REVIEW

Nanoparticles as contrast agents for in-vivo bioimaging:current status and future perspectives

Megan A. Hahn & Amit K. Singh & Parvesh Sharma &

Scott C. Brown & Brij M. Moudgil

Received: 18 August 2010 /Accepted: 7 September 2010 /Published online: 6 October 2010# Springer-Verlag 2010

Abstract Nanoparticle-based contrast agents are quicklybecoming valuable and potentially transformative tools forenhancing medical diagnostics for a wide range of in-vivoimaging modalities. Compared with conventional molecular-scale contrast agents, nanoparticles (NPs) promise improvedabilities for in-vivo detection and potentially enhancedtargeting efficiencies through longer engineered circulationtimes, designed clearance pathways, and multimeric bindingcapacities. However, NP contrast agents are not withoutissues. Difficulties in minimizing batch-to-batch variationsand problems with identifying and characterizing keyphysicochemical properties that define the in-vivo fate andtransport of NPs are significant barriers to the introduction ofnew NP materials as clinical contrast agents. This manuscriptreviews the development and application of nanoparticles andtheir future potential to advance current and emerging clinicalbioimaging techniques. A focus is placed on the application ofsolid, phase-separated materials, for example metals andmetal oxides, and their specific application as contrast agentsfor in-vivo near-infrared fluorescence (NIRF) imaging,magnetic resonance imaging (MRI), positron emission to-

mography (PET), computed tomography (CT), ultrasound(US), and photoacoustic imaging (PAI). Clinical and preclin-ical applications of NPs are identified for a broad spectrum ofimaging applications, with commentaries on the futurepromise of these materials. Emerging technologies, forexample multifunctional and theranostic NPs, and theirpotential for clinical advances are also discussed.

Keywords Nanoparticles . In-vivo imaging . Clinical .

Characterization .Multifunctional . Theranostic

Introduction

Noninvasive imaging and minimally invasive in-vivo bio-imaging techniques are valuable tools in the arsenal of clinicaldiagnostics. Many types of bioimaging are available, span-ning from techniques that enable whole-organism anatomicalimaging (e.g., magnetic resonance imaging, MRI) to othersthat provide specific molecular imaging (e.g., optical fluores-cence) at subcellular resolution. Such tools are expected to bepivotal for advancing early-stage cancer diagnosis, guidedstem cell therapies, drug delivery, pathogen detection, genetherapy, image-guided surgery, and cancer staging [1], inaddition to many other clinically relevant procedures,diagnostics, and therapies.

Nanoparticles (NPs) are a class of materials generallyranging in size from 1 to 100 nm that are emerging aspotentially powerful probes for in-vivo imaging in medicaland biological diagnostics. Several NP-based contrastagents have been developed to overcome issues that plagueconventional contrast agents; improvements in chemicaland photostability of NP fluorophores, and contrast agentdetection limits, have been demonstrated in a broad array ofimaging modalities. The ideal NP agent must fulfill a

Published in the special issue Nanomaterials for Improved AnalyticalProcesses with Guest Editors Miguel Valcárcel and Bartolomé M.Simonet.

M. A. Hahn (*) : P. Sharma : S. C. Brown :B. M. MoudgilParticle Engineering Research Center, University of Florida,205 Particle Science and Technology Building,P.O. Box 116135, Gainesville, FL 32611, USAe-mail: mhahn@perc.ufl.edu

A. K. Singh : B. M. MoudgilDepartment of Materials Science and Engineering,University of Florida,205 Particle Science and Technology Building,P.O. Box 116135, Gainesville, FL 32611, USA

Anal Bioanal Chem (2011) 399:3–27DOI 10.1007/s00216-010-4207-5

number of stringent requirements: it should be easilydispersible and stable (i.e., resist aggregation) in a varietyof local in-vivo environments and not be affected bydifferences in solvent polarity, ionic strength, pH, ortemperature when these conditions are not intended formeasurement; it should exhibit limited nonspecific bindingand be resistant to reticuloendothelial system (RES) uptake,and have programmed clearance mechanisms; and it shouldhave high sensitivity and selectivity for the target (e.g.,antigen, cell, tissue) with good contrast quality (high signal-to-noise ratio, SNR) and sufficiently long circulation timesin the blood if administered intravenously. Ideally, thesematerials will be suitable for long-term quantitative imagingat low doses and be safely cleared from the body afterimaging is complete.

Important areas in which NP-based contrast can proveadvantageous include tumor imaging for guided surgery,imaging of gene expression in vivo to elucidate diseasedevelopment, and efficacy of anti-cancer drugs. Forexample, a combination of in-vivo imaging can be used toimage a tumor pre-operatively utilizing MRI and intra-operatively using optical imaging that can then aid image-guided tumor resection. In-vivo imaging could track tumorresponse to chemotherapy treatments in real time—savingtime and money while diminishing patient discomfort andside effects—which would be an immensely powerful rolein the drug development of cancer therapeutics.

The performance of the bioimaging modality used, andthe contrast agents or probes, is dependent on the type ofinformation desired, the characteristics of the biologicaltarget, and the size and thickness of the subject. Differenttechniques are more powerful and safer on humans (e.g.,MRI) in a clinical setting, rather than fluorescence imaging,which is better suited toward small animal imaging forcancer and other disease models. Each type of in-vivoimaging technique has its own advantages and limitations,which include spatial and/or temporal resolution, sensitiv-ity, SNR, penetration depth in tissue, and quantitativeaccuracy. Table 1 highlights the most commonly used in-vivo imaging modalities. There is a high desire for earlierdetection and characterization of disease development anddetermining treatment effectiveness, rather than the endeffects of disrupted molecular processes (e.g., malignantcancers and metastases).

One area of research which has led to significantadvancement in achieving specific molecular targeting isthe development of biomarkers based on NP constructs.Regardless of composition, surface functionalization of thenanomaterial is often required to enable targeting andstealth for long circulation times with minimal nonspecificbinding. There is a plethora of entities that can beincorporated on to a NP’s surface, with covalent bondingpreferred over electrostatic interactions: DNA, RNA, and T

able

1Com

parisonof

common

lyused

bioimagingtechniqu

es(adapted

from

Refs.[225]and[226])

Technique

Typical

NPlabel

Signalmeasured

Resolution

Depth

Sensitiv

ity(m

oles

oflabeldetected)

Throughput

Cost

Mainlim

itatio

n

NIRF

QDs,dye-dopedNPs,upconvertin

gNPs,

SWNTsandothercarbon-based

nanomaterials

Light,particularly

inthenear-infrared

1–3mm

<1cm

10−1

2High

Low

Poordepthpenetration

MRI

Iron

oxideNPs,Gd(III)-doped

NPs,NP-based

CESTandhyperpolarized

probes

(e.g.,

129Xe)

Alteratio

nsin

magnetic

fields

50μm

Nolim

it10

−9–1

0−6

Low

High

Low

sensitivity,cannot

follo

wmanylabels

PET

NPsincorporatingradioisotopes(e.g.,

18F,

11C,

64Cu,

124I)

Positron

from

radionuclid

es1–

2mm

Nolim

it10

−15

Low

High

Can

detect

only

one

radionuclid

e,requires

radioactivity

SPECT

NPsincorporatingradioisotopes(e.g.,

99mTc,111In)

γ-rays

1–2mm

Nolim

it10

−14

Low

High

Requiresradioactivity

CT

IodinatedNPs,gold

NPs,iron

oxide-doped

nanomaterials

X-rays

50μm

Nolim

it10

−6Low

High

Poorresolutio

nof

soft

tissues

US

Microbubbles,nanoem

ulsions,silicaNPs,

polystyreneNPs

Sound

50μm

Several

cm10

−8High

Low

Poorim

agecontrast,

works

poorly

inair-containing

organs

PAI

Goldnanoshells,gold

nanocages,gold

nanorods,

gold

NPs,SWNTs,dye-dopedNPs

Sound

50μm

<5cm

10−1

2High

Low

Inform

ationprocessing

andmachinesstill

beingoptim

ized

4 M.A. Hahn et al.

oligonucleotides (aptamers); peptides, proteins, peptidomi-metics, enzymes, antibodies, and antibody fragments;tumor cell receptors, for example folate and Her2, orligands against particular antigens or epitopes; carbohy-drates; and agents to reduce the chance of an immunogenicresponse while increasing the circulation time in the blood,and stealthily avoiding the RES and promoting dispersi-bility and solubility (e.g., poly(ethylene glycol) (PEG),polymers, phospholipids, dextran, latex). No matter whatthe surface moiety, its activity must not be altered onceanchored to the NP surface.

This review will focus on several principle types ofNPs currently affecting or with the ability to improveupon powerful in-vivo imaging techniques, for examplenear-infrared fluorescence (NIRF) imaging, MRI, posi-tron emission tomography (PET), computed tomography(CT), ultrasound (US), and photoacoustic imaging (PAI).Table 1 provides an overview of the various techniquesthat are discussed, highlighting current NP-based contrastlabels for each technique. Multifunctional and theranosticNPs, and emerging technologies and their potential forclinical use, will also be discussed. Many types of NPsare under investigation as potential contrast agents for in-vivo imaging; however, the scope of this review will belimited mainly to rigid, sparingly soluble NPs, forexample those composed of metals, metal oxides, semi-conductors, and ceramics, and will not discuss in detailother “softer” NPs, for example those formed fromliposomes, dendrimers, polymers, proteins, and viruses.In addition to the online resource http://www.mi-central.org, information on numerous imaging techniques can befound in reviews by Massoud et al. [2] and Debbage et al.[3], the latter concentrating on molecular imaging usingnanoparticles.

