quantumdot bioconjugates for in vitro

Cancer Biomarkers 4 (2008) 307–319 307IOS Press

Quantum dot bioconjugates for in vitrodiagnostics & in vivo imaging

Yun Xing and Jianghong Rao∗Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University School of MedicineStanford, CA 94305, USA

Abstract. Semiconductor quantum dots are tiny light-emitting nanocrystals (2–10 nm) that have captivated researchers in thebiomedical field in the last decade. Compared to organic dyes and fluorescent proteins, quantum dots (QDs) have unique opticalproperties such as tunable emission spectra, improved brightness, superior photostability, and simultaneous excitation of multiplefluorescence colors. Since the first successful reports on biological use of QDs a decade ago, QDs and their bioconjugates havebeen successfully applied in various imaging applications including fixed cell labeling, imaging of live cell dynamics, in situtissue profiling, fluorescence detection, sensing and in vivo animal imaging. In this review, we will cover the optical propertiesof QDs, the biofunctionization strategies, their in vitro diagnostic applications and in vivo imaging applications. In addition, wewill discuss the making of a new class of QDs – the self-illuminating QDs and their in vivo imaging and sensing applications.We will conclude with the issues and perspectives on QDs as in vivo imaging probes.

Keywords: Molecular probes, quantum dots, in vivo imaging, optics

1. Introduction

The development of a wide spectrum of nanoscaletechnologies is beginning to change the foundations ofdisease diagnosis, treatment, and prevention. Thesetechnological innovations, referred to as nanomedicineby the National Institutes of Health (Bethesda, MD,USA), have great potentials to lead to major advancesin disease detection, diagnosis, and treatment and even-tually personalized therapy and disease management.The basic rationale is that metal, semiconductor, andpolymeric nanoparticles possess novel optical, elec-tronic, magnetic, and structural properties that are oftennot available from single individual molecules or bulksolids [1]. Recent research has developed function-al nanoparticles that are covalently linked to biologi-cal molecules such as peptides, proteins, nucleic acids,or small-molecule ligands [2–4]. Medical applicationshave also emerged, such as the use of superparamag-

∗Corresponding author. Fax: +1 650 736 7925; E-mail: [email protected].

netic iron oxide nanoparticles as a contrast agent forlymph node prostate cancer detection [5] and the useof polymeric nanoparticles for targeted gene deliveryto tumor vasculatures [6]. In this review, we will focuson the use of semiconductor quantum dots for in vitrodiagnostics and in vivo animal imaging.

QDs are light-emitting semiconductor nanorystalswith novel optical and electrical properties. Com-pared with organic dyes and fluorescent proteins, semi-conductor QDs offer several unique advantages, suchas size- and composition-tunable emission from vis-ible to infrared wavelengths, large absorption coef-ficients across a wide spectral range, and very highlevels of brightness and photostability [7]. By far,QDs have found applications ranging from bioanalyt-ical assays, to live cell imaging, fixed cell and tissuelabeling, biosensors and in vivo animal imaging [4].The long-term photostability and superior brightnessof QDs make them appealing for live animal target-ing and imaging. Additionally, recent studies haveshown that QDs have two-photon action cross sectionsof magnitude larger than those of conventional fluo-rescent probes now in use, a further plus for deep tis-

ISSN 1574-0153/08/$17.00 2008 – IOS Press and the authors. All rights reserved

308 Y. Xing and J. Rao / Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging

sue imaging [8]. In this paper, we will cover quantumdots optical properties, biological functionlization, invitro diagnostics applications and in vivo animal imag-ing applications, and end with a discussion on the is-sues/limitations and future perspectives.

2. Optical properties of quantum dots

The classic and most commonly used quantum dotsconsist of a CdSe core and a shell layer made of ZnSor CdS. Fluorescence properties are determined by thecore materials and the shell layer removes surface de-fects and prevents nonradiative decay, leading to a sig-nificant improvement in the particle stability and fluo-rescence quantum yields. For biological imaging ap-plications, these hydrophobic dots can be made watersoluble by exchanging with bifunctional ligands (most-ly thiol or phosphine mono or multidentate ligands) orcoating with amphiphilic polymers that contain botha hydrophobic segment or side chain (mostly hydro-carbons) and a hydrophilic segment or group (suchas polyethylene glycol [PEG] or multiple carboxylategroups) Fig. 1a [3]. Additionally, biomolecules suchas antibodies, peptides can be attached to the QDs toachieve specific labeling and targeting.

