Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid...

Review ArticleBimodal Imaging Probes for Combined PET and OIRecent Developments and Future Directions for HybridAgent Development

Uwe Seibold12 Bjoumlrn Waumlngler2 Ralf Schirrmacher3 and Carmen Waumlngler1

1 Biomedical Chemistry Department of Clinical Radiology and Nuclear Medicine Medical Faculty Mannheim ofHeidelberg University Theodor-Kutzer-Ufer 1-3 68167 Mannheim Germany

2Molecular Imaging and Radiochemistry Department of Clinical Radiology and Nuclear MedicineMedical Faculty Mannheim of Heidelberg University 68167 Mannheim Germany

3McConnell Brain Imaging Centre Montreal Neurological Institute McGill University Montreal QC Canada H3A 2B4

Correspondence should be addressed to Carmen Wangler carmenwaenglermedmauni-heidelbergde

Received 11 February 2014 Accepted 18 March 2014 Published 16 April 2014

Academic Editor Patrick Riss

Copyright copy 2014 Uwe Seibold et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Molecular imagingmdashand especially positron emission tomography (PET)mdashhas gained increasing importance for diagnosis ofvarious diseases and thus experiences an increasing disseminationTherefore there is also a growing demand for highly affine PETtracers specifically accumulating and visualizing target structures in the human body Beyond the development of agents suitablefor PET alone recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI)as this combination of modalities can provide clinical advantages PET due to the high tissue penetration of the 120574-radiation emittedby PET nuclides allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body OI on thecontrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumormargins and infectedlymph nodes during surgery without bearing a radiation burden for the surgeon Thus there is an emerging interest in bimodalagents for PET andOI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseasesThis short review summarizes the available hybrid probes developed for dual PET andOI and discusses future directions for hybridagent development

1 Introduction

Within the last decades the development of new radiotracersfor PET imaging has experienced an enormous progress dueto its enormous specificity and sensitivity in the visualizationof target tissues Thus a rising number of valuable com-pounds applicable in cardiologic neurologic and especiallyoncologic imaging were developed However PET alone dis-plays a limited spatial resolution of 1ndash3mm in clinical practiceand also is not able to allow a morphological correlation ofthe tracer accumulation which is however especially crucialin case of tumor diagnosis localization and staging Thusalmost all clinical PET systems sold within the last yearsare combinations of PET and computed tomography (CT)

systems integrating the strengths of bothmodalities the highspecificity and sensitivity of PET making already functionalchanges in tissues visible at a very early stage of disease andthe detailed morphologic information provided by CT [1]Most recently also combined clinical PETMRI (magneticresonance imaging) systems are commercially available TheMRI modality provides an even higher resolution and softtissue contrast than CT allowing for a functional imagingwithout causing any additional radiation burden to thepatient In combination with the very high sensitivity andspecificity of PET an almost ideal combined imaging modal-ity is obtained for the whole-body imaging of patients [2]although the number of hybrid agents applicable in PETMRimaging is very limited so far

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 153741 13 pageshttpdxdoiorg1011552014153741

2 BioMed Research International

Despite these favorable properties of PETCT and alsoPETMRI systems in whole-body imaging for the identifi-cation of target structures these modalities exhibit certainlimitations after having specifically identified and localized atumor target tissue the resection of the tumormass is difficultdue to the intricate intraoperative identification of tumormargins and smallmetastases Additionally the identificationof the sentinel lymph node (SLN) which is often resected forhistology is not trivial For this purpose another combinationof imagingmodalities could be of special interest namely thecombination of PET with optical imaging (OI)

Although OI is a modality with restricted applicabilityfor whole-body in vivo imaging due to the limited tissuepenetration of the light emitted by the fluorescent probeit is a valuable methodology for surface imaging applica-tions such as intraoperative image-guided surgery due toits favorable spatial resolution and sensitivity [3ndash5] Thusa combined bimodal imaging consisting of an initial PETscan using 120574-radiation with a high tissue penetration rangeto identify and localize tumor lesions throughout the bodyand a subsequent intraoperativeOI in order to identify tumormargins and infected lymph nodes can result in a significantclinical improvement [6ndash8] Especially in breast and prostatecarcinomas as well as melanomas the prognosis stronglydepends on the presence of lymph node metastases [9ndash11]However the secure intraoperative identification of sentineland infected lymph nodes is crucial for efficient diagnosisand treatment but is difficult if the surgeon can only rely onabnormal visual appearance and palpation to discriminatebetween lymph nodes and surrounding tissues or to identifyinfected nodes The use of a specific tumor-accumulatingagent which can be visualized during surgery by opticalimaging techniques emitting light which can penetrate tissuein a reasonable range (so that a target node can be detectedeven if not already fully surgically exposed) would mean asignificant improvement for surgery (Figure 1)