Near-infrared fluorescence (NIRF) imaging

Fluorescence imaging is a powerful molecular imagingtechnique in which specific probes (i.e., fluorophores) areexcited by incident radiation, usually in the visible or NIR,and emit energy at a (usually) lower energy than that withwhich they were excited. Despite its extremely high-sensitivity detection and location of individual cells,mRNA, DNA, proteins, peptides, receptors, low-expressing cellular markers, and epitope distributions, itlacks the ability to provide anatomical resolution. In fact, itsresolution is limited to 2–3 mm [2]. However, fluorescenceand autofluorescence thoroscopic and endoscopic techni-ques are emerging as powerful diagnostic tools foridentifying disease and abnormal structural features onbody cavity surfaces. Regarding noninvasive imaging,fluorescence in the visible region is acceptable only for

thin tissue sections; the requirement for deeper penetra-tion depths for most clinical applications is drivingfluorescence-based techniques into the NIR region(650–950 nm). In this NIR window, the absorption ofwater, hemoglobin, and lipids are at their minimum whileautofluorescence and tissue scatter are low, enablingmaximum light penetration; therefore, high SNRs andsensitive detection limits result. Typically <1 cm, lightpenetration depth depends on the type of tissue imaged:skin and muscle are more transparent than organs havinglots of vasculature (e.g., liver and spleen) because ofabsorption by hemoglobin. However, new advances inoptical microscopy imaging techniques have increasedlight penetration depths [4]. The fluorophores must bebright with, preferably, large Stokes shifts and highfluorescence quantum yields in the NIR, photostable, andresistant to degradation in biological systems. Reviews onNPs used in in-vivo fluorescence imaging are available [3,5, 6]. A targeted approach is usually used for in-vivoimaging, with a moiety for a particular cellular targetconjugated to the NP surface.

Quantum dots

With their broad absorption spectra, large absorption crosssections, narrow and tunable emission spectra, highfluorescence quantum yields, and high photostability,inorganic semiconductor nanocrystals, or quantum dots(QDs), are a popular choice for fluorescence imagingapplications. Their optical properties enable multiplexing,where different colors of QDs are used in a single assaywith only one excitation source. Figure 1 demonstrates thepower and versatility of use of multiple QDs in a singleassay to visualize several lymphatic drainages in amouse. QDs that emit in the NIR include II–VI, IV–VI,and III–V compounds, for example CdSe, CdTe, HgTe,PbS, PbSe, PbTe, InAs, InP, and GaAs, alloys of thesecomponent materials, and even core/shell structures,which can tune the emission further and alter fluores-cence lifetimes. An interesting probe is self-illuminatingQDs, using fluorescence resonance energy transfer(FRET) from bioluminescent proteins conjugated to theQD [7]. However, potential toxicity from the heavy metalions may preclude their use in clinical bioimaging andmay limit their uses to in-vitro and diagnostic assays.However, they have been used successfully in a sentinellymph node mapping procedure that utilized intraoperativeNIRF imaging [8].

Dye-doped silica nanoparticles

NIR dye-doped silica NPs are becoming popular choices ofcontrast agent for a number of reasons: silica NPs are

NP contrast agents for bioimaging 5

optically transparent, water dispersible, biologically inert,nontoxic in the amorphous form, with well establishedconjugation strategies to modify the surface to proteins,peptides, and other ligands for cellular receptors usingsilane chemistry. Utilizing this matrix, numerous NIRfluorophores can be encapsulated, reducing the potentialtoxicity of these fluorescent probes and shielding theNIR emitter from the aqueous environment, where thedye usually suffers from low fluorescence quantum yield,degradation, and insufficient photostability. Dyes emit-ting in the NIR that can be incorporated into silica NPsinclude polymethines (e.g., Cy5.5, Cy7), indocyaninegreen (ICG), Alexa Fluor 750, and IRDye78, amongothers. Encapsulating thousands of dye molecules withinone silica NP provides a tremendous advantage: a singleNP loaded with dye molecules is much brighter andmore stable than its single-molecule counterpart. Dye-doped silica NPs are usually synthesized by a sol–gelprocess (i.e., Stöber) or a microemulsion system bysimply adding the dye (or a modified form of the dye) tothe silica-forming solution. In addition, the surface area

of these NPs can be increased by making mesoporoussilica NPs; this method enables loading an additionalcomponent into the resulting pores, for example atherapeutic agent capable of photothermal ablation or adrug able to be released at the appropriate time andlocation.

Upconverting nanomaterials

Upconverting NPs are a relatively new class of compoundsbeing developed as agents for in-vivo fluorescence imaging.Doped with rare-earth ions, these materials absorb NIR light(usually 980 nm) and emit upconverted light at a higherenergy, usually in the green or far-red/NIR, with longfluorescence lifetime (μs to ms) [9]. An example of such asystem is yttrium oxide (Y2O3) NPs doped with erbium andyttrium, which have excellent photostability in the NIR andlow toxicity [10]. In another study, Zhang and coworkersused upconverting polyethyleneimine-coated NaYF4:Yb,Erand NaYF4:Yb,Tm NPs excited with NIR laser light todemonstrate the imaging of visible fluorescence through

Fig. 1 In-vivo five-color lymphatic drainage imaging was able tovisualize five distinct lymphatic drainages. In-vivo and intrasurgicalspectral fluorescence imaging of a mouse injected with five carboxylQdots (565, blue; 605, green; 655, yellow; 705, magenta; 800, red)intracutaneously into the middle digits of the bilateral upperextremities, the bilateral ears, and at the median chin, as shown in

the schematic diagram. Five primary draining lymph nodes weresimultaneously visualized with different colors through the skin in thein-vivo image and are more clearly seen in the image taken at thesurgery. (Reprinted, with permission, from Ref. [227], Copyright 2007American Chemical Society)

6 M.A. Hahn et al.

mouse skin and even thigh muscle up to 10 mm deep[11]. However, upconversion may not be an advantage ifthe fluorescence is in the visible region, which suffersfrom the aforementioned tissue penetration issue, butmay be satisfactory for imaging thin tissue sections. Arecent study reports YF3:Yb

3+/Er3+ NPs having upcon-version luminescence in the NIR rather than in thevisible, which enables greater penetration of the light[12]. Some upconverting NPs also have NIR fluorescenceat energies lower than their excitation wavelength, thusaffording them even more advantages for bioimagingapplications [13]. An overview of luminescent NPs usingsuch doped host lattice systems can be found inBachmann et al. [14]. Other examples include NdF3/SiO2 core/shell NPs having excitation and emission in theNIR range, and efficient deep tissue imaging of small animals[9]. All of these materials can be dispersed in aqueoussolutions and conjugated to relevant biomolecules fortargeting purposes.

Carbon nanomaterials

Carbon-based nanomaterials are also potential NIRcontrast agents for in-vivo imaging. Single-walled carbonnanotubes (SWNTs) can have emission in the second IRwindow (1000–1350 nm), which would enable evendeeper light penetration [15]. However, the toxicity ofSWNTs is hotly debated, and reproducible synthesis andfunctionalization are lacking, as are the methods to obtainhigh-purity samples. Carbon dots were found to haveemission in the visible region when passivated by polymerchains [16]. These materials are being investigated foroptical imaging agents using both one and two-photonexcitation [17–19]; however, with their visible fluores-cence, small animal or thin tissue imaging remainspossible but clinical applications will most likely remainelusive. Colloidal diamond NPs (i.e., nanodiamonds) areyet another nanomaterial being investigated as potentialin-vivo fluorescent probes. With fluorescence originatingfrom N–vacancy, Si–vacancy, and Ni–N complexes, theyare biocompatible, not cytotoxic, and have a highlyreactive surface that is easily functionalized with biolog-ical entities. Fluorescence arising from Si–vacancy defectsis preferred over the N–vacancy defects, because theformer emits narrowly at 738 nm; however, generatingthese particular defects after synthesis (which involvesdetonation of carbon-containing precursors) has not yetbeen demonstrated. The N–vacancy defects can beproduced, but this method usually involves proton-beamirradiation from an accelerator, followed by high temper-ature annealing—a highly costly procedure. A compre-hensive review of use of nanodiamonds as biolabels wasrecently compiled by Barnard [20].

Other probes and NIRF techniques

Other NIR fluorescence-based contrast agents under devel-opment for in-vivo imaging include ICG-doped calciumphosphate NPs that are cleared by a hepatobiliary mecha-nism [21]; the appeal of degradation into innocuousbiological byproducts combined with an FDA-approvedfluorophore is understandable. Luminescent porous siliconNPs emitting at ~800 nm are also attractive candidates,excitable by NIR or two-photon excitation, and they werefound to be biodegradable [22]; however, their synthesisrequires use of hydrofluoric acid, and it takes approximate-ly two weeks to activate their NIR luminescence—potentialdrawbacks for commercial contrast agents. One area forgrowth includes the use of “smart” probes, those that are“off” until reaching the desired target and then turn “on”[23]; these molecular beacons are commonly used withfluorescent agents via FRET or quenching mechanisms.

In addition to the development of the actual fluorescentcontrast agents, other fluorescence-based imaging techniquesare being developed that minimize the effects of autofluor-escence that plague bioimaging. One method utilizes time-gated fluorescence imaging, which separates short-livedautofluorescence (fluorescence lifetime of a few ns) from theemission of fluorescent probes like QDs, with their longfluorescence lifetimes of a few hundred ns to 1 μs. Similarly,fluorescence lifetime imaging (FLIM) is another potentialgrowth area based on separation of probes on the basis of theirlifetimes [24]. Bioluminescence imaging is popular for smallanimal imaging because no external excitation light source isrequired, thus eliminating autofluorescence, but this methodis not likely to be used for humans because of the necessaryinjection of a substrate such as luciferin. With high spatialresolution and using a normalized Born approximation,fluorescence molecular tomography (FMT) is emerging asa powerful imaging technique for resolution of tumors inmice using NIR fluorophores; the subject is imaged overseveral projections (i.e., different angles of source/detectorpositions rotated through 360°), and a three-dimensionalimage is mathematically reconstructed from the ratio offluorescence intensity (at the fluorophore’s emission wave-length) to intrinsic intensity (at the wavelength of excitation)[25]. This technique has been used to visualize thedistribution and localization of GFP-expressing T cells deepwithin mice [26] and even to quantify the number of GFP+T cells in lymphoid organs in mice [27].

