Advanced Review

In vivo near-infrared uorescence imaging of cancer with nanoparticle-based probes

Xiaoxiao He,1,2 Kemin Wang2 and Zhen Cheng1
The use of in vivo near-infrared uorescence (NIRF) imaging techniques for sensitive cancer early detection is highly desirable, because biological tissues show very low absorption and autouorescence in the NIR spectrum window. Cancer NIRF molecular imaging relies greatly on stable, highly specic and sensitive molecular probes. Nanoparticle-based NIRF probes have overcome some of the limitations of the conventional NIRF organic dyes, such as poor hydrophilicity and photostability, low quantum yield, insufcient stability in biological system, low detection sensitivity, etc. Therefore, a lot of efforts have been made to actively develop novel NIRF nanoparticles for in vivo cancer molecular imaging. The main focus of this article is to provide a brief overview of the synthesis, surface modication, and in vivo cancer imaging applications of nanoparticlebased NIRF probes, including dye-containing nanoparticles, NIRF quantum dots, and upconversion nanoparticles. 2010 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol
2010 2 349366

ancer early detection is critical and expected to contribute signicantly to the success of cancer therapy and improvement of patients survival rates.1 A prevalent need in early detection and localization of cancer is for noninvasive imaging of the cancer with various modalities that can be useful for presenting the tumor anatomical structure as well as the tumor metabolism and biochemistry. One of the biggest obstacles of current diagnostic imaging methods in detection of cancer is the lack of specicity and sensitivity. Although various forms of nonuorescence in vivo imaging techniques, including magnetic resonance imaging (MRI),2 2-deoxy-2-[18 F]uoro-dglucose ([18 F]FDG)-positron emission tomography (PET),3 X-ray,4 radiography,5 ultrasound,6 and so on,7 are being used in clinic, they are not highly selective for cancer. In addition, many of these modalities are expensive.8
Correspondence 1 Molecular

to: zcheng@stanford.edu

Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program and Stanford Cancer Center, Stanford University School of Medicine, Stanford, CA 94305, USA State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, Hunan University, Changsha 410082, PR China DOI: 10.1002/wnan.85

Optical imaging approaches, on the other hand, are emerging as promising high-resolution modalities for the cancer detection. The optical signals can provide the molecular information of biological tissues and are related to the change of some signicant physiologic parameters.911 Among the optical imaging technologies, uorescence imaging is a desirable modality for early detection of cancer because of its high sensitivity and multiplex detection abilities involving simultaneous application of several different molecular probes.8 This technology essentially depends on the probes emitting in the near-infrared uorescence (NIRF) spectrum window (650900 nm wavelengths) where biological tissues display low absorption. The most common organic NIR uorophores used for in vivo NIRF imaging are polymethines (e.g., Cy5.5 and Cy7).12 Recently, some new NIRF probes with improved uorescence quantum yield, high photostability, low aggregation, and low cytotoxicity have also been developed, such as boron-dipyrromethene derivatives,13 Nile blue analogs,14 oxazine dye AOI987,15 and the activatable probes.16,17 Progress in NIRF in vivo cancer molecular imaging has rapidly increased in recent years because of the development of novel NIRF probes and optical imaging instruments.1618 However, conventional organic uorophores suffer from several signicant
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limitations. First, few NIR uorochromes are highly water soluble, stable and could be easily coupled covalently to biological molecules. Second, when biomolecules or other tumor targeting ligands are conjugated with uorophores, there is usually only small number of uorophores presented per ligand, so the detection sensitivity is relatively low. Third, conventional uorophores suffer with rapid photobleaching, which is not t for long-term molecular imaging. Finally, the in vivo toxicities of the majority of uorophores are unknown, so far only one uorochrome, indocyanine green (ICG), is approved by the Food and Drug Administration (FDA) for clinical use.8,19 Advances in nanotechnology have produced signicant contributions to the biomedicine. One of the most exciting and rapidly progressing areas of nanotechnology in biomedical research is the development of nanoparticle-based probes for in vivo cancer molecular imaging. These probes can target tumor either through the enhanced permeability and retention effect (EPR effect) of the tumor microvasculatures or by the specic binding between the tumor cell receptors and biomolecules-modied nanoparticles.20 By using nanoparticle-based imaging probes, passive accumulation and active targeting of cancer receptors have both become great interests to the eld of medical imaging, because of their potential to detect early stage cancers. Nanoparticles for different cancer imaging strategies, including photoacoustic imaging,21,22 MRI,23,24 Raman imaging,25,26 NIRF imaging,27,28 have been actively pursued. Nanoparticle-based NIRF probes such as NIR dye-containing nanoparticles, NIR inorganic uorescent semiconductor nanocrystals, upconversion nanoparticles (UCNs), Au nanocluster and Ag nanocluster are recent additions to the list of new NIRF probes. In this article, we will mainly review the synthesis, surface modication, and primary application for cancer in vivo molecular imaging of three types of well-developed nanoparticlebased NIRF probes. They are NIRF dye-containing nanoparticles, NIRF quantum dots (QDs), and UCNs.

Core: near-infrared dyes

Shell: inorganic materials or polymer

FIGURE 1 | Schematic representation of a typical coreshell NIRF

dye-containing nanoparticle design.

photostability and biocompatibility due to the shell protection of the NIRF dyes from outside quenching and degrading factors; (2) improved nanoparticle dispersibility in aqueous medium for the hydrophilic shell materials; (3) improved uorescence signal from high payloading dye molecules per nanoparticle; and (iv) easy and straightforward bioconjugation of biomolecules to the functional groups of the shells.3032 Three most commonly used NIRF dyecontaining nanoparticles for in vivo cancer imaging namely NIRF dye-doped silica nanoparticles, NIRF dye-doped calcium phosphate nanoparticles (CPNPs), and NIRF dye-containing lipoprotein nanoparticles are described below in detail.