The novel optical property of QDs arises from theso-called “quantum confinement” effect of the semi-conductor materials. This refers to the size- andcomposition-dependence of the semiconductor bandgap energy. For nanocrystals smaller than the so-calledBohr excitation radius (a few nanometers), energy lev-els are quantized, with values directly related to the sizeof the nanoparticle. This dependence of light emissionon the particle size allows the development of new fluo-rescence emitters with precisely tuned emission wave-lengths (Fig. 1b). For example, the semiconductor cad-mium selenide (CdSe) has a bulk bandgap of 1.7 eV(corresponding to 730 nm light emission). QD of thismaterial can be tuned to emit between 450–650 nm bychanging the nanocrystal diameter from 2 to 7 nm. Thecomposition of the material may also be used as a pa-rameter to alter the bandgap of a semiconductor. QDwith a diameter of 5 nm can be tuned to emit between610–800 nm by changing the composition of the alloyCdSexTe1−x [9]. This property allows the productionof virtually “unlimited” number of fluorophores usingthe same material.

Compared with organic dyes and fluorescent pro-teins, QDs have several advantages and unique ap-plications. Firstly, QDs have very large molar ex-

tinction coefficients in the order of 0.5–5 × 106

M−1cm−1 [10], about 10–50 times larger than that (5–10 × 104 M−1cm−1) of organic dyes. Therefore, QDsare able to absorb 10–50 times more photons than or-ganic dyes at the same excitation photon flux (that is,the number of incident photons per unit area), leadingto a significant improvement in the probe brightness;making them brighter probes under photon-limited invivo conditions (where light is severely attenuated byscattering and absorption). In theory, the lifetime-limited emission rates for single QDs are 5–10 timesslower than those of single organic dyes because oftheir longer excited state lifetimes (20–50 ns). In prac-tice, however, fluorescence imaging usually operatesunder absorption-limited conditions, in which the rateof absorption is the main limiting factor of fluorescenceemission (versus the emission rate of the fluorophore).As a result, individual QDs have been found to be 10–20 times brighter than organic dyes [11]. Secondly,QDs are several thousand times more resistant againstphotobleaching than organic dyes (Figure 1d) and arethus well-suited for continuous tracking studies over along period of time. In addition, the longer excited statelifetimes of QDs can be used to separate the QD fluo-rescence from background fluorescence, in a techniqueknown as time-domain imaging [12]; since QDs emitlight slowly enough that most of the background aut-ofluorescence emission is over by the time QD emissionoccurs. Thirdly, the large Stokes shifts of QDs (mea-sured by the distance between the excitation and emis-sion peaks) can be used to further improve the detectionsensitivity. The Stokes shifts of semiconductor QDscan be as large as 300–400 nm, depending on the wave-length of the excitation light. Organic dye signals witha small Stokes shift are often buried by strong tissue aut-ofluorescence, whereas QD signals with a large Stokesshift are clearly detectable above the background. Afurther advantage of QDs is that multicolor QD probescan be used to image and track multiple molecular tar-gets simultaneously. This feature is very desirable be-cause most complex human diseases such as cancerand atherosclerosis involve a number of genes and pro-teins. Tracking a panel of molecular markers at thesame time will lead to better understanding, classifyingand differentiating complex human diseases than a sin-gle biomarker each time. Multiple parameter imaging,however, represents a significant challenge for magnet-ic resonance imaging, positron emission tomography,and computed X-ray tomography. By contrast, fluo-rescence optical imaging provides both signal intensityand wavelength information, and multiple wavelengths

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or colors can be resolved and imaged simultaneously(color imaging). In this regard, QD probes are particu-larly attractive, because their broad absorption profilesallow simultaneous excitation of multiple colors andtheir emission wavelengths can be continuously tunedby varying particle size and chemical composition. Fororgan and vascular imaging in which micrometer-sizedparticles could be used, optically encoded beads (poly-mer beads embedded with multicolor QDs at controlledratios) could allow multiplexed molecular profiling invivo at high sensitivities [13,14].

3. Biofunctionization

To make QDs more useful for molecular imagingand other biological applications, QDs need to be con-jugated to biological molecules without disturbing thebiological function of these molecules. Biomoleculesincluding peptides, proteins and olgio nucleotides havebeen successfully linked to QDs. Several successfulapproaches have been used to link biological moleculesto QDs, including non-specific adsorption, electrostat-ic interaction, mercapto (-SH) exchange, and covalentlinkage (Fig. 2) [15]. Firstly, biological molecules con-taining thiol groups can be conjugated to the QD surfacethrough a mercapto exchange process [16–19]. Unfor-tunately, since the bond between Zn and thiol is not verystrong and is dynamic. Biomolecules attached to QDsin this way can readily dissociate from the nanoparticlesurface, causing QDs to precipitate from the solution.

It has also been reported that simple small molecules,such as oligonucleotides [20,21] and various serum al-bumins [22], are readily adsorbed to the surface of wa-ter soluble QDs. This adsorption is nonspecific anddepends on ionic strength, pH, temperature, and thesurface charge of the molecule. Mattoussi and cowork-ers presented a method of conjugating proteins to QDsurfaces using electrostatic interactions. The proteinof interest was engineered with a positively chargeddomain (poly histidine), which in turn interacted elec-trostatically with the negatively charged surface of di-hydrolipoic acid (DHLA)-capped QD. The protein-QDconjugates prepared in this way were stable and thefluorescence quantum yield was even higher than thatfrom the nonconjugated QDs [23]. Electrostatic inter-actions generally are not sufficiently specific, however,given the complexity of biological milieu. Therefore,conjugates made this way are not suitable for in vivo orex vivo cell labeling due the possible interference frompositively charged proteins.