Two-photon absorption (TPA) is emerging as a powerfultool in fluorescence imaging. It is an excellent option forexcitation, because it can discriminate fluorophores atdifferent depths owing to its sensitive temporal and spatialresolution; excitation occurs only at the focal point [5]. Asmentioned previously, TPA can be used with numeroustypes of fluorescent nanomaterials, for example quantum

NP contrast agents for bioimaging 7

dots, carbon dots, and silicon NPs. In addition, goldnanoshells, consisting of a silica core with a thin layer ofgold shell surrounding it, can be excited by TPA and maybe candidates for future biological imaging applications [5].

Using NIR fluorophores for image-guided surgery iscurrently underway. Clinical instrumentation includes theSpy system from Novadaq (Bonita Springs, FL, USA), thePhotodynamic Eye from Hamamatsu, and the Fluorescence-Assisted Resection and Exploration (FLARE) system devel-oped by Khullar and coworkers. The latter researchers haveactually used NIR probes for fluorescence-guided sentinellymph node (SLN)mapping and nodal treatment in preclinicaland clinical trials [28]. SLN mapping is a growing area inwhich NIR agents can make an impact by enabling easyidentification of the location of the sentinel lymph node tocheck for malignancies and to determine if removal of thelymph nodes is necessary. In addition, drug delivery tolymph nodes or metastases by use of NP devices can localizechemotherapy drugs, which may improve cancer prognosesand outcomes [29]. Combining fluorescent probes withmicroendoscopy could lead to imaging deep within tumors[24].

Magnetic resonance imaging (MRI)

MRI is a noninvasive and nonionizing imaging method thatprovides physiological and pathological information aboutliving tissue, usually by measuring water proton relaxationrates. MRI offers high soft tissue contrast and is capable ofdeep tissue imaging with high spatial resolution (~50 μm). Itsinherent drawback is its low sensitivity—millimolar concen-trations of protons are needed—so the technique oftenrequires use of exogenous contrast agents. These probes canalter relaxation processes when used in small amounts (of theorder of nmolL−1 to μmolL−1 concentrations). MR contrastagents can broadly be divided into two classes: those thatincrease the T1 signal in T1-weighted images (positivecontrast agent, bright contrast), and those that reduce the T2signal in T2-weighted images (negative contrast agent, darkcontrast). The effectiveness of a particular probe is definedby its longitudinal (r1) and transverse (r2) relaxivities—enhancement of water proton relaxation rates by 1 mmolL−1

solutions of contrast agent is measured and compared withthe intrinsic tissue or other MRI contrast agents (e.g.,Feridex). Although T2 agents enable highly sensitivetracking of labeled cells, they suffer from poor contrast thatis sometimes weaker than the dark contrast produced byhypointense areas developed from pathogenic conditions.Also, the “blooming effect” associated with T2 contrastagents makes staging of lesions difficult, because thesignal from abnormal areas blends in with the back-ground signal [30]. In contrast, the signal produced by T1

(paramagnetic) contrast agents, which does not displaythis “blooming effect,” can easily be detected with highspatial resolution, but the major challenge in developingT1 probes is achieving the high sensitivity obtained withT2 agents.

Iron oxide nanoparticles

As with other imaging modalities, NP-based probes havebeen developed for MRI to achieve high tissue contrast andto improve imaging sensitivity. The most popular materialstudied for T2 (superparamagnetic) contrast agents is ironoxide NPs, which are generally coated with dextran, PEG,or other polymers, and are used for clinical MRI [31, 32].Based on their size, these NPs are classified as magneticiron oxide nanoparticles (MION, μm), superparamagneticiron oxide (SPIO, hundreds of nm), and ultra-smallparamagnetic iron oxide (USPIO, <50 nm). SPIO contrastagents have been used clinically for diagnosis of liverdiseases [33], whereas USPIO probes are generally used forlymph-node imaging, angiography, and blood-pool imaging[32]. Besides their clinical use, MRI contrast agents basedon iron oxide nanoparticles have been developed forstudying biological processes: Weissleder and coworkershave contributed significantly in this research area anddemonstrated use of these particles for molecular andcellular imaging applications [34–37].

As shown in Fig. 2, the efficiency of iron oxide probes issize-dependent and increases with higher particle crystal-linity [31]. However, these nanoparticles are generallysynthesized at low temperatures, have poor crystallinity,and lack monodispersity in particle size, as is common withother nanomaterials [38]. Thus, further optimization andimprovement in synthetic processes is needed to make theseprobes useful for molecular imaging. Yet another challengefor this class of contrast agents is the development of robustmethods for dispersion in aqueous media and surfacefunctionalization for biological targeting. The inherentnegative contrast associated with iron oxide NPs limitstheir use in low-signal regions of the body or in organs withintrinsically high magnetic susceptibilities, for example thelungs. To solve this problem, specific methods based oneither pulse sequences [39, 40] or design of nanoparticles[41] have been developed by researchers to generate brightcontrast from iron oxide NPs.

To fulfill the high resolution and high sensitivityrequirements for in-vivo imaging applications, metalalloy-based T2 contrast agents with improved magneticand physicochemical properties have been developed.Some examples of the bimetallic ferrite NPs in thiscategory are CoFe2O4, MnFe2O4, and NiFe2O4 NPs [42].However, the biological fate and long term toxicity of thesenew nanomaterials have yet to be assessed.

8 M.A. Hahn et al.

Gadolinium-based agents

According to the Solomon–Bloembergen–Morgon theory,there are three important requirements for the design of highlysensitive paramagnetic NPs: large number of labile watermolecules coordinated to the metal; optimum residencelifetime at the metal site; and slow tumbling motion of theNP containing the contrast agent [43]. A well investigatedprobe for T1 contrast, Gd(III), has been incorporated intovarious nanomaterials, for example silica and perfluorocar-bon nanoparticles, carbon nanotubes [30, 31], and nano-diamonds [44], which all yield high MR contrast because ofa high payload of gadolinium ions and a slow tumblingmotion of particles. For a Gd(III) complex attached tonanodiamonds, a 10-fold relaxivity increase was observedcompared with the monomeric Gd(III) complex [44]. Inanother example of a T1 contrast agent, gadolinium chelateswere grafted on to mesoporous silica NPs to yield particlescapable of drug delivery, thus granting them a therapeuticfunction [45]. However, gadolinium loading on theseconstructs is limited by the number of anchoring sites

available on the surface of the NPs. To solve this problem,various paramagnetic nanoparticles, for example Gd2O3,GdF3, and GdPO4 [31] have been proposed to yield highmagnetic moments because of the abundance of paramag-netic ions on their surfaces. Transition metal oxide (e.g.,MnO) NPs have recently been developed by various groupsfor T1-contrast imaging of brain tumors, in addition to theliver and kidney [30]. In another report, hollow MnO NPswere synthesized that can carry drug molecules in theircavities for simultaneous imaging and therapy applications[46].

With relaxivities that depend on the biological environ-ment, “smart” T1 MR probes that respond to their surround-ings have been pursued extensively [43]. This class of probeprimarily consists of Gd(III)-based complexes; incorporationof these smart probes into NPs will further enhance theirutility for molecular imaging applications.

Other probes for additional MR techniques

Chemical exchange saturation transfer (CEST), a methodused in NMR, is now currently being used as a techniquefor generating contrast in MRI [47]. In fact, MRI utilizingcontrast agents for CEST could potentially be used forsimultaneous visualization of multiple biological events.Upon application of a suitable radiofrequency (RF) pulse,CEST agents reduce the intensity of the bulk water signalby saturation transfer through their chemical exchange sites[47]. CEST contrast can originate endogenously fromsugars, amino acids, nucleosides, and other diamagneticmolecules (DIACEST). Because CEST contrast depends onthe frequency difference (Δω) between the protons associ-ated with the contrast agent and the protons of bulk water, alarge Δω is desirable as it gives rise to greater flexibility inselecting an RF pulse and enhanced contrast [43]. Althoughhigh Δω can be achieved, this approach is not attractive forclinical translation because of the high magnetic fieldsrequired. Various lanthanide-based paramagnetic complexes(PARACEST) with large Δω (~50 ppm for Eu(III)complexes compared with <5 ppm for diamagneticmolecules) are currently being developed for highersensitivity and improved contrast in MRI [43]. Variousnanocarriers with efficient PARACEST contrast includethose based on paramagnetic liposomes and perfluorocarbonNPs [31].