NIRF Dye-Doped Silica Nanoparticles

Among the inorganic materials, amorphous silica appears to be a biocompatible and nontoxic material and has high potential applications in the bionanotechnology. The surface hydroxyl groups can be chemically modied with amines, carboxyls, or thiols using conventional silane-based chemistry. These chemically modied silica shells can be easily conjugated with the amine groups of the targeting biomolecules. Up to now, the incorporation of visible dyes, such as a metallorganic dye, tris(2,2 bipyridyl)dichloro ruthenium (II) (Rubpy), organic dyes, uorescein isothiocyanate (FITC), and tetramethylrhodamine isothiocyanate (TRITC), into the silica nanoparticles using a reverse microemulsion-based synthesis approach has been widely reported and used for biosensing and bioimaging. For example, Tan et al. used Rubpy dye-doped silica nanoparticles to label human leukemia cells and CCRFCEM acute leukemia cells.31,33,34 Various dye-doped silica nanoparticles, including Rubpy dye, FITC3-aminopropyl-triethoxysilane (APTS), and TRITCdoped silica nanoparticles have been synthesized to detect SmIgG+ B lymphocytes, HepG liver cancer
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NIRF DYE-CONTAINING NANOPARTICLES

NIRF dye-containing nanoparticles are polymer or inorganic matrix-based particles containing a high payload of NIRF organic dyes.29 The NIRF dyes are usually embedded inside the particles either noncovalently or covalently to form a functionalized nanoparticles with a typical coreshell structure (Figure 1). For in vivo cancer imaging applications, NIRF dyecontaining nanoparticles are particularly attractive because of the following advantages: (1) enhanced
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cells, and the early stage apoptosis, respectively.3537 Moudgil et al. developed FITC-doped silica nanoparticles covalently attached to folic acid for imaging of human lung cancer cells.38 However, these visible dye-doped silica nanoparticles are unlikely to be used for in vivo cancer imaging. For in vivo imaging, silica nanoparticles can be doped with NIRF dyes, such as Cy5.5, Cy5, and ICG. There are two synthetic routes for the NIRF dye-doped silica nanoparticles reported in the literatures, water-in-oil (W/O) microemulsion method and solgel process method. The W/O microemulsion is a thermodynamically stable system which is composed of water, oil, and surfactant. Typically, the NIRF dyes are suspended in the water core of the microemulsion, and the shell is formed by the hydrolysis and condensation reaction of tetraethylorthosilicate (TEOS) in the presence of ammonium hydroxide (NH4 OH) catalyst. Wang et al. prepared Cy5 dye-doped coreshell silica uorescent nanoparticles, employing polylysine-modied Cy5 as the core material and silica coating produced from the hydrolysis of TEOS in the W/O microemulsion.39 Zhou et al. synthesized Cy5-doped silica coreshell nanoparticles using Au colloid conjugated with Cy5-labeled oligonucleotide as the core.40 The solgel process is a technique that is used to prepare transparent oxide glasses by hydrolysis and polycondensation of alkoxides, which is also an attractive method for preparing NIRF dye-doped silica nanoparticles.4143 For in vivo applications, it is highly desirable that the surface of the NIRF dye-doped silica nanoparticles is appropriately modied with targeting biomolecules such as cancer specic antibodies, peptides, aptamers, etc. The biological modication of silica nanoparticles with different functional groups has been reviewed recently and will not be described here.32 Researchers have demonstrated the in vivo distribution and nontargeted cancer imaging of NIR dye-doped silica nanoparticles. The biodistribution study of ICG-doped mesoporous silica nanoparticles (MSN-TA-ICG) in rats revealed that the nanoparticle with a rather strong and stable uorescence displays prominent uptake in the liver.42 Scherman et al. found that Lewis lung carcinoma (3LL) could be visualized with the PEG-modied NIR persistent silica nanoparticles in a subcutaneous xenograft model of 3LL tumor in C57BL/6 mice, because of the high extravasation ability of the nanoparticles. These results provide an encouraging foundation toward the further development of NIRF dye-doped silica nanoparticles as NIRF probes for in vivo cancer imaging.43
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NIRF Dye-Doped CPNPs

By virtue of its presence throughout the body (e.g., bones, teeth), calcium phosphate (CP) is generally regarded as safe by the FDA.44 It has been widely used to synthesize CPNPs, a novel, nonviral system for effective delivery of targeted genes or drugs.45,46 Moreover, CPNPs also present an interesting class of inorganic matrices for doping with uorescence dyes that show promising properties for cell and animal imaging.4649 For example, Adair et al. synthesized ICG-doped CPNPs with carboxylate functional groups by employing a double reverse microemulsion approach. The prepared nanoparticles were further modied with methoxypolyethylene glycol amine (mPEG-amine) through an ethyl-N(3-dimethylaminopropyl)-N-hydrochloride carbodiimide reaction. It was found that the PEGylated ICG-doped CPNPs accumulated in the breast adenocarcinoma xenografts (5 mm in diameter) at 24 h after intravenous tail vein injection, mainly due to the EPR effects. In contrast, the in vivo uorescence of the free ICG is relatively short due to its uorescence quenching in physiological environments with rapid aggregation and clearance from the body (Figure 2(a)(c)). The clearance pathway of PEGylated ICG-CPNPs is the initial hepatic localization and eventual total clearance through the biliary tree with minimal acute renal involvement as demonstrated from the excised organs imaging at 10-min postsystemic injection (Figure 2(d)). The ex vivo tissue imaging further demonstrated that detectable penetration and emission depths are seen with ICG-CPNPs at 3 cm in porcine muscle tissue compared to the weakly uorescent free uorophore at only 2 cm, which highlighted the ICG-doped CPNPs hold the promise for solid tumors early detection.49