A more stable linkage is obtained by covalently link-ing biomolecules to the functional groups on the QDsurfaces using cross-linker molecules (Fig. 2) [7,24–27]. This method is the most commonly used approachfor making biofunctionized QDs for in vitro cell label-ing and in vivo imaging purposes. Most water solubi-lization methods result in QDs covered with carboxylicacid, amino or thiol groups. Under these situations,it is easy to link QDs to biological molecules whichalso have these functional groups. For example, thecross-linker 1-ethyl-3-(3-dimethylaminopropyl) car-bodiimide (EDC) is commonly used to link -NH2

and -COOH groups, whereas 4-(N-maleimidomethyl)-cyclohexanecarboxylicacid N-hydroxysuccinimidees-ter (SMCC) can be used to cross-link -SH and -NH2

groups. Using these methods, there have been nu-merous reports of conjugating QDs with various bi-ological molecules, including biotin [24], oligonu-cleotides [28], peptides [29], and proteins includingavidin/streptavidin [30], albumin [3], adaptor proteins(e.g. protein A, protein G) and antibodies [27,30]. Inaddition, the native functional groups (-COOH, -NH2

or -SH) on a water – soluble QD surface can be furtherconverted to other functional groups to allow more ver-satile conjugation of QDs to biomolecules (site-specificconjugation, molecules that are sensitive to EDC orSMCC modification) (Fig. 2). For instance, carboxylicacids on QDs have been converted to hydrazides, al-lowing attachments of biomolecules containing sugargroups [27]. Recently, Zhang et al. presented anotherstrategy for site-specific conjugation of biomoleculesto QD surface [31]. This study utilized the specific andstable binding between HaloTag protein (HTP) and itsligand. QDs were first functionilized with HaloTag lig-ands, and the protein of interest (e.g Renilla luciferase,Lu8) was genetically fused to a HTP. When mixed to-gether, QDs and Luc8 can be immobilized on QDsthrough the HTP-HaloTag ligands linkage. In mostcases, the biological functions of these molecules havebeen preserved during the conjugation process.

4. In vitro diagnostic applications

Significant opportunities exist at the interface be-tween biomarkers and nanotechnology for molecularcancer diagnosis. In particular, nanoparticle probes canbe used to quantify a panel of biomarkers on intactcancer cells and tissue specimens, allowing a correla-tion of traditional histopathology and molecular signa-tures for the same material. A single nanoparticle is


Fig. 1. Quantum dots structure and novel optical properties a) Structure of a multifunctional QD probe, showing the capping ligand TOPO,an encapsulating copolymer layer, tumor-targeting ligands (such as peptides, antibodies or smallmolecule inhibitors) and polyethylene glycol(PEG) [11]; b) Size-tunable emission spectra of QDs. This image shows ten distinguishable emission colors of ZnS-capped CdSe quantum dotsexcited with a near-UV lamp. From left to right (blue to red), the emission maxima are located at 443, 473, 481, 500, 518, 543, 565, 587, 610,and 655 nm [1]; c) Excitation (dotted line) and fluorescence (solid line) spectra of fluorescein (top) and a typical water-soluble QD (bottom) [24]and d) Superior photostability of QDs as compared to organic dyes [30]. (Colors are visible in the online version: www.iospress.nl)

large enough for conjugation to multiple ligands, lead-ing to enhanced binding affinity and exquisite speci-ficity through a “multivalency” effect. These featuresare especially important towards the analysis of cancerbiomarkers that are present at low concentrations or insmall numbers of cells. In this section, we will dis-cuss some in vitro diagnostic applications of QDs as afluorescent labeling agent.

The ability to study molecular and cellular events byusing fluorescent probes has broadly impacted manyareas in biomedical research including cell/molecularbiology, drug screening, and molecular diagnostics.However, traditional fluorophores such as organic dyesand fluorescent proteins suffer from several intrin-sic problems including rapid photobleaching, spectralcross-talking, narrow excitation profiles, and limitedbrightness/signal intensity. In contrast, the novel op-

tical properties of QDs overcome many of the prob-lems and offer new applications which are either dif-ficult or impossible with traditional fluorophores. Forinstance, due to their broad excitation profiles and nar-row/symmetric emission spectra, high-quality QDs arewell suited for multiplexed tagging or encoding, inwhich multiple colors and intensities are combined toencode thousands of genes, proteins, or small-moleculecompounds 3[3].