The recent development of hyperpolarization techniquesoffers novel opportunities for using NMR-active heteronucleisuch as 13C and 15N in MRI. Use of nuclei other thanprotons drastically improves the SNR, because of theinherently low natural abundance of these heteronuclei inbiological tissue. A hyperpolarized state has a higher numberof spins aligned with the magnetic field compared with thenormal Boltzmann distribution, resulting in an increase in

Fig. 2 (b–e) Nanoscale size effects of Fe3O4 (MEIO) nanoparticleson magnetism and MR contrast effects. (b) Transmission electronmicroscopic (TEM) images of 4, 6, 9, and 12 nm sized MEIOnanoparticles. (c) Mass magnetization values, (d) T2-weighted MRimages (top: black and white, bottom: color). (e) Relaxivity coefficientr2 of the nanoparticles. (Reprinted, with permission, from Ref. [228],Copyright 2005 American Chemical Society, and Ref. [229],Copyright 2008 Wiley–VCH)

NP contrast agents for bioimaging 9

the NMR signal intensity. In contrast with standard MRIcontrast agents, the hyperpolarized molecules themselves arethe source of the NMR signal and, therefore, the signalintensity and SNR is directly proportional to their concen-tration and polarization level [43]. 129Xe has been shown tobe a potential tissue-specific CEST contrast agent because ofits exquisitely sensitive chemical shift. The extremely lowsensitivity of the 129Xe nucleus was improved by five ordersof magnitude simply by hyperpolarizing it before imaging[48, 49]. Ongoing efforts in this area explore the slow rate ofexchange between caged and free xenon for furtherimproving the sensitivity of these probes [50]. For in-vivoMRI, hyperpolarized 13C has been used extensively becauseof its relatively higher sensitivity and the availability ofdedicated RF coils [43].

In addition to technical challenges related to the design ofimproved pulse sequences, the need to design contrast agentswith higher sensitivity and target specificity is critical.Particular attention should be given to the developmentof NP-based CEST and hyperpolarized probes, becauseof their promising potential in MR-based molecularimaging applications. However, incorporating the agentsthat are used with CEST and hyperpolarized techniques intonanomaterial carriers is not straightforward, and moreresearch is needed to determine the best way to take advantageof these more advanced magnetic imaging techniques.

Positron emission tomography (PET)

PET is an imaging technique that relies on emission fromradioisotopes in the form of positrons without the need forexternal excitation. Approved by the FDA as a clinicalmolecular imaging technique with a resolution of 1–2 mm[2], it lacks the anatomical resolution of a technique such asMRI. However, having the highest sensitivity of allimaging modalities enables quantification of the localconcentration of radionuclide tracer, with the possibility ofdetecting a single abnormal cell labeled with only a fewtrace isotopes [3]. Furthermore, the penetration depth of thistechnique is unlimited, so the probe can always be imaged,irrespective of the location of the target. Especiallyimportant in cancer imaging and research, PET is capableof detecting molecular changes that are occurring in thebody before the macroscopic disease is observed [51, 52]and of monitoring disease progression after treatment (i.e.,tumor response to therapy) [53].

Radioisotope-based agents

Common isotopes that can be chelated on to or incorporat-ed within NPs (in an analogous way to the gadolinium ionsused for MRI) include 18F, 11C, 15O, 13N, 64Cu, 124I, 68Ga,

82Rb, and 86Y. Oftentimes PET tracers have been incorporatedwith another modality in NPs, most notably CT [54, 55].Figure 3 illustrates the power of this dual technique using 18F-doped cross-linked iron oxide (CLIO) NPs to image the liverand blood pool of a mouse. In fact, since the inception of thefirst PET–CT scanner [56], commercial instruments thatprovide solely PET imaging have been rendered virtuallyobsolete. A source of information from the Academy ofMolecular Imaging on PET and PET–CT technology can befound online at http://www.ami-imaging.org.

Other contrast agents

Other modalities that have been combined with PETinclude NIRF agents within silica NPs [57, 58] or QDs[59] and MRI agents in conjunction with iron oxide

Fig. 3 Dynamic PET/CT imaging of BALB/C mouse injected with18F-CLIO. Fused PET/CT coronal images at 2 h (a), 7 h (b), and 16 h(c) postinjection of 18F-CLIO. PET only coronal images at 2 h (d), 7 h(e), and 16 h (f) postinjection of 18F-CLIO. CT only coronal image(g). Three-dimensional rendering of fused PET-CT images at 2 h (h)and 16 h (i) postinjection. The green arrow indicates blood poolregion of interest (ROI) and the asterisk indicates liver ROI.(Reprinted, with permission, from Ref. [209], Copyright 2009American Chemical Society)

10 M.A. Hahn et al.

nanomaterials [60, 61], with the latter being a powerfulcombination of sensitivity and anatomical resolution withdual instrumentation unveiled in 2007 and 2008 [62, 63].However, the amount of PET tracer must be carefullycontrolled compared with the amount of MRI contrastagent, because of PET’s extreme sensitivity and MRI’s lackof it. Single-photon emission computed tomography(SPECT), a similar technique, relies on the detection ofgamma rays from radioisotopes such as 99mTc, 111In, 123I, and131I; however, SPECT is an order of magnitude less sensitivethan PET [2], but it has been used in combination with CTby loading SWNTs with 125I [64]. One advantage of SPECTover PET is that numerous radionuclides can be multiplexedfor use in the former because different isotopes emit differentenergies of gamma rays, whereas the various isotopes for thelatter all emit the same energy in the form of positrons [2].

The major advantages of PETare its extreme sensitivity andFDA approval; therefore, the transition from cell culture tosmall animal to clinical setting is quite feasible. Theseadvantages come at a high price, quite literally. A cyclotronsource is the only way to generate the positron-emittingisotopes; therefore, the setting in which the PET imaging takesplace must be located near such a source or at least have a rapidpathway to get the radionuclides to it. In addition, hospital orclinical settings must have appropriate areas for storage andhandling of these radioactive materials [3]. Exposure to thisionizing radiation, for the patient and the clinicians adminis-tering the agents, should be limited and may be a severedrawback to the widespread use of PET. Nevertheless, itremains a technique that will continue to be developed andpotentially improved upon with the help of NP carriers.

X-ray imaging and computed tomography (CT)

Clinical X-ray imaging works by the principle of photonattenuation differences between materials according tothe well established Bourguer–Lambert–Beer exponentialabsorption law. For clinical applications, both X-rayenergy specific mass attenuation coefficients, and massenergy absorption coefficients of contrast materials areimportant to evaluate the relative penetration attenuationand energy deposition (e.g., ionization) of imaging X-rays caused by these materials. The mass attenuationcoefficient is a measure of the X-ray opacity of amaterial, whereas the mass energy absorption coefficientprovides an indication of the fractional amount ofionization that occurs in the sample caused by incidentX-rays [65]. When designing X-ray probes for clinicalapplications, the resulting contrast is highly dependent onthe energy of the incident X-rays, which varies with theimaging purpose. Hence, contrast agents that perform wellin projectional radiographic imaging may exhibit lacklus-

ter performance in CT imagery. Figure 4 provides the massattenuation coefficients as a function of incident X-ray energyfor several relevant elements, illustrating this effect. Althoughmass attenuation coefficients are important tools for rational-izing the composition of radio-opaque nanomaterials, massenergy absorption coefficients rely on several assumptionsthat are not necessarily valid for NP systems; as with any data,due care is warranted before extrapolating information derivedfrom the bulk material [66].

Although X-ray imaging has been a clinical workhorsefor more than half a century, little activity was devoted tothe development of new engineered nanomaterials asX-ray contrast agents until the last decade, when thenumber of publications involving nanomaterials as X-raycontrast agents has more than quadrupled. The firstwidespread clinical use of nanoparticulates as X-raycontrast agents in humans goes back to the 1930s whenThorotrast [67], a suspension of 3 to 10-nm thoriumdioxide nanoparticles, was applied as an IV radiographiccontrast agent; because of long-term radiation effectsand significant carcinogenicity of the 232Th, however,the clinical application of Thorotrast was abandonedwithin 20 years. Because of the questionable biocom-patibility of many high atomic number (Z) elements, thenumber of materials explored for in-vivo applicationshas been limited; today safer hydrophilic iodinatedmolecules are universally used as radiographic contrastagents. However, a renewed interest in NP-based agentshas emerged with the promise of more detailed andquantitative imaging, and potential for therapeuticapplications.

Iodinated nanoparticles

The widespread clinical use of iodinated compounds hasspurred the development of iodinated nanomaterials. There

Fig. 4 Calculated values of the mass attenuation coefficient for differentmaterials from NIST (database: http://physics.nist.gov/PhysRefData/XrayMassCoef/tab3.html)

NP contrast agents for bioimaging 11

is much research on, essentially, incorporation of iodinatedorganic compounds into a NP, with designs ranging fromemulsions [68, 69], liposomes [70], and lipoproteins [71] tonano-milled insoluble compounds [72–75] and polymericNPs [76, 77], many of which have been successfullyapplied in vivo [70, 71, 73–77]. The design principle formany of these nanomaterials has simply been to enhancelocalized iodine concentrations, resulting in higher localcontrast compared with conventional water-soluble com-pounds. In addition to modifying particles to alter physio-logical fate and transport, doping with an iodinatedcompound is used to enhance X-ray contrast for thepurpose of creating multifunctional particles [69, 71, 78].Despite iodine having a lower atomic number than bothgold and bismuth, it has a superior elemental mass attenuationcoefficient and incident X-ray energies that are relevant forprojectional radiographic imaging. A recent comparison of theperformance of gold NPs with that of iodinated contrastagents demonstrated that under conditions used for coronaryangiography, both materials performed equivalently [79].Although these nanomaterials may be viewed as a simpleevolution of conventional iodinated compounds, the strategyof adding iodine to NPs with fairly well understood fate andtransport properties is promising.