NIRF Dye-Containing Lipoprotein Nanoparticles

Lipoproteins, biochemical assemblies of proteins and lipids, are responsible for the transport of lipids and other hydrophobic substances in the aqueous environment. The main classes of lipoproteins are high-density lipoproteins and low-density lipoproteins (LDL), varying in their size of 811 nm and 1825 nm in diameter, respectively. Due to their biocompatibility, biodegradability, high-payload capacity, and nonimmunogenicity, the lipoproteins are regarded as ideal particles to be used in biomedical eld.5052 In general, there are three ways to incorporate molecules into lipoprotein particles, as shown in Figure 3.5255 The rst method is the direct conjugation of the molecules to the amino acid residues of
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(a)
i ii iii i

(b)
ii iii

3 hours

24 hours

(c)
i ii iii Liver

(d)

Heart Stomach

Spleen Kidneys Small instestine 96 hours Large instestine

Organs excised at 10 minutes

FIGURE 2 | NIR transillumination images (Ex 755 nm, Em 830 nm) taken at various times track uorescence signals and pharmacokinetic
distributions for the indocyanine green calcium phosphate nanoparticles (ICG-CPNPs) and controls delivered systemically via tail vein injections in nude mice implanted with subcutaneous human breast adenocarcinoma tumors. Hash (-) marks next to each mouse indicate the position of the 5-mm tumors. (a) NIR transillumination images taken at 3 h to track uorescence signals for the indocyanine green calcium phosphate nanoparticles (ICG-CPNPs) and the controls delivered systematically via tail vein injections in nude mice implanted with subcutaneous human breast adenocarcinoma tumors. Two control samples, (i) carboxylate-terminated CPNPs without ICG encapsulant and (ii) free ICG, match the particle concentration and uorophore content (1013 particles/mL and 105 M, respectively) of a (iii) PEGylated ICGCPNP sample. (b, ii) No uorescence signal is detected from the free ICG at 24-h postinjection, while the PEG-ICG-CPNP sample (c, iii) retains signicant signal even after 96 h. (b, iii) Fluorescence signal is unmistakably localized in tumors 24 h after administration with PEGylated ICG-CPNPs. The excised organs in panel (d) illustrate the biliary clearance route 10-min postinjection of PEG-ICG-CPNPs. Fluorescence signal is not seen from the stomach or spleen with minimal renal involvement. (Reproduced with permission from Ref 49. Copyright 2008 American Chemical Society).

apolipoproteins. The second is the incorporation of the molecules into the phospholipid monolayer via an intercalation mechanism. The last method is the lipoprotein reconstitution into the apolar core (core loading). By using these methods, the lipoproteins as nanocarriers have been reported for MRI.50,56 Recently, the incorporation of NIRF probes into LDL has been well demonstrated by Zheng et al. First, LDL labeled with NIRF dyes has been prepared by using tricarbocyanine cholesteryl laurate (TCL17) and 1,1 -dioctadecyl-3,3,3 , 3 -tetramethylin-dotricarbocyanine iodide (DiR), respectively. Ex vivo biological studies on a lipoprotein receptors overexpressing tumor model, human hepatoblastoma G2 (HepG2), conrmed that these NIRF probes were internalized selectively by the tumor, and
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the tumor could be detected with high sensitivity by a low-temperature 3D redox scanner.57,58 Although the NIR dye-labeled LDL has proven to be a useful approach for delivery of diagnostic agents to tumors, its application is largely limited to LDL receptor overexpressed tumors. In fact, it has a limited use for cancer detection and therapy because many tumors do not overexpress the LDL receptor, whereas some normal tissues do. A folate receptor (FR)-targeted LDL nanoparticles containing NIRF dye were further prepared by conjugating folic acid to the lysine residues of the apolipoprotein B (apoB)-100 proteins. Cellular localization of the targeted LDL was monitored in FR-overexpressing KB cells, FR-nonexpressing CHO and HT-1080 cells, and LDL receptor overexpressing HepG2 cells, respectively. Folic acid-conjugated LDL
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(B)

(B) Phospholipid monolayer

(B) (C) Apolar core (C) (C) (C) (B) (A)

FIGURE 3 | Schematic representation of the

Apolipoproteins

(A)

Incorporated molecules

three ways to incorporate molecules into lipoprotein particles. (a) Direct conjugation of the molecules to the amino acid residues of apolipoproteins. (b) Incorporation of the molecules into the phospholipid monolayer via an intercalation mechanism. (c) Reconstitution into the apolar core.

was taken up and accumulated in KB cells while minimal uptake was seen in CHO and HT1080 cells or HepG2 cells. These studies demonstrated that the folic acid conjugation blocked the binding of the normal LDL receptor with LDL, resulting in cancer cells recognition through their special binding of FRs and folic acid.59 Furthermore, the in vivo conrmation of the FR-modied, NIRF dye-containing LDL nanoparticles rerouting principle and application of this concept for enhanced cancer optical imaging in live animals has also been presented for the rst time. The schematic illustrations and the in vivo cancer imaging application of this functionalized LDL nanoparticles are shown in Figure 4.60 While many factors and processes that compete for the uptake of FR-modied NIRF dye-containing LDL nanoparticles, the KB tumor showed much stronger uorescence than the HT1080 tumor due to the efcacy of the FR targeting. These results indicate that the rerouting strategy for targeting LDL to alternate receptors for in vivo cancer applications is a viable and promising approach. This research also provides a general approach for targeting cancers by modifying the LDL nanoparticles with other types of binding biomolecules.

