findingthat nearly identical SERS spectra were obtained from the encodedgold nanoparticles in vitro and in vivo. We believe the pegylated gold

particles were intact and stable in systemic circulation as well as afterbeing taken up into intracellular organelles. No toxicity or otherphysiological complications were observed for the animals after2–3 months of gold particle injection.

DISCUSSION

SERS nanoparticle tags can be delivered to tumors by both a passivetargeting mechanism and an active targeting mechanism (Supple-mentary Fig. 7 online). In the passive mode, nanometer-sizedparticles are accumulated preferentially at tumor sites through anenhanced permeability and retention effect52,53. For active tumor-targeting, ligand molecules such as antibodies and peptides are oftenused to recognize specific tumor antigens. In this work, we used anScFv to specifically recognize the EGFR on the tumor cell surface. Todifferentiate active tumor targeting from passive accumulation, weprepared three types of nontargeted or control SERS nanotags, andexamined their behavior in terms of in vitro cellular binding as well asin vivo distribution. The first type of control particle was prepared bycoating colloidal SERS nanoparticles with a plain PEG layer. Theseparticles gave intense and stable SERS signals, with little or no bindingto live or fixed tumor cells (Fig. 4). Their in vivo distribution was alsodifferent from that of EGFR-targeted nanoparticles. These controlparticles accumulated in the liver and the spleen, but their tumoruptake was 10 times less efficient than that of the EGFR-targetednanoparticles (Fig. 6). For the second type of control nanoparticle, thePEG-coated nanotags were conjugated with a nonspecific antibody(mouse IgG1 isotype). This mouse IgG1 isotype (clone IS5-21F5) isspecific for keyhole limpet hemocyanin, an antigen that is absent inhuman cells lines. The IgG1-labeled SERS tags were incubated withEGFR-positive cells (Tu686 cell line) for 30 min. After washing steps,no distinct fingerprint spectral signature of DTTC was observed underthe same experimental conditions, indicating minimal nonspecificbinding. For the third type of control nanoparticle, the PEG-coatednanotags were linked with smaller nonspecific proteins to match themolecular size of our ScFv peptide (25 kDa) used for EGFR targeting.In one example, we used a 27-kDa recombinant His-tagged green

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Figure 6 Biodistribution data of targeted and nontargeted gold nano-particles in major organs at 5 h after injection as measured by inductively

coupled plasma-mass spectrometry (ICP-MS). Note the difference in tumor

accumulation between the targeted and nontargeted nanoparticles. The

s.d. (error bars) were calculated based on four animals (n ¼ 4) in each

study group.

Figure 5 In vivo cancer targeting and surface-

enhanced Raman detection by using ScFv-

antibody conjugated gold nanoparticles that

recognize the tumor biomarker EGFR. (a,b) SERS

spectra obtained from the tumor and the liver

locations by using targeted (a) and nontargeted

(b) nanoparticles. Two nude mice bearing human

head-and-neck squamous cell carcinoma (Tu686)xenograft tumor (3-mm diameter) received 90 ml

of ScFv EGFR-conjugated SERS tags or pegylated

SERS tags (460 pM). The particles were

administered via tail vein single injection.

SERS spectra were taken 5 h after injection.

(c) Photographs showing a laser beam focusing

on the tumor site or on the anatomical location of

liver. In vivo SERS spectra were obtained from

the tumor site (red) and the liver site (blue) with

2-s signal integration and at 785 nm excitation.

The spectra were background subtracted and

shifted for better visualization. The Raman

reporter molecule is malachite green, with

distinct spectral signatures as labeled in a

and b. Laser power, 20 mW.

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fluorescent protein (GFP). This protein ligand was produced from apGFP-his-tag plasmid that was constructed in our laboratory using thePCR-directed insertion of oligonucleotides (encoding six histidineresidues into a bacterial pGFP-expressing plasmid vector). Therecombinant product was purified and conjugated to SERS nano-particles using the same procedures as for the ScFv peptide. Inaddition to its fluorescence properties, this protein provides a goodmodel for our EGFR-targeting peptide because of their similar sizeand weight. A quantitative comparison of the in vivo uptake data forthe protein-functionalized particles and the plain PEG-coated particlesis provided in Supplementary Figure 4. Overall, these nontargetednanoparticles have similar in vivo distribution behavior, with minimalpassive accumulation in xenograft tumors.