Gold nanoparticles

Within the last decade, there has been substantial interest ingold NP-based contrast agents for in vivo X-ray imaging.Various gold [42, 80–87], gold–dielectric hybrid [88–90],and multimodal materials [83, 91], have been fabricatedand tested for X-ray contrast performance. These particleshave been shown to have in-vivo functionality as CTcontrast agents for cancer [82, 84, 91], tissue-specific [87],and blood-pool imaging [86]. In fact, gold NPs have beenshown to match or exceed the performance of conventionaliodinated contrast agents under conditions relevant formammography and relevant for CT and trans-torso imaging[79], as expected from gold’s higher k-edge energy shownin Fig. 4. Recently, it was found that gold NP-induced X-ray contrast in CT imaging is further enhanced as the goldNP size is reduced [92]. Gold nanomaterials are currentlybeing explored in multiple clinical trials. The depth ofresearch on the fate, transport, and toxicology of gold-basedNPs make them a promising next generation candidate forX-ray contrast materials, and their use to potentiatecombined radiotherapy [93, 94] adds yet another dimensionto the application of these materials.

Other contrast materials

Although the bulk of the current literature involvesiodinated and gold-based NPs as contrast agents, other

NPs consisting of bismuth sulfide [95] and compositeceramics containing iron oxide [96] and lanthanide materi-als [97] have been reported. Bismuth sulfide NPs haverecently been shown to have superior performance to iodineon a molar basis [95]. Although the initial report suggestslimited toxicity, the overall similar mass attenuationcoefficients for bismuth and gold, in addition to bismuth’shigher k-edge transition, indicate that bismuth-based nano-materials may not perform significantly better than gold-based materials. Hence, the toxicological risks from thepresence of bismuth may preclude clinical use of thesematerials; however, more research describing any biologicalside effects is still needed. Other forms of contrast agents,for example those used in MRI, in particular iron oxide andgadolinium-doped materials, have also been shown to haveX-ray contrast properties. Toxicologically and practically,the biodegradability and known absorption, distribution,metabolism, and excretion (ADME) profiles of iron oxideNPs in humans make them attractive alternatives toiodinated and gold-based materials for non-critical, low-energy X-ray imaging applications. Although the clinicalapplications of X-ray contrast materials based on gadolin-ium and other lanthanides are limited, primarily because ofthe associated toxicological risk, gold-based X-ray poten-tiated therapy may realistically complement establishedimage-guided X-ray therapy in the near future. Use of thesematerials in the clinic will depend strongly on added value,with efforts being made to combine therapeutic componentswith these contrast agents to broaden their potential marketvalue. Design criteria for such materials are currently beingestablished, and the promise of X-ray potentiated therapyseems to be real.

Ultrasound (US)

As with X-ray imaging, US is a well established clinicalimaging modality. A relatively inexpensive and versatilemethod, it is based on the pulse–echo principle, wherebysound waves having frequencies greater than 20 kHz areemitted and received. Ultrasound has been used formolecular imaging and is capable of resolving nanoscalefeatures, albeit at limited penetration depths using non-clinical wave frequencies. The clinical application of USinvolves sound waves in the range of 2–3 MHz forpediatric imaging and 5–12 MHz for adult imaging,providing spatial resolution in the range 0.2 to 1 mm.

Traditionally, contrast in US is provided by the variableability of sound to propagate through media, resulting inreflection and refraction of the sound waves. The extent ofthis reflection and refraction is based on the mismatch ofacoustic impedance between materials, which is defined asthe speed of sound through the material multiplied by its

12 M.A. Hahn et al.

density. Because sound travels poorly through gaseousphases, several microbubble-based contrast agents havebeen developed and are applied clinically to enhance theechogenicity of vasculature and organ-specific regions [98,99]. These microbubbles are composed of surfactant,protein, and/or polymer shells containing gas cores, forexample air, perfluorocarbons, or nitrogen. The last two arepreferred because their minimal plasma solubility leads toimproved longer-term imaging performance. In addition tomicrobubbles, perfluorocarbon emulsions have been usedas US contrast agents [100]. Whereas microbubbles sufferfrom instability to insonification pressures, marked attenu-ation, “blooming” effects, and short circulation times,emulsion-based US contrast agents are mechanically moreresilient to these effects, despite their reduced echogenicity.

Although reports of use of nanobubbles [101–105] andnanoemulsions [106] as US contrast agents have beenfrequent in recent years, these contrast agents rarely exist astrue nanoparticles, because they are typically between 150and 1000 nm in diameter. As with microbubbles, thesematerials are typically composed of a perfluorocarbon gas(or liquid in the case of nanoemulsions) encapsulated by asurfactant, protein, and/or polymer shell. Because of theirlower scattering cross sections and the often less-than-optimum mechanical properties of the shell, the perfor-mance of these materials is often inferior to that ofcomparable microbubbles. A recent theoretical evalua-tion of US contrast agents indicates that the optimumbubble size for current imaging practice is in thevicinity of 2–3 μm [107]. However, because of theclinical significance of US and potential labeling advan-tages of nanomaterials, there is a continued interest indeveloping smaller ultrasound contrast agents. Solidparticles composed of silica and polystyrene have alsobeen used as US contrast agents to visualize mice livers,although the size of the particles ranged from 500 to3000 nm, which may not necessarily classify them asnanoparticles [108].

As with other imaging modalities, there is an emergingtrend to combine both imaging and therapy. Rapoport et al.have used a polymeric micelle and perfluorocarbon nano/microbubble system that encapsulated a drug, which wasreleased locally within tumor cells; US was also used todetermine the efficacy of this drug therapy [105]. However,the imaging performance of nanoscale US contrast agentsmay become less significant when compared with theirpotential therapeutic potency. Sonodynamic therapy,ultrasound-induced apoptosis, sonoporation/sonotransfec-tion, ultrasound-induced drug/gas delivery, and focusedultrasound-induced thermal ablation are currently beingexplored for therapeutic clinical applications [109]. Reportsof US contrast agent-based theranostics are emerging, andtheir incorporation into multimodal probes.

Photoacoustic imaging (PAI)

PAI, also known as laser optoacoustic imaging, is anemerging noninvasive, nonionizing, imaging modality thatcombines the high sensitivity of optical methods with theexcellent resolution of acoustic methods [110, 111].Illuminated by a short-pulsed laser, the biological sampleabsorbs this light on the basis of the characteristics of itscomposition. This excitation is followed by a transientincrease in temperature (~10 mK) and subsequent thermo-elastic expansion of the absorbent, generating an ultrasonicacoustic signal which is detected by wideband transducerssurrounding the object and used to determine its geometry.PAI is generally performed using two main techniques,photoacoustic microscopy (PAM) and photoacoustic com-puted tomography (PAT) [112]. PAM employs a coupled,focused ultrasonic detector–confocal optical illuminationsystem to generate multidimensional tomographic imageswithout the need for reconstruction algorithms, whereas thedetectors in PAT scan the laser-illuminated object in acircular path and use inverse algorithms to construct three-dimensional images.

In-vivo imaging, especially with optical techniques,suffers from the issues of hemoglobin absorption and tissuescatter, which limit overall light penetration depth. PAI canovercome this primary challenge because of the lowerultrasonic scattering coefficients (by 2–3 orders of magni-tude) of absorbents compared with their optical equivalents;therefore, the propagation of the photons in the diffuseregime [113] enables PAI up to ~50 mm deep with aresolution of <1 mm [114]. Further, because PAI’ssensitivity is primarily based on the optical absorptionproperties of the specimen, all absorbed photons producephotoacoustic signals, whether or not they were scattered.Selection of the appropriate central frequency and band-width of the ultrasound enables variation of the penetrationdepth and spatial resolution, so samples of various thick-nesses and sizes can be studied using the same technique.In fact, the depth issue is completely eliminated whenmicrowaves or radio waves (referred to as thermoacoustictomography (TAT)) are used as illumination sources [115].

Using only endogenous contrast, PAI has been used toimage blood vessels [116], tumors [117, 118], tumorangiogenesis [119], and hemoglobin oxygenation [120].Using laser PAT, lesions 18 mm in size have been detectedin a human breast 23 mm below the laser source [114].Exogenous agents such as dyes [121, 122], which absorband fluoresce at desirable wavelengths, have also beenused for cancer staging by PAM. For example, methyleneblue was injected into the lymphatic system and accumu-lated in the sentinel lymph node, which was then identifiedand imaged with an axial resolution of 144 μm and apenetration limit of 30 mm [121]. PAI using multi-

NP contrast agents for bioimaging 13

wavelength illumination further increases the potential toimage multiple chromophores such as biomarkers, intrin-sic, and exogenous agents [123, 124]. Functional imagingis routinely performed on biological specimens on thebasis of endogenous contrast, and engineered nanomate-rials are also being used to perform molecular imaging invivo.

Gold-based nanomaterials

Gold-based nanomaterials stand out as the most significantclass of materials being explored for PAT applications.Some of those commonly used for PAT include sphericalgold NPs [125], nanorods [126, 127], nanocages [128],agglomerates [129], and even hollow nanoshells [130], andcomposite materials such as gold nanoshells having silicacores [131, 132], cobalt/gold core/shell NPs [133], gold-speckled silica [134], polymer–gold hybrids, and goldnanobeacons [135]. The reasons for the increasingattention to gold nanoconstructs are many: their size-dependent and shape-dependent plasmonic properties[136, 137] enable them to absorb and scatter light in thevisible to NIR region, which may render them suitable forimage-guided therapy [138] and photothermal ablation oftumors [139–141]. Because of the prevalent use of gold-based compounds in medicine (e.g., chrysotherapy[142]), the benign toxicity profile of NPs [143–145],and ongoing clinical trials [146], the possibility ofapproval of these gold-based materials for clinical andmedicinal applications is greatly enhanced. Conjugationof biologically relevant entities to gold surfaces is wellestablished [147–149], and most of these nanomaterialshave multimodality capabilities incorporating the othertechniques outlined here, for example PET [150], CT[141], MRI [151], conventional microscopic opticaltechniques [152], reflective confocal microscopy [153],multi-photon plasmon resonance microscopy [154], opti-cal coherence tomography (OCT) [155] (including phasesensitive OCT) [156], scattering [157], surface enhancedRaman spectroscopy (SERS) [158], and diffuse opticalspectroscopy [159].