4.1. In vitro cell labeling

Wu et al. were among the first demonstrating thatQDs can be used to specifically and effectively labelmolecular targets at the subcellular level [30]. In thisstudy, QDs encapsulated within a polymer-shell werebiofunctionized with molecules (streptavidin and im-


Fig. 2. Common methods used for the QD biofunctionization, including direct linkage to the TOPO coated QDs (Biomolecules can be linkedinclude thiolated DNA (ligand exchange) or peptides with adhesive domains (see [4] for details), electrostatic interaction [23], and covalentlinking (see text for references).

munoglobulin) and applied to target cell surface recep-tor, cytoskeletal components (actin and microtubules)and nuclear antigen in both fixed and living cells. QDsof two different colors (630 nm and 535 nm) wereused simultaneously and compared to an Alexa dye.Their results showed that QDs were considerably morephotostable than the Alexa dye. Since then, QD-bioconjugates (previously commercially available fromQuantum dot corporation, now Invitrogen) have beenused in a myriad of applications including in situ tissue

profiling [27,32] and fluorescent in situ hybridizationapplications [33].

4.2. In situ tissue diagnostics

Diagnostic and prognostic classifications of humantumors are currently based on immunohistochemistry(IHC), a technique that has been used in clinicalmedicine for over 80 years [33]. However, the immu-noenzyme -based IHC method has a single color nature


and is incapable of multiplexed molecular profiling.The feature of multiplexing is becoming increasing im-portant for cancer diagnosis since more and more stud-ies are showing that a panel of biomarkers rather than asingle one is needed to accurately determine the stage ofthe disease. Multiplexing has the advantage of reduc-ing variability between tissue slices and is especiallyappealing in the case of precious specimens. The sec-ond issue with IHC is that it remains semi-quantitativeand subjective, resulting in considerable inter-observervariation of the results. Immunofluorescence with or-ganic fluorophores overcomes some of these problems(multiplexing, quantitative) but so far failed to prevailbecause of the poor photostability of these dyes. More-over, multiplexed fluorescent labeling with convention-al fluorophores is complicated with the need for a so-phisticated laser system due to the non-ideal spectraproperties of organic dyes. With the incorporation ofQDs, quantitative and multiplexed fluorescent labelingbecomes easy and handy. For instance, the simultane-ous excitation of multiple colored QDs eliminates theneed of a complex laser system and their superior pho-tostability allows the samples to be stored and imagedmany times for later analysis. Recognizing these ad-vantages, several groups have successfully developedprocedures for multiplexed fluorescent labeling of tis-sue specimens using QDs [27,32–35]. It is worth tonote that in order for successful translation of QDs toclinical practice, it is important to gear the technologydevelopment toward real clinical specimens, the ma-jority of which are archived, formalin fixed paraffin-embedded tissues and might be several decades old.Since the clinical outcomes of these tissues are alreadyknown, it is of great value to use these specimens forexamining the relationship between molecular profileand clinical outcome. Compared with cells or freshlyharvested animal tissues, archived human specimensneed special treatment such as harsher antigen retrieval(e.g. EDTA buffer (pH 8.0) vs. citrate buffer) and theincubation, and their background autofluorescence isgenerally stronger. It has been demonstrated that up to5 biomarkers can be labeled and imaged simultaneous-ly in the same tissue specimen [27,32]. Combined withspectral analysis and automated image processing soft-ware, quantitative molecular profiling of clinical tissuespecimens is likely to become the first clinical trans-lation of QDs; since unlike in vivo imaging, in vitroprofiling of tissue specimens do not face the problemssuch as toxicity and opsonization issues.

Although QDs have demonstrated great potential forin vitro tissue diagnostics and clinical translation seems

not far away, several issues need to be solved in orderfor this technology to achieve widespread adaptationor significant clinical success. Firstly, systematic vali-dation studies are urgently needed to verify the resultsfrom QDs labeling with conventional techniques suchas western blotting and PCR. Some of the groups havealready started and the initial results looked promis-ing [33]. Secondly, more work on QD surface engineer-ing and biofunctionization are needed to fully exploitthe multiplexing potential of QDs for molecular profil-ing. This could include new conjugation chemistriesthat allow well-controlled bio-ligand orientation andthe number of bio-ligands per nanoparticle and “friend-ly” chemistries which impart minimal detrimental ef-fects to the ligand. For instance, the affinity of an anti-body should not be significantly affected. Thirdly, theinclusion of housekeeping markers such as beta-actinor GAPDH is needed for standardization of the resultsand making the quantification more meaningful. Last-ly, more studies are needed to establish robust protocolsand experimental procedures to define the key factorsand steps involved in QD immunohistochemical stain-ing and data analysis. In particular, there are no consen-suses on methods for QD– antibody (QD–Ab) biocon-jugation, tissue specimen preparation, multicolor QDstaining, image processing and data quantification [27].