The development of new combined imaging techniqueshowever also requires the development of the respectivehybrid imaging agents that are suitable for all involvedimaging modalities Thus considerable research has beenconducted in this field of hybrid contrast agents over the lastyears [3 4 12ndash15]

For combined PET and optical imaging in principle theuse of two separate molecular markers one for PET andone for OI (instead of using a hybrid imaging agent) wouldalso be possible However this is no optimal approach asboth agents are likely to exhibit differing biodistribution andpharmacokinetic properties (especially in cases of relativelysmall specifically accumulating biomolecules such as pep-tides) Hence to achieve reliable results that are comparablebetween both imaging modalities a hybrid marker has to beapplied

It has to be kept in mind that optical imaging is not fullyquantifiable as it is surface-weighted due to absorbance andscattering of the photons by tissue penetration (especiallywhen imaging deep tissues exhibiting a low imaging agentaccumulation) and thus cannot be fully correlated to PETimaging data [16ndash19] As PET however is fully quantifiableand used for whole-body imaging whereas OI is used for

intraoperative imaging purposes only in order to identifytumor tissues (tumormargins small metastases and infectedlymph nodes) the quantification of optical signals is nocritical criterion

For OI different classes of reporter probes detectableby optical imaging techniques can in principle be used ina hybrid PETOI agent (i) fluorescent proteins that can bedetected by bioluminescence imaging (BLI) (ii) 120574-emittingradionuclides that can be visualized by Cherenkov lumi-nescence imaging (CLI luminescence that can be observedwhen a particle travels faster than light in the examinedmedium) (iii) fluorescent small dye molecules that canideally emit near infrared light and (iv) quantum dots whichare semiconductor nanocrystals consisting of CdTe or CdSematerials and whose emission characteristics can be tailoredby particle size For all these probes that can be used in thedevelopment of a hybrid PETOI agent substances emittinglight of the near-infrared and infrared spectrum (700ndash900 nm) aremost useful as light of thesewavelengths exhibitsthe highest tissue permeability of several mm to cm in vivo[20 21]

Large proteins such as GFP (green fluorescent protein) orRFP (red fluorescent protein) are in principle applicable inthe synthesis of a hybrid compound However they are struc-turally demanding and would most possibly have a severeimpact on the pharmacokinetic properties of the resultingimaging agent Thus it is only conceivable to use these com-pounds in combination with particle carriers Furthermorethe quantum yield of these proteins is rather limited and theydo not enable near-infrared photon emissions [22] furtherrestricting the use of fluorescent proteins in hybrid opticalimaging agents

In contrast CLI using different positron-emitting radio-nuclides has been proposed as a favorable optical imagingtechnique for imaging-guided surgery [23] This techniquedoes not require the conjugation of an additional fluorescentcompound in order to obtain a bimodal imaging agent Thisis favorable as an additionally conjugated fluorescent dyecanmdashif susceptible to the radiolabeling conditions appliedmdashinterfere with the radiosynthesis or result in a significantalteration of the pharmacokinetic properties of the result-ing hybrid compound Unfortunately using the Cherenkovluminescence imaging approach one of the most valuableproperties of combined PETOI probes to be applied in intra-operative imaging namely the consecutive detection via PETand the subsequent later resection of the tumor cannot beutilized Using a hybrid compound consisting of a fluorescentdye in addition to a radionuclide the optical intraoperativeimaging can be performed delayed in time after identifyingand localizing the tumorous tissue by a whole-body PETscan By this procedure the radionuclide at least partiallydecayed before surgery resulting in no or only low radiationburden to the surgeon during intraoperative imaging andresection In contrast using CLI for intraoperative imagingcan result in a significant radiation burden as is indicatedby a recent study systematically investigating the potential ofCLI in a preclinical setting In this workmdashwhen imaging an124I activity depot located subcutaneously in 4mmdepthmdashan