In addition to their in vivo biocompatibility and safety, the goldnanoparticles are dual-modality probes for optical and EM imaging,and could be used as a model system to design nanoparticles formolecular imaging and targeted therapy. A major task toward this goalis to develop ‘smart’ nanoparticles that are able to avoid nonspecificorgan uptake15–17, and to target specific cells and organs in vivo8–12.Because monodispersed gold colloids are available in a broad sizerange from 2 nm to 250 nm, nontoxic gold nanoparticles could beused to gain a better understanding of how particle size and surfacecoating affect their in vivo biodistribution and targeting. Furthermore,therapeutic drugs can be conjugated to gold nanoparticles by covalentor noncovalent methods, allowing the development of integrateddiagnostic and therapeutic nanoparticle agents.

We have developed a class of biocompatible and nontoxic goldnanoparticles for in vivo tumor targeting and surface-enhancedRaman detection. Various spectroscopic encoders such as crystal violetwere not displaced, but were stabilized by a coating layer of thiol-PEG.This nanoparticle platform allows facile conjugation of tumor-targeting ligands to heterofunctional PEG linkers. By using an ScFvantibody as a targeting ligand, we designed SERS nanoparticles foractive targeting of both cancer cells and xenograft tumors in animalmodels. With their excitation and emission spectra in a clear infraredwindow, these particles were 4200 times brighter than near-infrared-emitting quantum dots, and allowed spectroscopic detection of smalltumors (0.03 cm3) at a penetration depth of 1–2 cm. The pegylatedgold nanoparticles are also dual-modality optical and EM probes54,and thus provided an important tool to study the effects of particlesize and surface coating materials on biodistribution and organtargeting. Furthermore, their plasmonic light–absorbing propertiescould be used for tumor imaging and treatment based on thephotothermal effect48,55–57.

METHODSLive animal studies. The protocols were approved by the Institutional Animal

Care and Use Committee (IACUC) of Emory University.

Reagents. Ultrapure water (18 MO cm–1) was used throughout the work. The

following chemicals were obtained from commercial sources and were used

without further purification: 60-nm citrate-stabilized gold particles at a

concentration of 2.6 � 1010 particles per milliliter (Ted Pella Inc.), near-

infrared-emitting quantum dots (QD705, Invitrogen), malachite green iso-

thiocyanate (MGITC) (Invitrogen), diethylthiatricarbocyanine iodide (DTTC)

(Exciton), mPEG-SH (MW B5 kDa) (Nektar Therapeutics), HS-PEG-COOH

(MW B3 kDa) (Rapp Polymers). The human carcinoma cells line Tu686 was

established from a primary tumor in base of tongue. Human carcinoma cell

line NCI-H520 was purchased from the American Type Culture Collection

(ATCC). Cell culture media, fetal bovine serum, hemacytometer, and cell

culture supplies were purchased from Fisher Scientific. All other reagents were

obtained from Sigma-Aldrich at the highest purity available.

Preparation of pegylated SERS nanoparticles. A freshly prepared reporter

solution (3–4 mM) was added dropwise to a rapidly mixing gold colloid at a 1:6

reporter solution/colloid volume ratio, which facilitated even distributions of

the reporter molecules on the gold particle surface. The molar ratio of reporter

molecules to gold particles was optimized for maximal SERS intensities and

minimal colloid aggregation. For example, the optimized surface coverage

values were 14,000 malachite green isothiocyanate molecules per 60-nm gold

particle, and about 15,300 crystal violet molecules per gold particle of the same

size. It should be noted that the above parameters (that is, stock reporter

concentration, volume ratio of stock reporter solution to gold nanoparticle

solution, and the rate of reporter addition to gold) all affected the aggregation

state of the resulting tags. When reporter solution was added to gold colloid, we

observed higher SERS signals than when adding gold to reporter.

After 10 min, a thiol-PEG solution (10 mM) was added dropwise to the

Raman-encoded colloids, with a minimum ratio of 30,000 PEG-SH molecules

per 60-nm gold particle. This surface coverage corresponded to a complete PEG

monolayer on the gold particle surface, and was necessary to stabilize gold

colloids against aggregation under various conditions. Simple geometric

calculations showed that each thiol-PEG molecule occupied a footprint area

of 0.35 nm2 on the gold surface, consistent with the literature data reported for

PEG-SH in a brush conformation. Importantly, addition of 10- to 20-fold

excess PEG-SH did not result in any changes in colloid stability or in the

thickness of the polymer coating layer.