In addition to tissue diagnostics, there are alsoemerging applications of QDs for in vitro detection as-says and many of them are fluorescence resonance en-ergy transfer (FRET) based DNA hybridization appli-cations [36–38]. In terms of detection sensitivity, QDsare less competitive than other nanoparticles such asthe gold nanoparticle based bio-bar-codes developedby Mirkin and co-workers [39]. The unique advantageof QDs over other nanoparticles/nanostructures for invitro detection assays is their multiplexing capabilityand the eventual winning diagnostic application willmost likely to be the one that utilizes this feature suchas the QD-tagged microbeads technology for DNA hy-bridization reported by Han et al. [14].

5. In vivo imaging applications of quantum dots

While fluorescence imaging is often limited by thepoor transmission of visible light through biologicaltissue, there is a near-infrared (NIR) optical windowin most biological tissue that is suitable for deep-tissueoptical imaging [40]. Only a few organic dyes emitbrightly in this spectra region, and they suffer from pho-tobleaching. On the contrary, the novel optical proper-


ties of QDs allows the synthesis of bright and stable flu-orescent labels that emit in the near-infrared spectrumby adjusting their size and composition [41]. Becausevisible QDs are more synthetically advanced, most an-imal imaging studies implementing quantum dots haveused CdSe/ZnS QDs that emit visible light and a fewrecent studies started use near-infra red dots [41,42].Although still far from its mature stage, these studieshave demonstrated the great performance and promiseof QDs as fluorescent imaging agent in living animals.

5.1. Non-targeted animal imaging

Quantum dots have been used for non-targeted imag-ing in various animal models. Ballou and coworkersinjected PEG-coated QDs into the mouse blood streamand investigated how the surface coating would affecttheir circulation lifetime [43]. In contrast to small or-ganic dyes, which are eliminated from the circulationwithin minutes after injection, PEG-coated QDs werefound to stay in the blood circulation for an extendedperiod of time (half-life more than 3 h). This long-circulating feature is due to the relatively large size ofPEG-coated QDs, which falls within an intermediatesize range: they are small enough and sufficiently hy-drophilic to slow down opsonization and reticuloen-dothelial uptake, but are large enough to avoid renalclearance. Amazingly, these QDs maintained their flu-orescence even after four months in vivo. In 2003, Lar-son et al intravenously injected green QDs (550 nm) in aliving mouse and visualized them dynamically throughthe skin (in capillaries hundreds of micrometers deep)by using two-photon microscopy (Fig. 3a) [8]. In addi-tion to the superior brightness and photostability, thisstudy also found that QDs have two-photon excitationcross sections as high as 47,000 Goeppert-Mayer units,by far the largest of any label used in multiphoton mi-croscopy. Two-photon excitation allows greater tissuepenetration due to excitation within the NIR spectralrange (e.g. 900 nm), but few fluorophores are brightenough for these purposes; QDs, with their large multi-photon excitation cross section, appear as ideal probesfor multiphoton microscopy imaging.

Sentinel lymph node (SLN) SLN mapping is a tech-nique that the assessment of nodal status for the spreadof neoplasms. The underlying hypothesis of SLN map-ping is that the first lying node to receive lymphaticdrainage from a tumor site will contain tumor cells ifthere has been direct lymphatic spread. ConventionalSLN mapping involved using a blue dye (for visual-ization of lymphatic vessels) and radioactive tracer to

improve detection rate [44]. In 2004, Kim et al. [41]reported a non-invasive, real-time SLN mapping andresection by using near-infrared QDs. Advantages ofthis method arise from QD’s novel optical properties in-cluding strong NIR fluorescence, superior photostabil-ity in body fluid and the relatively larger size (∼15 nm)which prevents leakage into neighboring regions andensures retention of the particles in the SLNs. In thisstudy, the authors prepared a novel core-shell nanos-tructure called type II QDs with fairly broad emissionat 850 nm and a moderate quantum yield of ∼13%.In contrast to conventional QDs (type I), the shell ma-terials in type II QDs have valence and conductionband energies both lower than those of the core mate-rials. As a result, the electrons and holes are physi-cally separated and the nanoparticles emit light at re-duced energies (longer wavelengths). These NIR QDsmade water soluble by oligomeric phosphine coatingand injected intradermally in the left paw of a livingmouse. Their results showed rapid uptake (5 min) ofQDs into nearby lymph nodes and could be imaged vir-tually background-free (Fig. 3b). The same study alsodemonstrated the feasibility of image-guided surgery ina big animal model. 400 pmole of NIR QDs injected in-tradermally permits sentinel lymph nodes 1 cm deep tobe imaged easily in real time (and removed surgically)using excitation fluorescence rates of only 5 mW/cm2.The versatility of this technique was further proven bysuccessful applications for SLN mapping in the lungand the gastrointestinal tract [44].