BioMed Research International 3

Tumor

SLN

Lymphaticflow

Single injection ofthe bimodal imaging

agent

First step whole-body PET imaging for tumor

localization and surgical planning

Second step intraoperative fluorescence image-guided surgery of tumor margins

and SLN

120574

120574

120582em

120582ex

Figure 1 Schematic depiction of the operation principle of a PETOI hybrid compound After being applied to the patient in a single injectionan initial whole-body PET scan is performed identifying and localizing tumor and potential metastases thus serving as a tool for surgeryplanning During the following surgical intervention the same compoundmdashhaving accumulated in the target tumor areas over timemdashcan beused as a marker for intraoperative image-guided surgery of the respective malignant tissues

activity concentration of at least 03mCimL (111MBqmL)was necessary to obtain a detectable signal [24]

In most of the reported bimodal hybrid compoundsfor PETOI small fluorescent dyes or quantum dots arethus applied as they produce no ionizing radiation and arerelatively stable under physiological conditions [25ndash27] Thisallows for an image-guided surgery even after the decay ofthe radionuclide In addition small fluorescent dyemoleculesexhibit the advantage of being relatively small in size and thusresult in a less prominent influence on the binding parametersof the carrier molecule which is especially important for thederivatization of small and medium-sized biomolecules

2 Examples of Dually Labeled AgentsApplicable in Hybrid In Vivo PET andOptical Imaging

Besides hybrid agents for combined PETOI also markersfor dual SPECTOI have been developed over the last yearscomprising dually labeled antibodies [28 29] peptides [30ndash35] a nontargeted small molecule [36] and nanoparticles[37ndash40] However as PET ismdashin contrast to SPECTmdashfullyquantifiable and exhibits a much higher sensitivity than thelatter the main focus in this young field of bimodal probedevelopment for use in nuclear medicine and optical imaginglies on the development of PETOI agents having a greaterpotential for a possible clinical application

21 Nontargeted Small Molecules Apart from targetedand nontargeted probes based on different biomoleculeor nanoparticle carriers developed for a mostly tumor

target-specific accumulation the synthesis of several smallmolecule-based bimodal labels was reported These areintended to be used directly without any further targeting forimaging (Figure 2 1ndash3) or could serve as a basis for a futurebimodal labeling of biologically active compounds such asantibodies and other proteins (Figure 2 5ndash8)

The imaging agents 1ndash3 [41ndash44] depicted in Figure 2 arebased on porphyrin or phthalocyanine derivatives whichcan show a significant accumulation in tumor tissues andcan be used as photosensitizers thus being applicable inphotodynamic therapyThese porphyrin and phthalocyaninederivatives were radiolabeled with 124I and 64Cu in differentpositions respectively and subjected to tumor xenograftmicefor in vivo evaluation of their PET andor optical imagingcharacteristics Due to the missing tumor targeting entitythe observed tumor accumulations were faint and also ahigh unspecific accumulation of the compounds in nontargetorgans such as liver spleen gut lung and bloodwas observed[41 42 44] limiting the usefulness of these compounds for invivo tumor imaging The 18F-labeled Cy55 derivative 4 wassynthesized in a proof of concept approach to demonstratethe applicability of a new secondary 18F-labeling precursorfor the radiolabeling of even sensitive molecules such ascyanine dyes and was thus not investigated regarding its invivo characteristics It could however be useful as hybrid labelif functionalized for a bioconjugation and introduced into atargeting vector [45]

In contrast to compounds 1ndash4 hybrid agents 5ndash8 [46ndash49] are not intended to be used directly for an in vivoapplication but for conjugation to a specifically accumulatingagent such as a peptide antibody or antibody fragment bydifferent reactive functional groups (active esters maleimide


N HN

NNH

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OOO

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124I-PS41

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64 Cu-PcS42

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OO

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NH2

18F-Cy554

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18F-BODIPY N-hydroxysuccinimide ester5