Nanoparticle characterization. UV-Vis absorption spectra were recorded on a

Shimadzu (UV-2401) spectrometer using disposable polyacryl cuvettes. Trans-

mission electron micrographs (TEM) were taken by using a Hitachi H7500

high-magnification electron microscope. The nanoparticle sample (5 ml) was

dropped onto copper 200-mesh grids that were pretreated with UV light to

reduce static electricity. After 30 min, the solvent was drained with a filter paper

and a phosphotungstic acid stain solution (1% by weight, adjusted to pH 6)

was applied for 30 s. Fresh tumor tissue specimems were fixed in 0.1 M

cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4 1C. The tissue

was rinsed three times in 0.1 M cacodylate buffer for 15 min, post-fixed with

1% OsO4 buffer, and then dehydrated and embedded in a resin (Epon).

Ultrathin sections (B60 nm) were produced with an ultratome machine, and

were placed on copper grids for TEM imaging.

DLS data were obtained by using a Brookhaven 90Plus particle size analyzer

instrument. Each sample was measured three times consecutively. SERS spectra

were recorded on a compact Raman system using 633 nm (3 mW) or 785 nm

(40 mW) excitation (Advantage Raman Series, DeltaNu). In vivo SERS spectra

were collected using 785-nm laser excitation on a handheld Raman system

(Inspector Series, DeltaNu). The laser beam diameter was 35 mm at the

focal point, so the probe volume was estimated to be B23 nl at 633 nm

excitation and B19 nl at 785 nm excitation. SERS intensities were nor-

malized to the Raman spectra of cyclohexane and polystyrene to correct

for variations in optical alignment and instrument response. The spectral

resolution was B5 cm–1 for both the Advantage and the Inspector

Raman systems.

For imaging of single SERS nanoparticles and quantum dots, a narrow

bandwidth laser excitation filter (633 ± 3 nm) and a long-pass emission filter

(655LP, Chroma Tech) were employed with an Olympus IX71 inverted

microscope. The images were taken with 750 ms exposure time and were the

average of 50 images by using an electron-multiplying (EM) CCD camera

(Hamamatsu, Model C9100-12) attached to the microscope. The use of long

exposure times and image averaging cancelled out any signal fluctuations of

single nanoparticles. For quantitative comparison of SERS and quantum dot

signal intensities, the wavelength dependence factor was corrected by using the

CCD camera response curve.

Conjugation with ScFv ligands. ScFv B10, an antibody fragment specific for

human EGFR, was isolated from the YUAN-FCCC human naive phage display

library by using established solid phase biopanning methods. Large quantities

of ScFv were purified from bacterial extracts under native conditions using a

Ni2+ NTA-agarose column (Qiagen). Protein purity greater than 95% was

determined by using sodium dodecyl sulfate (SDS)-PAGE. The heterofunc-

tional linker HS-PEG-COOH (430 ml and 1 mM) was added dropwise to 2.2 ml

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Au-MGITC (or Au-DTTCI) solution in a polypropylene tube under rapid

mixing. The number of carboxy groups per gold particle was controlled to be

B5,000 by changing the amount of linker molecules used. After 15 min

of mixing, the gold nanoparticles were exposed to a large volume of PEG-SH

(1.6 ml at 10 mM) to fill the areas not covered by the heterofunctional PEG,

yielding well-shielded and stable particle surfaces. Before covalent ligand

conjugation at the carboxylic acid functional groups, the gold particles were

purified by three rounds of centrifugation (1,000g) and resuspension in PBS.

To activate the -COOH groups on the particle surface for covalent

conjugation, freshly prepared ethyl dimethylaminopropyl carbodiimide

(EDC) solution (5 ml) at a concentration of 40mg/ml) and sulfo-NHS (5 ml

at 110 mg/ml) were mixed vigorously at 25 1C for 15 min. Excess EDC and

sulfo-NHS were separated from the activated nanoparticles by three rounds of

centrifugation (1,000g) and resuspension in PBS using Nanosep 10K MWCO

OMEGA membrane (Pall Life Sciences). The purified gold particles with

activated carboxyl groups were then reacted with the ScFv antibody

(11.2 nmol) at 25 1C for 2 h, and the reaction mixture was stored at 4 1C

for overnight. Excess ScFv ligand was removed by three rounds of centrifuga-

tion and resuspension in PBS using 100K MWCO OMEGA membranes. Based

on protein absorption measurement at 280 nm, we estimated that there were

about 600 ScFv molecules per gold particle. This value was further confirmed

by using a fluorescently labeled ScFv ligand to determine the conjugation ratio

at higher sensitivity. The fully functionalized nanoparticles were characterized

by UV-Vis, TEM and DLS, and their colloidal stability and optical properties

were essentially the same as that of control nanoparticle tags.

Cellular SERS studies. Tu686 and H520 cells were cultured in DMEM/Ham’s

F-12 (1:1) and RPMI-1640 supplemented with 10% heat-inactivated fetal

bovine serum and antibiotics (streptomycin, penicillin G and amphotericin B),

respectively, and were maintained in a humidified incubator at 37 1C, 5% CO2.