Gold nanoshells, nanorods, and nanocages haveattracted the most attention for photoacoustic applications,because of the tunability of their peak absorption in theNIR region; gold nanoshells with a silica core, andnanorods, are the systems most investigated to date. Anin-vivo study imaging rat brain cortical blood vesselsreported a marked increase in blood vessel absorptionwith nanoshells, thus demonstrating successful applicationas a NIR PA contrast agent [160]. Gold nanoshells and theenhanced permeability and retention (EPR) effect wererecently used to image subcutaneous tumors by employinghigh-resolution PAM, with heterogeneous localization—

they were found mostly within the tumor cortex and werealmost absent from the tumor core [131]. In a comparisonof gold plasmonic nanostructures (surface plasmon reso-nance (SPR) tuned to 800 nm), Hu et al. noted that goldnanorods and nanocages have much larger absorption andscattering cross sections than gold nanoshells [161]; futuredevelopment of these materials may therefore increase.Currently, gold nanorods have similarly been used as NIRphotoacoustic contrast agents with high sensitivity [162].Manipulation of the aspect ratio enables tuning of the SPRof the resulting nanorods, which has led to multiplexingapplications [163, 164]; for example, Li et al. used antibody-conjugated gold nanorods with two different aspect ratios(peak absorptions at 785 and 1000 nm) to detect Her2 andCXCR4 target molecules by PAT [164]. A combination ofPAT and ultrasound has also been used to target and detectprostate cancer, using functionalized gold nanorods toprovide high photoacoustic contrast and anatomical detailsof the targeted tissue [126]. Gold nanorods have recentlybeen shown to be effective as tracers for noninvasive in-vivospectroscopic photoacoustic SLN mapping in a rat model[165]. In addition to the aforementioned studies, goldnanorods have been used in other bioimaging applications,for example detecting inflammatory response from cells[166], measuring quantitative flow in biological samples[167], and monitoring drug delivery [168]. Gold nanoc-ages [128, 169, 170] and hollow gold spheres [130] withNIR absorption profiles are also being explored as contrastagents for PAI.

Carbon nanomaterials

Carbon nanomaterials are being extensively used forpharmaceutical, biomedical [171, 172], and bioimagingapplications [173], including PAI and thermoacousticimaging [174]. The characteristic optical properties ofSWNTs, particularly those with optical properties in theNIR region [175], play an important role in photoacousticimaging [176]. In an in-vivo tumor-targeting study,SWNTs ~2 nm in diameter and 50–300 nm in length werecoupled to RGD peptides and were shown to bind to tumorvasculature, producing an ~8× higher photoacoustic signaland an ~4× higher Raman signal (ex vivo) than unconju-gated SWNTs [177]. Antibody-conjugated SWNTs tar-geted to integrins αvβ3-positive U87 human glioblastomatumors in mice similarly resulted in high photoacousticcontrast in vivo [178]. As an alternative and noninvasiveapproach to detection of the SLN, SWNTs have beenshown to result in significant signal enhancement fordetection by PAI [179]. Despite various in-vivo applica-tions of SWNTs, their absorption coefficients are relativelylow compared with those of gold nanoparticulates [180].To overcome this limitation, nanotubes are being modified

14 M.A. Hahn et al.

to enhance their NIR absorption and, thus, photoacousticcontrast. In one modification, nanotubes have been platedwith a thin layer (4–8 nm) of gold; use of these goldencarbon nanotubes (GNTs) resulted in a 100-fold increasein photoacoustic signal enhancement [180]. In a compar-ison of photoacoustic signals of GNTs with those of otherNIR contrast agents they were shown to exhibit higher PAsignals and correspondingly lower bubble-formationthresholds than those of pristine carbon nanotubes andgold nanospheres, with comparable properties to goldnanorods and nanoshells. The high photoacoustic sensi-tivity of GNTs has been demonstrated by employingfolate-conjugated GNTs as a secondary contrast agent toenable photoacoustic detection of magnetically capturedcirculating tumor cells, thus potentially enabling earlydiagnosis of cancer [181]. Antibody-conjugated GNTshave been used to target lymphatic vessels in vivo by PAI[180]. In another modification of SWNTs, ICG dyemolecules attached to the surface of the nanotubes byπ–π stacking interactions had 20-fold higher absorbancethan bare SWNTs (Fig. 5) [182]. Aided by the NIRabsorption property of the dye, SWNT–ICG nanomaterials

are reported to provide an ~300× improvement in photo-acoustic sensitivity compared with unmodified SWNTs invivo.

Dye-doped nanoparticles

Although dyes alone are sufficient to increase SNR, thusimproving image contrast, their encapsulation within NPsprovides additional advantages as mentioned previously[183]: loading numerous dye molecules into a protectiveNP matrix enables signal amplification, reduced chemicaland photodegradation, improved contrast, and the ability totarget specific biologically relevant sites. Besides the desiredabsorption profile, selection of the dye can be based on otherproperties, for example fluorescence imaging in addition toPAI or incorporating a therapeutic function, for examplephotodynamic therapy (PDT). ICG is the most commonlyused dye for photoacoustic imaging in molecular andnanoparticulate formulations. Currently the only FDA-approved dye for human applications, ICG’s absorption peakat ~780 nm lies within the biologically relevant NIR windowand enables deep tissue imaging. PAT has been used toimage objects containing blood and ICG that wereembedded at depths greater than 5 cm in chicken breasttissue [184]. However, the in-vivo applications of ICGare limited, primarily because of its rapid degradation inaqueous media and its short plasma half life of up to4 min [185, 186]. Encapsulation of ICG within differentNP matrices (e.g., organically modified silica (ORMO-SIL), poly(lactic-co-glycolic acid) (PLGA), and calciumphosphate) has been shown to improve its stability andblood circulation time [160], thereby enabling in vivoapplications.

Current challenges and opportunities

Characterization and toxicology

One obstacle to overcome when using NPs is the lack ofreproducibility in both synthesis of the actual material andfunctionalization to render it biologically active. Batch-to-batch variations within the same laboratory, betweenlaboratories, and even between different techniquesemployed in synthetic and modification procedures arecommon, often yielding the same NPs but with slightlydifferent characteristics (e.g., purity of size distribution,number of conjugated biomolecules). In part, these diffi-culties arise from complex characterization of thesematerials off-line, on-line, and in-situ. There is a need forconsistency and scale-up in NP production and functional-ization, especially to produce high yields at low cost ifcommercialization is desired. This area is one that is both

Fig. 5 Photoacoustic detection of SWNT-ICG in living mice. (a)Mice were injected subcutaneously with SWNT-ICG at concentrationsof 0.82–200 nmolL−1. The images represent ultrasound (gray) andphotoacoustic (green) vertical slices through the subcutaneousinjections (dotted black line). The skin is visualized in the ultrasoundimages, and the photoacoustic images show the SWNT-ICG distribu-tion. The white dotted lines on the images illustrate the approximateedges of each inclusion. (b) The photoacoustic signal from eachinclusion was calculated using 3D regions of interest and the“background” represents the endogenous signal measured fromtissues. Linear regression (R2=0.97) of the photoacoustic signal curveindicates that 170 pmolL−1 SWNT-ICG will give the equivalentbackground signal of tissues. (Reprinted, with permission, from Ref.[182], Copyright 2010 American Chemical Society)

NP contrast agents for bioimaging 15

challenging and has great potential for growth in the future,before extensive use of NPs can be realized.

As mentioned previously, the biggest knowledge gapexisting in this research area is the lack of comprehensivecharacterization of NPs that are subsequently used inbiological research and bioimaging. Currently most studies,whether in-vitro or in-vivo, involving the use of NPs inbiological systems and in bioimaging report only minimalparticle characterization. An in-depth understanding of thestructure–activity relationship of NP behavior in biologicalsystems is imperative if better contrast agents are to bedesigned; knowing what properties affect behavior will leadto more advances and improvements in contrast agents. Abase set of, admittedly extensive, characterized properties isrequired: size (actual diameter and hydrodynamic diameter)and size distribution; shape and shape distribution; surfacearea; surface charge; surface chemistry and reactivity;quantification of surface components; thickness and com-position of coating, elemental/chemical composition; crys-tal structure; porosity; and identification and levels of anyimpurities. Depending on the material composition andapplication, other techniques may be required, for exampleUV–visible–NIR absorption spectroscopy, fluorimetry, de-termination of absorption cross sections, fluorescencequantum yields, and fluorescence lifetimes for opticallyactive NPs. Magnetic NPs would require characterization oftheir magnetic properties, for example susceptibility andrelaxivity, whereas NPs for PAI would require tests of theirthermoelastic properties. Zhang and Yan have reviewedsome advances in the study of surface chemistry, includingNMR, Fourier transform infrared (FTIR) spectroscopy,liquid chromatography–mass spectrometry (LC–MS), andcombustion elemental analysis for characterizing moleculeson the NP surface [187]. However, filling this knowledgegap remains a daunting task.

Complicating matters further, different bioimaging techni-ques require different administered doses, based on thetechnique’s sensitivity, host biology, route of delivery, andthe targeting strategy used. When used in bioimaging, evenmore characterization is needed to fully understand and exploitthe structure–function relationship of engineered NPs, forexample particle number and dose administered (i.e., balanc-ing safety against good SNRs), behavior in the biologicalenvironment (e.g., dispersibility or aggregation), and inter-actions between the moiety on the surface of the functional-ized NP and the target of interest (i.e., binding kinetics andthermodynamics). Reproducibility of bioconjugation andstudy of the activities of those biomolecules once on the NPsurface are immense challenges but also research opportuni-ties. One of the most difficult properties to study is the numberand activity of the conjugated molecules on a NP’s surface.