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BODIPY-DOTAGA-isothiocyanate7

NN

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HO3SSO3H

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SO3

64 Cu

64 Cu-DO2A-IRDye800CW8

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OH

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OO

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HO3S

O3S

SO3H

SO3H

Cy55-Lys(SMCC)-CHX-6

A998400998400-DTPA

S=C=N

Figure 2 Structures of small molecule-based bimodal labels developed for hybrid imaging with PET and OI (fluorescent dyes are depictedin red and PET nuclides in green)

and isothiocyanate) By this approach the concomitantintroduction of the radio- and the fluorescent label into thebiomolecule is enabled and the resulting hybrid probe canbe used in a targeted in vivo imaging application Howeverthe application of such a hybrid label for the derivatization

of a specifically accumulating carrier molecule as well asthe subsequent radiolabeling and in vivo evaluation of theso obtained dually labeled imaging probe was so far onlyshown for 8 For this purpose an anti-EpCAM antibodywas first reacted with DO2A-IRDye800CW-sulfo NHS ester


NH

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R =

[64 Cu Cu] [64 ]GB173GB170 and 9 and 10

64 Cu-porphyrin-folate

11

SO3H

64 Cu64 Cu

NH2

NH2

O3S

HN

NH

Figure 3 Structures of hybrid small molecule agents intended for specific accumulation in tumor tissues

radiolabeled with 64Cu (8) and finally evaluated in a proofof concept study in PC-3 xenograft mice [49] Unfortunatelyonly near-infrared fluorescence imaging (NIR-FI) whole-body in vivo images and no PET data were shown in thisstudy which is most probably attributable to the foresee-able insufficient stability of the 64Cu-DO2A complex [50]resulting in very high liver and blood accumulations of theradionuclide compromising the in vivo PET data In additionalso the NIR-FI data point to a predominant liver and alsohigh kidney as well as lung accumulation at 40 h pi of thedual-labeled antibody limiting its potential for hybrid in vivotumor imaging Thus though not directly applicable in vivothese small molecule-based hybrid imaging probes reflect thehigh interest in this relatively new field of hybrid PETOIagent development

22 Dually Labeled Small Molecules and Peptides Intendedfor Target-Specific Accumulation and Bimodal Target Visu-alization by PETOI The introduction of a fluorescent dyetogether with a radiolabel (which is either covalently attachedor complexed by a chelator system) can result in a significantstructural alteration especially in case of rather small target-specific molecules Nevertheless attempts have been made tosynthesize such dually labeled small molecules and peptidesas they usually display fast pharmacokinetics and targetaccumulations and a rapid clearance from nontarget tissuesin principle resulting in favorable high-contrast images

Especially for the studied small molecules (Figure 3 9ndash11) the structural change by introducing two labels wasshown to result in high background and low specific tumoraccumulations The main excretory organ was in all casesthe liver for which a very pronounced uptake of the radi-olabeled substances was observed but also kidneys spleenand intestines showed high accumulations of the tracershampering a high tumor uptake and thus efficient tumorvisualization with PET [51 52]

Interestingly it could be shown in the study dealingwith the dually labeled cysteine cathepsin substrates [64Cu]9

and [64Cu]10 that the introduction of the NIR dye canresult in a favorable prolonged circulation compared to the64Cu-DOTA-modified analog not comprising a fluorescentdye [52] This positive effect of fluorescent dye conjugationcould be confirmed by another study [16] This prolongedcirculationwas described to result not only in a higher unspe-cific accumulation in all organs but also in a significantlyhigher and at least in part specific tumor accumulation [52]presumably due to a higher interaction probability of thehybrid agent with its target

Despite the disappointing results obtained with PETimaging of the 64Cu-labeled porphyrin-folate conjugate 11a clear tumor visualization was possible with fluorescenceimaging (FI) after 24 h pi which can be attributed to thefact that the tumors were rather large and located directlyunder the skin Furthermore the tumors weremdashfrom alltissuesmdashlocated nearest to the detector system minimizingthe absorbance and scattering of the fluorescent light emittedfrom the tumors whereas the photons emitted from theexcretory organsweremost probably strongly attenuated [51]