QDs have also been used for cell tracking studies [11,45–48]. QDs were delivered into live mammalian cellsvia three different mechanisms: non-specific pinocy-tosis, microinjection, and peptide-induced transport(e.g. using the protein transduction domain of HIV-1Tat peptide, Tat-PTD) [49]. A surprising finding wasthat two billions of QDs could be delivered into thenucleus of a single cell, without compromising its via-bility, proliferation or migration [45,47,49]. The abil-ity to image single-cell migration and differentiationin real time is expected to be important to several re-search areas such as embryogenesis, cancer metasta-sis, stem-cell therapeutics and lymphocyte immunolo-gy. These studies demonstrated the potential of usingQDs to track cell, tissue, and organ developments overextended periods of time, a task not possible with smallmolecule organic dyes with fast photobleaching.

5.2. Targeted in vivo imaging

The above studies demonstrated the capability ofQDs for living animal imaging, but have only demon-


Fig. 3. Animal imaging applications using quantum dots. a) Two-photon fluorescence imaging of capillaries at the base of the dermis withQD (1 µM) (top) and fluorescein (40 µM) (bottom) intravenously delivered to the animal (adapted from [8]). b) NIR QD fluorescence guideddissection of a lymph node in a pig. 400 pmol of NIR QDs were injected intradermally in the right groin [41]. c) Targeted tumor imaging usingquantum dots antibody conjugates [11]. d) Tumor imaging using QD705 RGD bioconjugates [42].

strated image contrast at the tissue/organ level. Thegoal of molecular imaging is to generate image contrastdue to the molecular difference in different tissues andorgans. This requires a probe that has a targeting moietyto generate contrast only in locations specified by thetargeting probe. Akerman et al. were among the first toexplore the possibility of using QD-peptide conjugatesto target tumor vasculatures in-vivo [19]. QDs coat-ed with peptides targeting the lung-vasculature, bloodvessel and tumor cell, or lymphatic vessels were in-jected systematically into mouse. Although they werenot able to image the QDs in living animal, histolog-ical sections of different organs after 5 or 20 min ofcirculation revealed that QDs homed to tumor vesselsguided by the peptides, but not to surrounding tissues,probably due to their larger size relative to organic dyes(which would stain surrounding tissues). Whole animalimaging of molecular-level detection was realized byGao et al. in 2004 using red fluorescent QDs conjugat-ed to antibodies specific to prostate-specific membraneantigen (PSMA) on a human prostate cancer inducedin a mouse [11]. QD conjugates used in this studycontained an amphiphilic triblock copolymer for in vi-vo protection, targeting ligands (anti-PSMA) for tumorantigen recognition, and multiple PEG molecules forimproved biocompatibility and circulation. The QD-tagged PSMA antibodies recognized and bound at the

tumor site and were clearly imaged in vivo (Fig. 3c).Controls experiments with QD-PEG (no targeting lig-and) or QD only confirmed the binding was specificand the accumulation of non-targeted QDs at tumorsites was marginal compared with the targeted ones.There are two possible mechanisms for the preferentialaccumulation of QDs at tumor sites: passive targetingdue to the enhanced permeability and retention (EPR)effect and active targeting because of the antibody, PS-MA, which is a cell surface marker for both prostateepithelial cells and neovascular endothelial cells. Be-cause the QDs emit in the visible range, a spectral un-mixing algorithm was used to separate QD signal frombackground autofluorescence. More recently, Cai et alused near-infrared QDs for tumor imaging by targetingangiogenesis, the formation of new blood vessels frompreexisting vasculture [42]. Amine-modified QD705(emission maximum at 705 nm) were conjugated toαvβ3 integrin targeting cyclic RGD peptide and inject-ed intravenously into living mice. Tumor fluorescencereached maximum at 6 hr post injection with good con-trast (Fig. 3d). Since angiogenesis is common to alltumors, this technique may aid cancer detection andmanagement in general. It is worth to note that in bothstudies, a significant portion of the injected QDs wentto the RES system, including the liver, spleen and lung.