The cells were grown to confluence in 35-mm dishes. Cell staining procedures

were performed under sterile conditions on a tabletop binding incubator at

25 1C. Live cells were gently mixed with the ScFv-conjugated SERS nano-

particles (15 pM in PBS) for 30 min, and then were harvested by gentle

scraping. The cells were subjected to four rounds of washing with ice-cold PBS,

and were resuspended in 500 ml PBS before SERS measurement. A portion of

the cells were incubated with pegylated control SERS tags to assess nonspecific

binding and internalization. An additional portion of the cells received neither

control SERS tags nor EGFR-SERS tags, and were used as controls to assess

background cell scattering. SERS spectra were normalized to cell numbers as

determined with a Coulter counter.

For quantitative comparison, we subtracted the pure cell scattering spectra

to generate difference spectra in Figure 3. All spectra were taken in cell

suspensions. Based on a cell density of 1 � 106 cells per ml, we estimated that

the laser detection volume contained B20 to 30 labeled cells. We did not

observe changes in either spectral signatures or intensities upon repeated

examination of the unfixed cell samples over a period of 3 d or upon cell

fixation in formaldehyde solution. These cell-suspension measurements

avoided the problems of nanoparticle tagging and cellular heterogeneities

and were found to be highly reproducible.

Tumor xenografts and in vivo SERS. A healthy nude mouse received

50 femtomoles of pegylated SERS nanoparticles administered at two locations:

(i) subcutaneous injection (1–2 mm under skin); and (ii) deep muscular

injection (1 cm under the skin). Different locations were examined by using an

NIR Raman spectrometer (Inspector Series, DeltaNu). The subcutaneous SERS

spectrum was obtained in 3 s, the muscular spectrum in 21 s, and the control

spectrum (obtained in an area away from the injection site) also in 21 s.

Based on protocols approved by the Institutional Animal Care and Use

Committee of Emory University, Tu686 cells (5 � 106) were injected sub-

cutaneously into the back flank area of B6- to 8-week-old female nude mice

(NC rathymic, nu/nu). The mice were divided into two groups for passive and

active targeting studies. When the tumor size reached 3 mm diameter, the nude

mice received 45 femtomoles of ScFv EGFR-conjugated SERS tags and

pegylated control SERS tags, respectively, by tail vein injection. After 5 h, the

mice were placed under anesthesia by injection of 70 ml of ketamine and

xylazine mixture solution and were examined by using a Raman spectrometer

with 20 mW laser power at 785 nm. The laser beam was focused to the tumor

or the liver anatomical region for both the targeted and nontargeted SERS

nanoparticles. With a focal length of B9 mm, SERS spectra were obtained in a

completely noncontact and noninvasive manner. After spectroscopic data

acquisition, the mice were killed to collect major organs for ICP-MS biodis-

tribution analysis. A small portion of each fresh tissue sample was also fixed

immediately in 0.1 M cacodylate buffer to prepare TEM thin sections

(Supplementary Figs. 5 and 6).

Briefly, major organ tissues were rinsed with ethanol three times and then

lyophilized and weighed into clean vials for acid digestion. After 2 d of strong

acid digestion, the samples were purified and diluted 35-fold for analysis by

ICP-MS (inductively coupled plasma–mass spectrometry). The experiments

were carried out in five independent runs for statistical analysis. Each run had

two mice with freshly prepared SERS tags, one with active targeting and the

other with passive targeting. One group of the animals was used for longer term

toxicity studies.

Note: Supplementary information is available on the Nature Biotechnology website.

ACKNOWLEDGMENTSWe are grateful to Gregory Adams at Fox Chase Cancer Center for providingthe ScFv B10 plasmid construct, to H.Z. Zhang for tumor cell injection, and toHong Yi for assistance with TEM. This work was supported by grants from theUS Air Force Office Multi-University Research Initiative, the National CancerInstitute Centers of Cancer Nanotechnology Excellence (CCNE) Program(U54CA119338 to S.N.), and the National Cancer Institute SPORE Program inHead and Neck Cancer (P50CA128613 to D.M.S.). Four of us (M.D.W., G.Z.C.,D.M.S. and S.N.) also acknowledge the Georgia Cancer Coalition (GCC) fordistinguished cancer scholar awards. The human carcinoma cells line Tu686was kindly provided by Peter G. Sacks (New York University College ofDentistry, New York, NY).

Published online at http://www.nature.com/naturebiotechnology/

Reprints and permissions information is available online at http://npg.nature.com/

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