Due to their larger molecular size peptides are inprinciple more likely to tolerate a derivatization with twodifferent labels in terms of receptor binding and in vivopharmacokinetics This theoretical tendency seems in fact tobe reflected in the results obtained for dually labeled peptidesFrom the dually labeled PET radionuclide and fluorescent dyecomprising peptides available for tumor imaging so far (12ndash16 Figure 4) 4 compounds namely 13ndash16 were evaluatedin vivo regarding their biodistribution properties and tumorvisualization abilities [16 17 25 26 53]

The results obtained in these studies seem to point to amore favorable biodistribution together with higher tumorto background ratios and higher tumor targeting specificityin case of larger dually labeled peptidic targeting vectorsIt could for example be shown that for Tyr3-octreotate(TATE) derivatized at the N-terminus with 64Cu-DOTA andat the C-terminus with a NIR dye (13) on the one handencouraging in vitro binding results to A427-7 tumor cell


O

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GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCG

O

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NH2

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H2N

64 Cu

64 Cu

64 Cu

68 Ga

68 Ga-protoporphyrin IX-RGD

64 Cu-DOTA-TATE-Lys(cypate)-NH2

68 Ga-DOTA-IRDye800CW-HWGF

12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S

64 Cu-BaAnSar-RGD2-Cy5564 Cu-DOTA-Lys(Cy55)-Gly-Gly-Tyr-25D

H3C

Figure 4 Structures of dually labeled PET radionuclide and fluorescent dye comprising peptides developed for in vivo tumor imaging withPET and OI


membranes with 119870119894values of 043 nM for TATE and 115 nM

for natCu-13 could be obtained but that the agent was onthe other hand not able to visualize the respective A427-7tumor in vivo in xenograft mice at 24 h pi with FI [26]Moreover the low tumor uptake (only reaching a tumor-to-blood ratio of about 2) could not be blocked by coldpeptide in biodistribution studies with 64Cu-13 pointingto an unspecific tumor accumulation caused by the EPReffect (enhanced permeability and retention effect a passivetumor-targeting process that results in an unspecific uptake ofcompounds due to a more permeable tumor vasculature andefficient diffusion through the tumor interstitium) Overalla significant accumulation of this compound could only beobserved in liver (16824 plusmn 1520IDg) spleen (8069 plusmn1808IDg) and lung (1428plusmn0738IDg) after 1 h pi (forcomparison tumor accumulation was 0287 plusmn 0046IDg)pointing to a too pronounced overall lipophilicity of thecompound for a successful in vivo application

Similar effects were shown for 14 which was developed tovisualize MMP2 and 9 in vivo [25] However in a heterotopicossification model activating MMP9 a target visualizationcould not be achieved by PET imaging Using NIR-FI theossification site could be visualized but no whole-bodyimages were shown limiting the informative value of theseimages

In contrast to these latter studies two examples of verypromising peptidic hybrid compounds for PET and OIwere reported One of thesemdashconsisting of an 120572]1205733 and120572]1205735-affine knottin peptide targeting tumor angiogenesisderivatized with Cy55 andDOTA via an amino acid spacermdashwas radiolabeled with 64Cu (15) and successfully used forspecific in vivo PET and NIR-FI of an integrin-positiveU87MG tumor in xenograftmice [17]These favorable resultswere achieved although the in vitro binding data indicatedan adverse influence of the derivatization of the peptidewith NIR dye and chelator compared to a monolabelingwith DOTA or NIR dye alone Interestingly comparing the64Cu-DOTA-monolabeled knottin peptide with the dual-labeled one regarding in vivo biodistribution with PET bothcompounds achieve tumor-to-background ratios (TBR) of sim45 However these comparable ratios were found at differenttime points the 64Cu-DOTA-monolabeled peptide reachesthis TBR already at 4 h pi whereas the same TBR is achievedby the dually labeled peptide 15 at 24 h pi indicatingmdashas described beforemdasha retention-prolonging effect of theconjugated NIR dye This is confirmed by the correspondingNIR-FI experiment comparing theNIR-monolabeled peptidewith the dually-labeled one 15 which both reach the TBR ofsim45 at 24 h pi