5.3. In vivo imaging using self-illuminating quantumdots

Self-illuminating quantum dots (also known as QD-BRET conjugates) are a class of new QDs that do notrequire external excitation light to fluoresce, instead,energy comes from a bioluminescence protein throughnon-radiative energy transfer from a neighboring bi-oluminescenct proteins. Bioluminescence resonanceenergy transfer (BRET) is a naturally occurring phe-nomenon whereby a light-emitting protein (the donor,e.g. R. reniformis luciferase) non-radiatively transfersenergy to a fluorescent protein (the acceptor, e.g. GFP)in close proximity. BRET is analogous to FRET ex-cept that the energy comes from a chemical reactioncatalyzed by the donor enzyme (e.g. R. reniformisluciferase-mediated oxidation of its substrate coelen-trazine) rather than absorption of excitation photons.Compared to fluorescence imaging, bioluminescencehas extremely high sensitivity for in vivo imaging pur-poses [50]. Recently, work in the Rao lab has demon-strated the feasibility of using QDs as the acceptorin a bioluminescence resonance energy transfer sys-tem [51]. In this study, QDs were covalently conjugatedby to the donor, Luc8 protein, an eight-mutation variantof the bioluminescence R. reniformis luciferase [52].The protein emits blue light with a peak at 480 nmupon the addition of substrate, coelentrazine. If a QDis in close proximity of the protein, it can be excitedand emit at its emission maximum (Fig. 4). The ad-vantage of using bioluminescence versus fluorescencelies in the fact that no external excitation is needed.This “self-illuminating” feature allows cancer imagingin deeper tissue where light is limited. Since no exci-tation light is needed, the autofluorescence problem isautomatically solved. In addition, since this couplingmethod is generic, any QDs with carboxyl groups (in-cluding those emits in the NIR region, e.g. 705, 800 nmQDs have been tested) can be used as a BRET receptorand therefore allows for multiplexed imaging. Com-pared with existing QDs, self-illuminating QD conju-gates have greatly enhanced sensitivity in small animalimaging, with an in vivo signal-to-background ratio of>1000 for 5 pmole of conjugates subcutaneously in-jected. One critical issue in making these self-emittingQDs is the size of the nanoparticle, since like FRET,BRET is also a distance-sensitive process. Increase inthe QD conjugate size results in greater distance be-tween the protein and the fluorescent semiconductorcore, and thereby decreases the energy transfer effi-ciency significantly. For instance, the authors have ob-

served that increasing the protein-nanoparticle distanceby only 2–3 nm causes the BRET ratio to drop from1.29 to 0.37 [51]. By attaching targeting moieties suchas tumor homing antibodies or peptides to the BRETassembly, it is possible to use BRET QDs for targetedtumor imaging in living animals.

More recently, QD-BRET was successfully appliedto proteolytic activity detection in buffer with a slight-ly different coupling scheme [53]. In this study, a 15amino acid peptide (GGPLGVRGGHHHHHH), con-taining the MMP-2 substrate and a six-histidine tag,was genetically fused to the C terminus of the BRETdonor, Luc8. In the presence of Ni2+ cations, the car-boxylic acids on the QDs will bind the metal ions andform complexes with the 6 x His tag on the Luc8 fu-sion protein. BRET will take place and produce lightemission from the QDs. The cleavage of the amidebond between Gly and Val by MMP-2 will release the6 x His tag from the fusion Luc8 and thus no BRETwill occur. In the presence of active MMP-2, the pro-tein is released from the conjugates and BRET did nothappen. Similar concept can be applied for in vivoprotease detection and imaging with a different cou-pling strategy which ensures site-specific conjugation(so that protease substrate positioned between QD andLuc8) and meanwhile makes the conjugates stable andresistant from interference from other proteins presentinside living body.

6. Future perspectives

Although QDs provide a class of exciting new fluo-rescent probes that opens up a lot opportunities for flu-orescence imaging, several issues remain and need tobe resolved before QDs can be widely used for targetedimaging of tumor or other diseases in human subjects.Some of these issues are applicable to all nanostructuresintended for molecular imaging purposes.

Due to their superior photostability and highlybrightness, QDs have been tested for in vivo imagingapplications from early on. Regardless of tremendousinterests and initial successes, the progress in recentyears is rather slow. This lack of breakthroughs reflectsthe nature of challenges in this aspect. In the rest ofthis section, we will go over each of the challenges andpoint out some future directions.


Fig. 4. QD-BRET based in vivo animal imaging. a) Schematic showing bioluminescence resonance energy transfer between Luc8 protein (donor)and QD (acceptor). Quantum dot (655 nm) is covalently linked to a BRET donor, Luc8. The bioluminescence energy of Luc8-catalyzed oxidationof coelenterazine is transferred to quantum dots in close proximity, resulting in QD emission (655 nm); b) In vivo imaging using QD-BRETconjugates (Image adapted from [51]).

6.1. RES uptake and blood circulation

The first issue is their relatively short circulationhalf-life, preventing long-term imaging or cell trackingstudies. Literature on the in vivo studies using QDsfor imaging have revealed that their circulation half-time is influenced strongly by the surface chemistryand that they are cleared from the circulation primar-ily by phagocytosis of the nanoparticle by RES in theliver spleen and lymph nodes [11,43]. Coating withPEG increases the circulation half-life, and attachmentto targeting moieties (such as antibodies) reduces thedose needed to generate contrast between normal andtumor tissue [11]. Even with these strategies incor-porated, the majority of nanoparticles still end up inthe reticuloendothelial system (RES). A recent studyusing commercial non-targeted amino (PEG) QD705for imaging glioma in rat suggested that maximal RESphagocytosis of QDs was reached between 3.4- and 8.5nmole doses [54]. Significant tumor uptake of QDswas observed for doses higher than 8.5 nmole. Such ahigh dose might pose health hazard to the living sub-jects. Therefore, there is an urgent need for engineer-ing strategies to improve the circulation half-time ofQDs, since the longer they can stay in the circulation