Small-animal imaging studies are a valuable tool whenstudying disease models, but they may not necessarily

predict the same behavior in humans. More complex issues(e.g., penetration depth, toxicity) must be explored beforeuse in the clinic. Repeated ad nauseam, toxicity studies ofnanomaterials are either lacking, especially long-termeffects, or inconsistent. Gadolinium chelates, ICG,polymer-coated iron oxides, and gold nanocomposites areeither approved or in clinical trials (Table 2). However, forthe remaining NP imaging agents under development forpossible clinical use, information on short-term and long-term toxicity, biodistribution, nonspecific uptake, androutes of elimination must be determined.

The need for benchmarking characterization techniquesand toxicity tests is imperative [188–194]. Deciding on aset of standard toxicity assays is difficult, especially whenresearchers from different laboratories use different tests toconfirm or deny toxicity. Toxicological characteristics mayinclude various in-vitro assays (often yielding differentconclusions), reactive oxygen species (ROS) and singletoxygen production, safe working concentrations (LD50

values), in-vivo studies, and biodistribution profiles [195].It must be remembered that the various toxicological testsstudy different aspects of toxicity (e.g., membrane perme-ability, apoptosis) and that commercial testing kits designedfor molecular toxins may not be the optimum tests for NPs,because the materials themselves may interfere with thesecommercial assays. In addition, results of in-vitro analysesmay not necessarily be valid in vivo.

A standard is needed against which all nanomate-rials can be tested, and an established set of tests thatshould be administered. The International StandardsOrganization (ISO) has outlined the work necessary,and the MINChar initiative has been formed toimprove toxicological studies of nanomaterials [188].NP characterization remains an almost overwhelmingchallenge; however, this area is a major opportunity forfurther development. The Nanotechnology Characteriza-tion Laboratory (http://ncl.cancer.gov), under the Na-tional Cancer Institute (NCI), is working with the FDAto develop standard tests to determine a NP’s safety andefficacy, and the properties of NPs which must becharacterized if they are to be used in conjunction withdrugs or therapeutics; this task is quite complex,considering that the tests should work for multipleparticle types, despite the fact that different NPs havedifferent characteristics and behavior (e.g., fluorescent,magnetic, metallic). Their current database groups NPsbased on size, surface charge, and solubility (Fig. 6)[196].

Biological considerations of NP treatment modalities

Important considerations when developing an imagingprobe are routes of delivery and bioavailability. Most

16 M.A. Hahn et al.

delivery routes now consist of intravenous injection; it is,therefore, crucial to optimize circulation time. The func-tionalized NPs must be able to pass through the blood-stream and reach their desired target intact. The necessary

functionalization of their surfaces may result in NPs that arelarger than their “core” components, especially if very thickshells of silica, PEG, or polymer are used to render themwell dispersed in aqueous solutions. This size increase may

Table 2 Selected NP-based therapeutics approved or in clinical trials (adapted from Refs. [196] and [223])

Product Nanoparticle drug component Delivery route Indication FDA status Company

Doxil PEGylated liposome/doxorubicinhydrochloride

IV Ovarian cancer Approved 11/17/1995FDA050718

Ortho Biotech

Amphotec Colloidal suspension of lipid-basedamphotericin B

Subcutaneous Invasive aspergillosis Approved 11/ 22/1996FDA050729

Sequus

Estrasorb Micellar NPs of estradiolhemihydrate

Topical emulsion Reduction of vasomotorsymptoms

Approved 10/9/2003FDA021371

Novavax

Abraxane Nanoparticulate albumin/paclitaxel IV Various cancers Approved 1/7/2005FDA021660

American PharmaceuticalPartners

Triglide Nanocrystalline fenofibrate Oral tablets Lipid disorders Approved 5/7/2005FDA021350

SkyePharma PLC

Megace ES Nanocrystal/megestrol acetate Oral suspension Breast cancer Approved 7/5/2005FDA021778

Par PharmaceuticalCompanies

Combidex Iron oxide IV Tumor imaging Phase III Advanced Magnetics

Aurimune Colloidal gold/TNF IV Solid tumors Phase II CytImmune Sciences

NB-00X Nanoemulsion droplets Topical Herpes labialis caused byherpes simplex I virus

Phase II NanoBio

AuroShell Gold-coated silica NPs IV Refractory head and neckcancer

Phase I Nanospectra Biosciences

CALAA-01 Cyclodextran-containing siRNAdelivery NPs

IV Various cancers Phase I Calando Pharmaceuticals

Cyclosert Cyclodextran NP IV Solid tumors Phase I Insert Therapeutics

INGN-401 Liposomal/FUS1 IV Lung cancer Phase I Introgen

SGT-53 Liposome Tf antibody/p53 gene IV Solid tumors Phase I SynerGene Therapeutics

Fig. 6 The physicochemicalcharacteristics of a nanoparticleaffect biocompatibility. Here wequalitatively show trends inrelationships between the inde-pendent variables particle size(neglecting contributions fromattached coatings and biologics),particle zeta potential (surfacecharge), and solubility and thedependent variable biocompati-bility—which includes the routeof uptake and clearance (green),cytotoxicity (red), and RES rec-ognition (blue). (Reprinted, withpermission, from Ref. [196],Copyright 2009 John Wiley andSons)

NP contrast agents for bioimaging 17

be crucial depending on the type of cancer, stage of cancer,location of tumor, and vasculature permeability. Stericinteractions of these larger NPs may enable only a smallfraction to bind to the target and/or affect bloodstreamcirculation: passing through thicker veins or arteries ismuch different from passing through thinner capillaries.

In addition to IV administration, intramuscular injec-tions, oral, transdermal, and inhalation routes are alsopossible, depending on the desired target (Table 2). TheNPs being used must be able to survive their particularroute of delivery; for example, oral routes require particlesthat can withstand the highly acidic environment of thestomach. If a region of the brain is the desired target, theNP agent must be able to cross the blood–brain barrier ifadministered intravenously. Any loss of biological activityof the component conjugated to the NP surface must bedetermined. The probe will not be effective if it cannot dothe desired function or bind properly to the desired target.

Developments in imaging techniques and instrumentation

Other types of bioimaging are becoming important. In-vivoRaman imaging has been developed using SERS probescomposed of Texas red and cresyl fast violet adsorbed onmetallic gold, silver, or platinum NPs [197]. OCT andspectroscopic optical coherence tomography (SPOCT) arebeing developed, as are probes for their use. Two-photonexcitation for optical imaging is also envisaged, with theadvantages of reduced tissue scattering and absorption, andincreased resolution. MRI/PET probes are also powerfulemerging tools, combining the sensitive, metabolicallyfunctional PET with the high-resolution, anatomical detailprovided by MRI. Their growth had been slow until thesuccessful development of MRI/PET scanners, becausePET detectors could not operate in the high magnetic fieldsrequired by MRI, so alternative instrument setups wererequired. However, the first MRI/PET scanners wereunveiled in 2007 and 2008 [62, 63]. Weissleder’s grouphas previously demonstrated the multimodal use of NPsthat were fluorescent, magnetic, and contained a radiotracerfor nuclear imaging [198]. This area will most likely seegrowth in the future. In addition to optical projectiontomography (OPT) and selective plane illumination micros-copy (SPIM), other imaging techniques are in development,including macroscopic and mesoscopic methods [4]. Thesesystems can increase light penetration depths comparedwith typical optical microscopy, thus enabling deep tissueimaging, but may come at the cost of spatial resolution.

Not only improvements in imaging probes, but alsoimprovements in the instrumentation and detection systemsthat make the imaging possible are required. Improvedimage-analysis software and expanded data storage canmake existing technologies even more powerful. System

performance (i.e., signal detection) must be optimized inorder to take advantage of newer imaging modalities, andenhancing the sensitivity of emerging imaging techniques isimperative. For fluorescence imaging, for example, detec-tors in the NIR range are often less sensitive than those inthe visible range; however, the use of CCDs and similarequipment has helped in this regard. Silicon detectors cancover the first NIR window, and InGaAs detectors are moreefficient in the second NIR window. The major problem inall optical imaging is tissue scattering; a method must bedeveloped that can deconvolute the scattering effectspropagated by imaged tissue [5, 92, 199] or otherwisecounteract this scattering by modification of imagingmodalities [4]. A cost/benefit analysis should be performedto balance the cost of the agents and detection systemsversus the improvement that those imaging techniques canprovide in terms of understanding disease progression,early detection, better prognoses, and improved patientquality of life and/or survival.

Design of improved NP probes

Additional types of NP contrast agents may see develop-ment in the future. Currently, “smart” probes that turn “on”when exposed to the target are being developed; forexample, superparamagnetic iron oxide NPs that containan optical probe which can be cleaved by proteases andthen fluoresce combine an MRI technique with a selectivefluorescent optical probe that turns “on” only when in thepresence of the desired target [23].

Most in-vivo imaging is focused on the detection andlocation of abnormal lesions (e.g., cancer), differentiating acluster of cells that are drastically different from surround-ing cells. If administered intravenously, the imaging agentmust selectively find and bind the tumor, which usuallyinvolves taking advantage of the EPR effect and targetedagents. Relying on the EPR effect alone is often time-consuming and may not provide enough contrast or signalintensity. Currently, fewer than 10% of engineered NPsreach tumor sites when both active targeting and EPR effectare combined; improvement in targeted delivery is thereforeneeded. In the future, NPs will hopefully be used ascontrast agents not simply to find tumors, but to elucidatebiological processes and cellular mechanisms, which maybe acute or transient processes, that are vital to understand-ing and perhaps even curing debilitating diseases such ascancer, Alzheimer’s, Parkinson’s, multiple sclerosis, rheu-matoid arthritis, and diabetes.