to photobleaching.61,62 Due to these unique optical properties, QDs have been widely explored in many biological applications, such as immunoassays, DNA analysis, in vitro and in vivo imaging, and so on.6167 The use of QDs in biological research has been focused on the uorescence labeling and uorescence resonance energy transfer (FRET). Use of QDs as uorescent biotags for biological tissue staining and disease diagnosis is one of the most attractive applications. So far, the synthesis, surface modication, characterization, and biological application of QDs in the visible spectral region have been reported and also reviewed in many articles.6870 For noninvasive in vivo imaging, the main barriers to the QDs in the visible spectral region are the autouorescence and the absorption properties of the tissues.71,72 Therefore, more and more attention is being paid on studying the NIR (650900 nm) emitting QDs and their application for in vivo cancer imaging.

Synthesis, Surface Modication, and Bioconjugation of NIRF QDs

Two synthetic routes have been reported and commonly used for the synthesis of NIRF QDs, organic synthesis, and aqueous synthesis. The organic synthesis method for the preparation of NIRF QDs was carried out via heating appropriate organometallic precursors in organic solvents [mixture of trioctyl phosphine/trioctyl phosphine oxide (TOP/TOPO)] at elevated temperature. For example, Nie et al. synthesized alloyed semiconductor QDs (CdSeTe)
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NIRF QDS
QDs are semiconductor crystals, exhibiting narrow and tunable emission bands along with broad excitation spectra and high molar extinction coefcient. They also display high uorescence quantum yields, very large effective Stokes shifts, and high resistance
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N Br

N C16H37

DIR

(a)
H2N HN O

C16H37 N N H N N

O H N O HOOC H NHS

FA-NHS

Imaging cargo (surface-loading) [DIR]

Phospholipid monolayer

Cholesterol ester core

ApoB-100

Targeting group [FA]

(b)
Pre-scan 5 min 30 min 2 hr 6 hr 24 hr 7000 6000 5000 HT1080 KB 4000 3000 2000

FIGURE 4 | (a) Schematic illustrations of rerouted low-density lipoproteins (LDL) nanocarrier (bottom) with folic acid (FA) targeting ligand
(middle) and 1,1 -dioctadecyl-3,3,3 , 3 -tetramethylin-dotricarbocyanine iodide (DiR)-based NIR uorescent label (top). (b) Real time in vivo uorescence images of KB/HT1080 dual tumor mice with i.v. injection of DiR-LDL-FA (5.8 M). (Reproduced with permission from Ref 60. Copyright 2007 American Chemical Society).

with both homogeneous and gradient internal structures. Cadmium oxide was used as an inexpensive precursor and mixed with hexadecylamine/TOPO solvents for improving size monodispersity and uorescence quantum yields.73 Chan et al. prepared CdS-capped CdTex Se1x QDs with uorescence emission wavelengths that could be tuned from 600 to 850 nm (Figure 5) by injecting a mixture of dissolved selenium and tellurium into a hot coordinating solvent of TOPO.74 NIRF QDs with different coreshells such as (CdSe)ZnS,75 InAsx P1x /InP/ZnSe IIIV alloyed,76
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CdSe1x Tex alloyed,77 and CdTe(CdSe)78 have also been synthesized by following the similar organic synthesis strategy. Although the NIRF QDs prepared in organic media through organometallic synthesis or its modied method are high quality and monodispersed, the resulting NIRF QDs are usually very hydrophobic. Alternatively, a exible, aqueous synthetic method can be used for the preparation of water-soluble NIRF QDs. Mao et al. presented a facile one-pot method to fabricate water-dispersed NIR-emitting (650800 nm) CdTeS
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(a)
TOPO CdS

(b)

FIGURE 5 | Schematic and optical properties

of CdS-capped CdTex Se1x alloyed near-IR-emitting quantum dots (QDs). (a) Biocompatible near-IR-emitting QDs contain a core structure (CdTex Se1x ) with a CdS and an organic shell. The outer organic shell contains functional groups for attaching biorecognition molecules to the QD surface. (b) Absorbance and uorescence spectra of CdS-capped CdTex Se1x near-IR-emitting QDs with different compositions of Te:Se in the core. (Reproduced with permission from Ref 74. Copyright 2006 American Chemical Society).
Intensity (A.U.) 325

CdTexSe1x

475

625

775

Wavelength (nm)