A very encouraging example of a dually labeled hybridcompound was described recently consisting of a cRGD-dimer (serving as tumor-targeting vector) and Cy55 whichis connected to the peptidic part via a sarcophagine-derivedchelator used for 64Cu-labeling [16] The radiolabeled com-pound 64Cu-16 was successfully used for the in vivo imagingof integrin-rich U87MG tumors in a xenograftmouse modelshowing a high tumor uptake together with a stable tumorretention (641 plusmn 028 651 plusmn 145 and 592 plusmn 157IDg

at 1 4 and 20 h pi resp) resulting in the highest tumor-to-background ratios of sim7 at 20 h pi As described beforethis NIR dye-labeled compound 64Cu-16 showed a pro-longed circulation together with a higher tumor accumula-tion compared to the corresponding nonfluorescent-labeledderivative [16] Furthermore 64Cu-16 was used for image-guided resection of the tumor in the same animal model andshowedmdashin contrast to the PET images displaying a homo-geneous tumor areal due to the physically limited spatialresolution of 64Cumdashthe presence of a metastasis near theprimary tumor impressively demonstrating the advantagesof intraoperative optical imaging and the synergistic effectsof PET combined with OI

These favorable in vivo imaging results found for 15and 16 are probably a result of two different effects thelarge size of the peptidic targeting vector relative to bothlabels and also the introduction of both labels in only oneposition of the peptidic moiety limiting their influence onthe overall biodistribution compared to two labels introducedin different positions of the peptide Thus due to the strongpotential influence of two labeling moieties introduced theligand design has to be carefully considered especially whenderivatizing peptides

23 Fluorescent and Radiolabeled Antibodies for CombinedPETOI Antibodies with their slow pharmacokinetics andvery high target specificity should be well suited as targetingvectors for a dual-labeling approach with a PET nuclideand a NIR dye as they exhibit a more complex structurethan small molecules and peptides resulting in a less strongalteration of structure binding characteristics and thusbiodistribution properties by the concomitant conjugation oftwo labels Several different antibodies have been derivatizedwith desferrioxamine [54ndash56] for 89Zr-labeling NOTA [57]or DOTA [18 58 59] for 64Cu-labeling and the NIR dyes800CW [54ndash59] or Alexa Fluor 750 [18] (Figure 5)

In all studies it could be demonstrated that the numberof introduced derivatization sites has a crucial effect on thebiodistribution characteristics of the obtained hybrid com-pounds One study for example describes the derivatizationof an anti-CD20 IgG with sim10 chelators and sim2 fluorescentdyes resulting in an unfavorable biodistribution of the hybridcompound in lymphoma-bearing mice showing a very highliver and spleen accumulation of the antibody Consequentlyonly a moderate tumor uptake was observed resulting in onlypoor tumor visualization in vivo [18] Reducing the numberof introduced labels radionuclide chelator and fluorescentdye to sim2 per trastuzumab molecule improved results couldbe obtained in 4T12neuR tumor-bearing xenograft miceallowing for a tumor visualization with PET as well as NIR-FI at 24 h pi although tumor-to-muscle ratios of only sim25were obtained [58] Reducing the number of both labelingmoieties to 1 per anti-CD105 antibody molecule the tumor-to-muscle ratios could be improved to sim7 in 4T1 tumorxenograft mouse models [56] However besides a tumoruptake of sim10IDg high liver spleen and blood uptakesof sim16IDg sim8IDg and sim11IDg were observed at48 h pi respectively impairing the in vivo imaging results


ON

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S

ON

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NNHO

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OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3

n = 05ndash10m = 07ndash5

m

n

=

=

Figure 5 Schematic depiction of dually labeled antibodies developed for in vivo hybrid PETOI of tumors

Nevertheless the observed tumor uptakes were no result ofthe EPR effect alone but also of a specific binding as theycould be blocked by about 50 by coapplication of unlabeledantibody