system the better chances are that they may be able toget to the tumor site. This might be achieved by min-imizing the opsonization or other components of theRES, and a few groups have started studying on the in-teraction between blood components and nanoparticlesin order to attack this problem [55,56]. The rationaleis that nanoparticles, once entered the blood stream,are immediately covered by plasma proteins; what theRES system “sees” and what defines the identity ofthe nanoparticle is largely the protein corona aroundthe particle, not the core material. Elucidation of theprofiles of adsorbed proteins on nanoparticles has thepotential to facilitate engineering a surface chemistrythat is less prone to opsonization and RES uptake. Thealternative approach is to make smaller QDs that com-pletely evade the RES system. A recent study by Fran-gioni laboratory [57] has shown that QDs with a hy-drodynamic size of 5.5 nm or smaller can evade theRES organs (no accumulation in the liver, spleen orlung) and be cleared by the renal system. QDs usedin this study were cysteine-coated [58] with no target-ing or other biofunctional groups, therefore it remainsa challenge, how to make biofunctionized QDs whilekeeping the size below 5.5 nm.


6.2. High quality NIR dots for deeper tissuepenetration

Although the superior brightness and photostabili-ty of QDs made them attractive candidates for in vi-vo animal imaging, most of the current QDs still emitwithin the visible range. The ideal QDs for deep tissueimaging, that is, high-quality QDs with near-infrared-emitting properties are not yet widely available. Recentdevelopments include a promising water-based synthe-sis method that yields particles that emit from the visi-ble to the NIR spectrum and are intrinsically water sol-uble, but the particles have yet to be tested in biologi-cal [59] environments. Most materials (e.g. PdS, PdSe,CdHgTe and CdSeTe) are either not bright or not stableenough for biomedical imaging applications. As such,there is an urgent need to develop bright and stablenear-infrared-emitting QDs that are broadly tunable inthe far-red and infrared spectral regions [11]. Theoreti-cal modeling studies by Lim et al. [60] indicate that twospectral windows are excellent for in vivo QD imaging,one at 700–900 nm and another at 1200–1600 nm. Inaddition to high quality NIR QDs, multiphoton fluores-cence microscopy and novel illuminating mechanismssuch as bioluminescence energy transfer can all be usedto achieve deeper tissue penetration.

6.3. Toxicity

One of the major issues that hinder the applicationof QDs to human subjects is the concern about theirsafety: cadmium and selenium are potential hazard forneurological and genitourinary toxicity. Indeed, in vi-vo toxicity is likely to be a key factor in determiningwhether QD imaging probes would be approved by reg-ulatory agencies for human clinical use. Cell culturestudies [61] indicate that CdSe QDs are highly toxic tocultured cells under UV illumination for extended pe-riods of time. This is not surprising because the energyof UV irradiation is close to that of a covalent chemi-cal bond and dissolves the semiconductor particles in aprocess known as photolysis, releasing toxic cadmiumions into the culture medium. In the absence of UVirradiation, QDs with a stable polymer coating are notlikely to be toxic to cells and animals. In vivo studiesby Ballou and coworkers also confirmed the nontoxicnature of stably protected QDs [43]. Still, there is anurgent need to systematically study the cellular toxici-ty and in vivo degradation mechanisms of QD probes.For polymer-encapsulated QDs, chemical or enzymaticdegradation of the semiconductor cores is unlikely to

occur. The polymer-protected QDs might be clearedfrom the body by slow filtration and excretion out ofthe body. This and other possible mechanisms must becarefully examined before any human applications intumor or vascular molecular imaging. While modifi-cations of QD surfaces can help QDs cleared from thebody within a reasonable time frane, an alternative is towill be more difficult to replace the toxic elements ofQDs while retaining replace the toxic elements of QDsbut retain similare optical properties (e.g. high quantumyield, stability) [15].

6.4. Perspectives

Quantum dots as a novel fluorescent probes haveproved to be tremendous useful in many areas of bi-ological and medical research, especially multiplexedtissue/cell labeling, live cell imaging as well as in vi-vo imaging. However, as an in vivo imaging agent,QDs are still at a very premature stage. Several issues(including toxicity, size issue and clearance by RES)remain before its potential can be fully exploited andapplied to human subjects. Although the performancesof visible QDs are greatly improved than convention-al fluorophores, an ideal QD fluorophore should emitin the near-infrared/far red region with high quantumyield and excellent stability. Meanwhile, techniquessuch as two-photon excitation [8] can be used to facili-tate the use of visible dots for better tissue penetration.In summary, quantum dots technology for in vivo can-cer imaging is still an area of active research and itsfurther development will benefit from the collaborationof chemists, biologists and material scientists.

Acknowledgements

The work in the authors’ laboratory was supportedby the Burroughs Wellcome Fund, the National Can-cer Institute Centers of Cancer Nanotechnology Ex-cellence (CCNE) 1U54CA119367-01, and the Depart-ment of Defense Breast Cancer Research Program Con-cept award.

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