alloyed QDs with high photoluminescence quantum yields via the hydrothermal method, employing 3-mercaptopropionic acid as stability reagent.79 Morga et al. prepared CdMnTe/Hg with a broad uorescence peak in the NIR (770 nm) in aqueous solution by adding thioglycolic acid as stability reagent.80 Harrison et al. synthesized a series of CdHgTe composite nanocrystals emission wavelengths ranging from 600 to 1350 nm using a wet-chemical colloidal technique, adopting CdTe nanocrystal precursors stabilized using 1-mercapto-2,3-propanediol (1-thioglycerol).81 NIRF QDs described above demonstrate unique optical properties of high quantum yield, size, and composition-tunable NIR uorescence emission and photostability after external excitation. A selfilluminating QD conjugates emitting wavelength from red to NIR has been rstly reported by Rao et al.8284 The conjugates are prepared by coupling QDs to a mutant of light-emitting proteins Renilla reniformis luciferase (Luc8). The self-illuminating QD conjugates rely on bioluminescence resonance energy transfers (BRET), which convert chemical energy into photo energy, resulting in a dramatic increase in QD exaction. As shown in Figure 6, upon the addition of substrate, coelenterazine, the protein Luc8 catalyzes the oxidation of coelenterazine and emits blue light with a peak at 480 nm and this energy transfers to the QDs in close proximity, leading to the QD emission. This self-illuminating QD conjugates provide a new type of material and strategy for in vivo imaging, and the Luc8 also can be replaced by other light-emitting proteins. Even though some NIRF QDs have been directly synthesized in water using thiols as stabilizers, high-quality QDs are usually synthesized at hightemperature in hydrophobic coordinating solvents, such as TOPO and TOP. These hydrophobic organic molecules not only serve as the reaction medium, but also as a stability reagent to prevent the formation
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of bulk semiconductors or the aggregation of synthesized QDs. Therefore, the QDs synthesized using the organic synthesis method are usually capped with a monolayer of the organic ligands (Figure 5(a)) and are highly hydrophobic. When these QDs are used in biological systems, they rst have to be transferred from the organic phase to the water phase. Two general strategies have been developed for phase transfer of NIRF QDs to aqueous solution, including solubilization by use of amphiphilic polymers and replacement with bifunctional ligands.72,77,8587 To the replacement with bifunctional ligands, the hydrophobic surface ligands are replaced with bifunctional ligands such as mercaptoacetic acid, 11-mercaptoundecanoic acid, or a natural thiol compound, glutathione. The thiol compound contains a thiol group that binds strongly to the QD surface as well as a carboxylic acid group that is hydrophilic and can be used for further bioconjugation. Except when used as passive targeting particles for cancer imaging,85 QDs require some degree of biological conjugation. Biomolecules, such as bovine serum albumin (BSA), v 3 integrin-binding peptide arginine glycineaspartic acid (RGD), monoclonal antibody, containing basic functional groups of amines or thiols can be directly conjugated to the surfacemodied QDs through a heterobifunctional linker [e.g., 4-maleimidobutyric acid N-succinimidyl ester, 1ethyl-3-(3-dimethylaminopropyl) carbodiimide].8890

In Vivo Cancer Imaging with NIRF QDs

Among all of the NIRF nanoparticle-based probes, QD-based probes are the most studied and developed for in vivo cancer imaging. Up to now, the NIRF QDs have been successfully used for imaging both tumor vasculature and tumor cell targets in various animal models. The application of QDs for in vivo vascular imaging is usually a nontargeted imaging approach.
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655 nm

(a)
Luc
NH CO

480 nm
COO

NH CO

NH CO

COO

8
OH

O2 +
OH

QD
CO

CONH

480 nm 655 nm

(b)

0.7 Absorbance_QD655 0.6 Absorbance (AU) 0.5 0.4 Luc8 Emission_QD655

4.E+06 3.E+06

0.3 0.2 0.1 0 350 2.E+06 1.E+06 0.E+00 700

400

450 500 550 600 Wavelength (nm)

650

For example, Morgan et al. used the CdTeMnHg-BSA QDs with uorescence peak at 770 nm as an angiographic contrast agent. After injecting these NIRF nanocrystals either subcutaneously or intravenously into athymic NCR NU/NU and C3H/HENCR MTV mice, blood vessels on the order of 100 m in diameter, located at a depth of several hundred microns were visualized.80 Because of their small size and poor accessibility, the visualization of the lymphatic vessels and studying of their function are relatively difcult. Kim et al. rstly reported the utility of NIRF QDs (CdTe/CdSe) for sentinel lymph node imaging in the mouse and pig models.91 By further exploiting two-color and multicolor NIRF QDs, multiplexing NIRF imaging (two-color and ve-color NIRF QDs) of lymphatic system has even been achieved.92,93 The beauty of molecular imaging is the ability to image the differences at molecular level between cancerous and normal tissues. This requires the NIR QDs to be modied with targeting moieties. The optical signal is generated only at the locations where there is an accumulation of targeted NIRF QDs. By attaching the RGD peptide to the amine-modied
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QD705, the in vivo targeting and imaging of tumor vasculature have been demonstrated. Tumor vasculature uorescence reached maximum at 6-h postinjection with good contrast using the athymic nude mice bearing subcutaneous U87MG human glioblastoma. At 27-h postinjection, no signicant uorescence signals from the tumors were observed in the mice injected with QD705 or RGD-modied QD705 (RGD-QD705), which indicated gradual clearance of both the QD705 and RGD-QD705 (Figure 7).90 The binding of nanoparticle conjugates to newly formed tumor neovasculature expressing v 3 integrins has been rstly observed in living subjects using RGDQD800 and intravital microscope.94 Since angiogenesis is common to all tumors, this technique opens up new perspectives for integrin-targeted NIRF optical imaging and may aid in cancer detection and treatment. Noninvasive imaging of growth factor receptor (epidermal growth factor receptor, EGFR) expression is also very important for cancer detection and therapy. Krishnan et al. developed EGF-conjugated QDs800 as nanoprobes to image EGFR expression
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PL intensity (c.p.s)

COOH

CO
OH CO

CO2 + Coelenteramide

CO
CO

CO

Coelenterazine

CO NH

CONH

CO OH

OH CO

480 nm

NH

H COO
COO
CONH

CONH

CONH

C O O
CONH
COO

6.E+06 5.E+06

OH

655 nm

FIGURE 6 | Design and spectroscopic

characterization of bioluminescent quantum dot conjugates based on bioluminescence resonance energy transfers (BRET). (a) A schematic of quantum dot that is covalently coupled to a BRET donor, luc8. The bioluminescence energy of luc8-catalyzed oxidation of coelenterazine is transferred to the quantum dots, resulting in quantum dot emission. (b) Absorption and emission spectra of quantum dot 655 (Ex 480 nm), and spectrum of the bioluminescent light emitted in the oxidation of coelenterazine catalyzed by luc8. (Reproduced with permission from Ref 82. Copyright 2006 Nature).