Multimodal bioimaging

A current popular approach to overcoming the limitationsimposed by a single imaging technique is to combine two

18 M.A. Hahn et al.

or more contrast agents into a single NP entity which canthen be imaged by those multiple techniques [1, 54, 200–202]. Combining the anatomical resolution of MRI with thesensitivity of optical imaging is common and could proveto be a powerful technique for finding and quantifying thesize of tumors, especially tumors or metastases that are toosmall for MRI detection alone. These MR/optical imagingagents have been used to monitor enzyme activity, in braintumor imaging, and to detect and monitor apoptosis andatherosclerosis [203]. Other types of multimodal contrastagents are also in development: PET/NIRF used with ICG[57] and QDs [59], SPECT/fluorescence [203], PET/MRI[198, 204–207], MRI/PAT [200], TAT/PAT used withSWNTs [123], and US/MRI [208], among others. Probeswith three modes of imaging are being considered, forexample MRI/NIRF/PET [55, 203, 209], PET–CT/MRI/NIRF [55], and even four modes incorporating MRI/PET/BRET/fluorescence [210].

An important concept to consider is that with anymultimodal system employed, the enhancement of onemodality must not be at the expense of another. For instance,concentrations of MRI agents must usually be higher thanagents for fluorescence imaging, because of the differentsensitivities of the two methods. Therefore, controlling theratios of the two types of agents is necessary: high MRI agentloading must be combined with a low fluorescent agentloading. Additionally, incorporating more than one modalitymay cause interferences between the two (e.g., iron-basedMRI agents quenching fluorescence agents) or complicatefabrication, resulting in higher production costs or commer-cial infeasibility. However, multimodal NPs would requireonly one dose of multiple agents to the patient, hopefullyreducing side effects from having to use multiple doses ofdifferent agents. This approach is likely to remain an area ofinterest in the future, but unambiguous data showingenhanced imaging with each technique incorporated in suchmultimodal NP agents remains to be seen.

Theranostic NPs

In addition to merely dual imaging, theranostic NPs areprominently finding their way into cancer research; theyprovide the diagnostic capability using an imaging modalityto detect a tumor, while supplying the component fortherapy against that disease, commonly utilizing photo-thermal ablation (PTA) or PDT. PTA works by exciting aNP with a large absorption cross section (e.g., gold), whichcauses localized heating that then kills the tumor cells intowhich the NPs have been injected. Gold nanoshellssurrounding a silica core have been used in photoablativetherapies, and an approach using OCT in combination withPTA has been used with gold nanoshells [211] andnanocages [212]. Iron oxide NPs with a gold shell enable

combined MRI and PTA, and PTA has been used withSWNTs [213]. Imaging strategies utilizing PTA will mostlikely see tremendous growth in the future, as goldnanoshells are already in clinical trials for cancer therapy.

PDT uses photosensitizers that, when excited by light,react with molecular oxygen in the biological environmentto produce ROS, which are cytotoxic to cells. Weissleder etal. have developed a multifunctional NP combining MRI,NIRF, and PDT [214]. If the photosensitizer requires visibleexcitation, two-photon absorption is an option to increasethe penetration depth of the excitation source. Even X-rayscan be used as the energy required for PDT: researchershave applied lanthanide-based materials and porphyrinswith PDT derived from X-ray bombardment, whichreleases UV photons. Gold NPs can also be applied asradiosensitizers at realistic doses with low energy X-raysources [169]; originating from these ionizing X-rays,Auger electrons directly cause single and double-strandedbreaks in DNA or produce free radicals when interactingwith water molecules, both resulting in local inactivation oftissue and cells. Theranostic NP probes can be used as theactual therapeutic itself (e.g., gold nanoshells) or simply asthe drug-delivery vehicle (e.g., liposomes). One materialthat may prove useful in combining a dual imaging andtherapy is mesoporous silica NPs; with their large surfaceareas and pore volumes, one modality can be incorporatedinto the silica matrix while loading the other modality intoits pores. This approach has already been done with anoptical imaging agent and an anticancer drug [215].

NP therapeutics can revive drugs that were previouslydiscontinued because of toxicity or solubility issues andhave the potential to mitigate side effects of the free drugby delivering it directly to the site of interest. Byencapsulating a concentrated drug payload, NPs preventexposure of healthy cells to the cytotoxic drug and mayprove more beneficial (e.g., lower toxicity and fewer sideeffects) at lower doses than the free drug. However, NPs aremuch more complex than the simple small-molecule drugs thatare easily characterized. In addition, the NP must remain intactuntil reaching the tumor site and then release the drugcontrollably through its desired mechanism—issues that willrequire further research and development. Kim et al. [201] andJarzyna et al. [54] have reviewed nanomaterials used formultimodal imaging and theranostic applications. Summarizedin Table 2, information on NP-based imaging agents andtherapeutics either approved or in clinical trials can be foundin articles by Adiseshaiah et al. [216] and McNeil [196].

Toward the future: cancer, therapy, and the public

In-vivo cancer imaging is likely to be the most prominentarea where nanomaterial research will be focused. The

NP contrast agents for bioimaging 19

statistics are staggering: the NCI has estimated that cancerexpenditure was approximately $104.1 billion in 2006, withbreast, colorectal, and lung being the most costly [217].According to the NCI over $3 billion was invested incancer research in 2009, when the top four cancersresearched were breast, prostate, colon/rectum, and lung[218]. According to estimates from the NIH, cancer will bethe third most heavily funded area in 2010–2011, precededonly by grants for clinical research and genetics [219].Therefore, early cancer and disease detection, and combin-ing detection and therapy, are potential areas for growth anddevelopment of NP contrast agents.

Considering the amount of money spent on the secondleading cause of death in the United States, this importantarea is a major opportunity for NP-based agents. Oncolo-gists are interested in using NPs for SLN mapping; thistechnique involves using an agent to locate the sentinellymph node before surgery and doing a biopsy to see if anymalignancies are present to warrant removal of all lymphnodes, which may or may not have cancerous lesions.Having been especially useful in breast cancer cases andsignificantly improving the detection of small metastases[220], this technique has been proposed as a standardprocedure for cutaneous melanoma, in which trials havebeen successful [221]. Tracers currently used in the UnitedStates are isosulfan or “blue dye” (Lymphazurin 1%, USSurgical, North Haven, CT, USA), which requires visualinspection, and 99mTc–sulfur colloid (approved by the FDAfor imaging liver and spleen), which requires a gamma raycounter; other radiotracers based on 99mTc are available inAustralia, Canada, and Europe. The technique is compli-cated because different procedures must be used fordifferent cancers and tumor locations, and the success ofmapping is linked very heavily to the experience and skillsof the surgeon performing the procedure. NP agents withbetter contrast or ease of detection would prove beneficialfor increasing the usefulness and success of SLN mapping,leaving it less dependent on the individual clinician.Besides the agents, other types of imaging, for exampleSPECT–CT, may help elucidate SLN location with tumorsdisplaying ambiguous lymph node drainage. Researchersare delving into other nanomaterials and imaging proce-dures for SLN mapping: Ravizzini et al. provide anoverview of various methods using CT, US, MRI, andfluorescence imaging for this procedure, with optical andMRI proposed as the future path for SLN mapping [29].

Not only diagnosis, but also treatment of cancer with NPtherapeutics is likely to see major development in thefuture. As shown in Table 2, many NP-based drugs areeither approved or in clinical trials [196, 216]. NP-basedtherapies for cancer, for example gold nanoshells combinedwith PTA, are in clinical trials by Nanospectra Biosciences(Houston, TX, USA). However, the biggest obstacles

toward the development of new NP contrast agents forbioimaging are government regulations and pure econom-ics. Getting an agent into clinical trials and approved by theFDA is a time-consuming and costly process, lasting up to18 years and costing up to $2 million [222]; procedures thatcan shorten the development time and/or reduce the cost ofbringing NP agents to market will therefore be mostlucrative. Examples include incorporating a NP on to analready-approved marker or combining the NP agent with atherapeutic. Bawa provides an overview of getting NP-based therapeutics through the drug development process,highlighting the importance of federal agencies such as theFDA and the USPTO [223]. A Nanoparticle Task Force hasbeen set up by the FDA to handle such regulatory issues[224].

Investment in a new NP for clinical imaging is highlydependent on a number of factors. Newer agents must beproved to be vastly superior to any currently inexpensiveversion that provides the same function; otherwise, therewill be no incentive to continue its development. Therefore,agents that incorporate additional functionality (i.e., thera-peutic agent, measure of disease progression, or evaluationof treatment effectiveness) with the in-vivo imagingmodality will most likely see growth, especially ifincorporated into an already-approved material or device.Also, investors in the development of new imaging andtherapies are generally hesitant unless the novel agent ispotentially lucrative with widespread clinical use: probesthat will benefit a large population of patients are morelikely to see development than those that target a raredisease or small subset of patients. Ultimately, it will takean interdisciplinary team, consisting of a variety ofresearchers with diverse backgrounds, for example materi-als scientists, chemists, biologists, electrical engineers,surgeons, clinicians, and regulators, to make the prospectof widespread NP use for clinical in-vivo imaging a reality.

Acknowledgments This work was supported by the NationalScience Foundation’s Division of Chemical, Bioengineering, Envi-ronmental, and Transport Systems (CBET) grant 0853707, theNational Science Foundation’s Nanoscale Interdisciplinary ResearchTeam (NIRT) Engineering Education and Center (EEC) grant0506560, the Center for Nano-Bio Sensors (CNBS) at the Universityof Florida, and the Bankhead Coley Florida Biomedical ResearchProgram.

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