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O O N O (CH2)3

O N O

CdTe
ZnS

1000 eq, pH = 8.5, 1h

CdTe
ZnS
O

-PEG-NH2
PEG

H N

O (CH2)3 N O

QD705

FIGURE 7 | A schematic of
quantum dot (QD)705-arginine glycineaspartic acid (RGD), PEG denotes poly(ethylene glycol) synthesis and in vivo NIR uorescence imaging of U87MG tumor-bearing mice (left shoulder, pointed by white arrows) injected with 200 pmol of QD705-RGD (left) and QD705 (right), respectively. All images were acquired under the same instrumental conditions. The mice autouorescence is color coded green while the unmixed QD signal is color coded red. Prominent uptake in the liver, bone marrow, and lymph nodes was also visible. (Reproduced with permission from Ref 90. Copyright 2006 American Chemical Society).
O

H N NHO NH O

NH2 NH

RGD-SH 1000 eq, pH = 7.5, 1h

CdTe
HO

HN O O HN N O H

N H

ZnS
HO

c(RGDyK) QD705-RGD

1h

4h

6h

27 h

in human colon cancer xenografts. The in vivo imaging results displayed that both QDs and EGF-QDs showed comparable nonspecic rapid tumor inux and clearance at 60-min postinjection. While pure QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors with extension of postinjection time from 1 to 6 h. The results indicated that the EGF-QDs could be used to image EGFR expression in tumor.95

UPCONVERSION NANOPARTICLES
As reviewed above, the limitations of NIR organic uorophores for in vivo imaging can be effectively overcome by using NIRF dye-containing nanoparticles or NIRF QDs, by taking advantage of their high quantum yields, bright photoluminescence, and good photostability.43,49,7678 However, these NIR uorescent nanoparticles used for uorescent biolabeling are mainly based on downconversion that exercises single photon uorescence by emitting
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low energy uorescence when excited by a highenergy light, typically within the UV short wavelength or visible range. IR light is less harmful to the whole body. It can minimize autouorescence from biological tissues and penetrate tissue up to several inches. Therefore, it is desirable to use uorescent nanoparticles that can be excited by IR light.43,96,97 UCNs, containing rare earth elements, which can convert a longer wavelength radiation (e.g., NIR light) to shorter wavelength uorescence (e.g., visible light), are emerging as a new class of uorescent nanoparticles for application in biotechnology.98101 Unlike the UV excitation or visible photon that causes a background signal from imaged tissues, the optical transparency of NIR in tissues results in improved signal-to-noise ratio in vivo imaging.

Synthesis and Surface Modication of UCNs

Among all the UCNs reported so far, the most efcient infrared-to-visible UCNs are Ytterbium (Yb)erbium (Er) or Ybthulium (Tm) co-doped with uorides
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such as sodium yttrium uoride (NaYF4 ) and lanthanum uoride (LaF3 ).100104 There are mainly three synthetic routes reported for the preparation of upconverting NaYF4 : Yb, Er/Tm nanoparticles with high-quality and strong uorescent performance: coprecipitation method, hydrothermal method, and thermal decomposition method. In a typical coprecipitation method, the component for precipitation is released slowly and evenly through the control of chelator-mediated complex reaction, resulting in homogenous appearance of precipitation in the solution. Ethylenediaminetetraacetic acid (EDTA) is an efcient chelator for rare earth ions. Its chelation constants (lg1) for Y3+ , Yb3+ , and Er3+ (collectively designated as Ln3+ ) are 18.09, 19.51, and 18.85, respectively.105 EDTA also prevents particles coagulation by shielding the rare earth metal ions, which is a necessity for the preparation of monodispersed particles. Yb and Er co-doped NaYF4 UCNs with narrow size distribution and high luminescent intensity were also prepared by a coprecipitation method in the presence of diethylenetriaminepentaacetic acid.106 The coprecipitation method has been widely used for synthesizing UCNs with particles diameter ranging from a few tens of nanometers to several hundreds of nanometers.100,106108 Hydrothermal synthesis of nanoparticles can be dened as a method for synthesis of nanoparticles in hot water under high pressure, which has been proven to be an effective and convenient process in preparing NaYF4 inorganic luminescent materials doped with different lanthanide ions.109112 On the basis of the conventional hydrothermal synthetic technique, solvothermal method, that is, using organic solvent or mixed organicwater solvent instead of water, is an improved method for UCN synthesis. For example, Hexagonal-phase NaYF4 : Yb, Er3+ phosphors with controllable size and morphology were synthesized in selected solvents aided by cethyltrimetylammonium bromide and EDTA.113 Xu et al. reported a novel two-phase solvothermal approach for the synthesis of -NaYF4 : Yb, Er/Tm/Ho upconversion nanocrystals using rare earth stearate as the precursor. The waterethanololeic acid solution containing Na and F ions was the liquid phase, and the precursor was the solid phase. Due to the good solubilization of oleic acid, precursor which was insoluble in water could be well dispersed in the liquid phase under agitation. With elevation of temperature, the rare earth ions released from the precursor reacted with Na and F ions at the solidliquid interface to form tiny particles possessing a monolayer of oleic acid.114
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It is well known that metal triuoroacetates could thermally decompose to generate the corresponding metal uorides and various uorinated and oxyuorinated carbon species.115 Capobianco et al. synthesized upconverting cubic NaYF4 UCNs doped with the Er3+ /Yb3+ or Tm3+ /Yb3+ ion couples via the thermal decomposition of sodium triuoroacetate in a high-boiling point octadecene and oleic acid. The prepared Er3+ , Yb3+ and Tm3+ , Yb3+ -doped NaYF4 particles exhibit green/red and blue upconversion luminescence, respectively, under 977-nm laser excitation with low power densities (Figure 8). The particles synthesized by this method were nonuniform with size distribution of 1060 nm.102 This synthetic procedure was further modied by introducing the lanthanide precursors slowly into the high-temperature reaction mixture through a steel cannula at a ow rate of ca 1 mL/min. The Er3+ /Yb3+ or Tm3+ /Yb3+ -doped upconverting cubic NaYF4 particles with a regular shape and average particle size of 27.6 1.6 nm were successfully synthesized by this modied thermal decomposition method.103 Most methods to produce the UCNs yield either uncoated nanoparticles that the luminescent lanthanide ions cannot be well protected from the high vibration energies of the organic solvent and other quenching factors located at the surface of the nanoparticles or cannot be well suspended in water. Therefore, preparation of UCNs with stable coating is a prerequisite for their application as bioprobes for in vivo imaging. Recently, core/shell nanoparticles with signicant enhancement of upconversion uorescence have been reported in many groups. Capobianco et al. synthesized NaGdF4 : Ce3+ , Tb3+ /NaYF4 core/shell nanoparticles. Upon addition of the NaYF4 shell to the core particles, the core/shell nanoparticles demonstrated higher quantum efciency under UV light and a greater resistance from cerium oxidation by comparison with core-only UCNs.116 By adopting the similar strategy, core/shell -NaYF4 : Yb, Er@NaYF4 and -NaYF4 : Yb, Er@-NaYF4 nanocrystals with controlled size were synthesized by Yan and Kong group.117,118 However, the prepared UCNs were still hydrophobic after coating with an undoped NaYF4 shell. These hydrophobic core/shell nanoparticles are needed to further render hydrophilic by using different approaches, such as coating with amphiphilic polymers, silica shell coating, and surface modication using biocompatible polymers.96,99,119124 For example, to render the nanocrystals soluble in water, Schafer et al. used hydroxyethyldiphosphonic acid (HEDP) to modify the NaYF4 : Yb, Er upconversion nanocrystals prepared in coordinating solvent of N(2-hydroxyethyl)ethylenediamine (HEEDA). HEDP is
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(a) i ii iii iv v

FIGURE 8 | (a) One weight percentage

colloidal solutions of nanocrystals in dichloromethane excited at 977 nm demonstrating upconversion luminescence. (i) NaYF4: 2% Er3+ , 20% Yb3+ solution showing its transparency. (ii) Total upconversion luminescence of NaYF4: 2% Er3+ , 20% Yb3+ solution. (iii and iv) NaYF4: 2% Er3+ , 20% Yb3+ upconversion viewed through green and red lters, respectively. (v) NaYF4: 2% Tm3+ , 20% Yb3+ solution. (b) Transmission electron microscopy (TEM) image of NaYF4: 2% Er3+ , 20% Yb3+ nanocrystals. Inset: High-resolution TEM image of a single nanocrystal. (c) Low-resolution transmission electron micrographs of NaYF4: 2% Er3+ , 20% Yb3+ samples showing uniformity of the particles. (Reproduced with permission from Ref 102,103. Copyright 2006, 2007 American Chemical Society).

(b)

(c)

a strong tetraprotonic acid which is highly soluble in water and in HEEDA.120 By using W/O microemulsion method for silica coating, Zhang et al. synthesized multicolor core/shell-structured UCNs. The hydrophobic NaYF4 nanocrystals were rst dispersed in cyclohexane, and surfactants and ammonia were added to form a W/O microemulsion. The multicolor upconversion nanospheres were then produced by encapsulating organic dyes or QDs in the silica shell. The upconversion uorescence was generated based on FRET from the NaYF4 nanospheres to these organic dyes or QDs.121

In Vivo Cancer Imaging with UCNs

The development of UCNs has added powerful tools to modern biomedical detection technologies. Up to now, the UCNs have already been used for the DNA detection,125 avidin detection,109 siRNA delivery,99 and cancer cells imaging, such as MCF-7 cells,121 HeLa Cells,96 and so on.126129 Further demonstrating the effectiveness of UCNs for in vivo imaging has also been performed on the digestive system of the nematode worm Caenorhabditis elegans,130 deep tissues in Wistar rat126 and vasculature imaging in a mouse model, respectively.131 These studies have clearly demonstrated that the upconversion emission signal from the nanoparticles is readily visible in vivo with excitation using a clinically relevant laser power
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density at about 980 nm. For example, in Zhan et al., the Polyetherimide (PEI)/NaYF4 : Yb, Er nanoparticles were injected subcutaneously at the groin and upper leg regions of the Wistar rats to image the deep tissues in animals. As shown in Figure 9, after the probe administration, the Wistar rats show visible uorescence from depth up to 10 mm. And a much stronger uorescence from deep injections at similar depths has been observed in the rat with skin removed than that of with intact skin. To the green-emitting QD, uorescence signal can be observed only through the translucent skin of the rat foot.126 The in vivo imaging of cancer with UCNs has not yet been reported. However, the well demonstrated tumor cell optical imaging and the effective imaging of deep tissues with UCNs in animal highlight the promise of UCNs as a platform for in vivo imaging of cancer.

CONCLUSIONS AND PERSPECTIVES

In this review, nanoparticle-based NIRF probes such as NIRF dyes containing nanoparticles, NIRF QDs, and UCNs have been briey discussed. Among the three types of nanoparticle-based NIRF probes, the NIRF dyes containing nanoparticles have the main advantage of high payload of NIRF organic dyes and protection of the dyes from outside harsh environment, resulting in high sensitivity and
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(a)

(b)

QD

QD

(c)

UV excitation

(d)

980nm excitation

QD

UCN

(e)

(f)

FIGURE 9 | In vivo imaging of rat: quantum

dots (QDs) injected into translucent skin of foot (a) show uorescence, but not through thicker skin of back (b) or abdomen (c); PEI/NaYF4 : Yb, Er nanoparticles injected below abdominal skin (d), thigh muscles (e), or below skin of back (f) show luminescence. QDs on a black disk in (a and b) are used as control. (Reproduced with permission from Ref 126. Copyright 2008 Elsevier).

photostability. However, the quantum yield and the wavelength tunability of the nanoparticles depend on the contained NIRF organic dyes. Up to now, NIRF organic dyes with better optical properties for inclusion in the nanoparticles are still needed. NIRF QDs exhibit high uorescence quantum yields, very long effective Stokes shifts, and high resistance to photobleaching. By changing the size or the particles, the emission bands of NIRF QDs can be tuned. In addition, the size of NIRF QDs is usually smaller than the NIRF dyes containing nanoparticles and UCNs. NIRF QDs have become the fastest growing probes in cancer molecular imaging, although the long-term toxicity of NIRF QDs is a big concern and needs to be carefully investigated. In view of the impact of the high-energy UV or visible photons on the tissues, UCNs are a promising alternative new class of nanoparticle-based NIRF probes, but the size of UCNs is relatively large. And their application of UCNs for in vivo cancer imaging is yet to be evaluated. Besides the nanoparticle-based NIRF probes described in this article, there are many other promising nanomaterialsbased probes under active investigation, such as Au and Ag nanoclusters, carbon nanotubes, etc. In addition, multimodal nanoparticles with NIRF and other imaging properties are novel materials
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for in vivo cancer detection, which could provide very powerful and highly complementary imaging information. The magnetic QDs for multimodal imaging have been well reviewed.132 The synthesis of uorescent and paramagnetic LDL nanoparticles,133 UCNs combined optical and magnetic resonance properties by co-doping with the rare earth ions Gd3+ and Er3+ /Yb3+ /Eu3+ ,129 and dual-function PET/NIRF probe based on 64 Cu labeled QDs134 have also been reported. It is beyond the scope of this review to conclude which nanoparticle-based NIRF probe is the most advantageous for in vivo cancer imaging. As reviewed in this manuscript, some successful small animal models of human cancer imaging have been performed using NIRF dyes containing nanoparticles and NIRF QDs, respectively. The well displayed tumor cell-targeted optical imaging and the effective imaging of deep tissues in animals also highlight that the UCNs are promising probes for in vivo imaging of cancer. However, to date, none of the nanoparticlebased NIRF probes came close to clinical application. There are still many critical roadblocks that need to be overcome before the nanoparticle-based NIRF probes can be ready for clinical translation.
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The toxicity of the nanoparticles is an importantissue that has to be resolved. It must be noted that the public still debates over the health and safety of nanoparticles. Drug corporations are also suspicious of the practical application of the nanoparticle probes. The toxicity of the manufactured nanoparticles is related to their size, concentration, structure, surface coating, and chemical composition. Renal excretion represents a desirable pathway for nanoparticle probes clearance from the blood. During kidney ltration, particles larger than 10 nm usually do not pass the lter. Therefore, the size of nanoparticle probes must be considered. An evaluation of the toxicity of nanoparticles could also be made on the basis of the chemical composition. FDA-approved biodegradable and biocompatible materials are more promising as starting materials for the nanoparticle-based NIRF probes preparation. Surface modication of nanoparticle-based NIRF probes is also critical for improving their aqueous dispersibility as well as obtaining appropriate surface functionality for bioconjugation. Dispersibility improvement can prevent aggregation and decrease the toxicity of nanoparticle-based NIRF probes. The bioconjugation can efciently enhance the targeting ability of nanoparticle-based NIRF probes for cancer in vivo imaging. Ideal coating strategies for nanoparticle-based NIRF probes should diminish toxicity effects and preserve the recognizing and binding capability of the modied nanoparticles, which are essential to be developed and investigated. Furthermore, the longer circulation times, target specicity and sensitivity are desired for the in vivo cancer

imaging using nanoparticle-based NIRF probes. The prolonged circulation time may help the efcient binding of the nanoparticle-based NIRF probes to the specic receptors of tumor. As we know, macrophages are widely distributed in many tissues of the body to recognize and clear altered and senescent cells. They are also able to endocytose foreign nanoparticlebased NIRF probes which provide an opportunity for the in vivo imaging of tumor tissues with rich presence of macrophages. This propensity of macrophages will also decrease the targeting specicity and sensitivity of nanoparticle-based NIRF probes for other tumor imaging. As reviewed in this manuscript, small animal models of human cancer in vivo imaging using nanoparticle-based NIRF probes have demonstrated that the live uptake is obvious even though the nanoparticle-based NIRF probes are modied with target molecules. To overcome these problems, attempts have to be made to obtain long-circulating and target-specic nanoparticle-based NIRF probes. In conclusion, development of NIRF nanoparticle probes for cancer imaging represents an exciting research area and may provide unique opportunities for human to ght cancer. The current evidence also suggests that there are many hurdles to overcome for advancing the nanoparticle-based NIRF probes in clinical applications. Overall, there are tremendous chances and challenges in this highly dynamic and relatively new rising eld. Development of nanoparticle-based NIRF probes is a promising approach for noninvasive cancer imaging at the early stage.

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