Other studies limiting the number of introduced labelsto a minimum of 05ndash09 equivalents of each labeling moietyper antibody found even more favorable biodistributionproperties such as a slowed clearance lower liver and higherandprolonged tumor uptakes resulting in a clear visualizationof the tumor mass in vivo at 48 h pi with PET as well as NIR-FI [54 57] Besides the observed impaired biodistributionproperties of high liver and spleen uptake in vivo whenconjugating several fluorescent dye molecules per antibodya conjugation of several dyes also results in a fluorescencequenching effect and thus a decreased overall fluorescenceintensity being detrimental to a successful in vivo NIR-FI ofthe target tissue [54 57] These studies furthermore investi-gated the correlation between organ uptakes determined byin vivo PET and in vivo or ex vivo NIR-FI The PET data inthese studies served as reference parameters as PET is fullyquantifiable It was found that the deviations in measuredorgan uptakes were higher for the NIR-FI data obtained invivo than obtained ex vivo Also the deviations were higherfor deeper tissues pointing to a significant scattering andabsorbance of the NIR light limiting the quantification oftissue uptakes by fluorescence imaging and necessitating thequantification of organ uptakes by PET This is howeverno limitation for optical imaging in terms of intraoperativeimaging settings where only qualitative images are requiredfor a successful tumor resection

Besides the described general findings regarding thenegative influence of a high number of conjugated labelingmoieties on the biodistribution properties of derivatizedantibodies one study systematically investigated the influ-ence of the number of conjugation sites on the biodistri-bution of dual-labeled antibodies [55] There the EGFRand VEGF targeting antibodies cetuximab and bevacizumabwere initially derivatized with on average 05 desferrioxaminechelators followed by an introduction of 05 to 5 800CWNIRdyes and the biodistribution of the resulting hybrid imaging

compounds was determined in vivo after 89Zr-radiolabelingIt could be shown that the antibody uptake into the liverproportionally increasedwith the number of conjugated dyeswhereas the tumor accumulation decreased to the sameextent

Thus in order to achieve optimal imaging results using adually labeled hybrid antibody for PET and NIR fluorescenceimaging the development of a small molecule serving as adual-label would be of advantageThismolecule could consistof a chelator for radiometal labeling the NIR dye and afunctionality enabling a concomitant conjugation of bothlabels in only one position of the antibody thus limiting itsstructural change to an absolute minimum

24 Nanoparticles as a Platform for Hybrid PETOI AgentsNanoparticlesmdashin contrast to biomoleculesmdashexhibit theadvantage of possessing a large surface which can easilybe modified with functional groups for the conjugationof targeting vectors radiolabels and fluorescent dyes Onthe other hand they also face several problems (i) theynecessitate a stable coating for functionalization (ii) exhibita long tissue retention (iii) only insufficient knowledge isavailable about their toxicity (especially in case of quantumdots consisting ofCd ions and other potentially toxicmetals)metabolism and excretion and (iv) they strongly accumulatein the reticuloendothelial system (RES) and thus in liverspleen bone marrow and lymph nodes Furthermore thestoichiometry of the conjugatedmoieties is difficult to controlor quantify after reaction Nevertheless most of the hybridcompounds developed for dual PET and OI so far are basedon nanoparticles as carriers

The group of nanoparticles applicable as structural basisfor hybrid PETOI agents consists of several different sub-groups polymer-based nanoparticles [60] lipid-based par-ticles such as micelles [61] and liposomes [62] carbon-based systems such as nanotubes [63] and also metal-basednanoparticles such as iron oxide [5 27 64ndash67] silica [8] andupconversion nanoparticles [68 69] as well as quantum dots(QDs) [70ndash73]


QDs are fluorescent semiconductor nanocrystals whosefluorescent properties can be influenced by the particlesize and composition Furthermore they exhibit high quan-tum yields and photostability [74] making them interest-ing fluorophores for the development of compounds forhybrid PEToptical imaging when stably radiolabeled witha positron-emitter (Figure 6(a)) Superparamagnetic ironoxide nanoparticles on the other hand are detectable byMRIenabling a triple-modality imaging with PETOI and MRIwhen derivatized with fluorescent dyes and radionuclides(Figures 6(b) and 6(c)) [27 64] QDs as well as iron oxidenanoparticles have to be coated with biocompatible materialsto render them amenable for an in vivo application Thiscoating can consist of differentmaterials such as SiO

2or other

inorganic material dextran micelles or polyethylene glycols(PEGs) and furthermore enables a chemical modificationof the surface of the particles with dyes radiolabels andtargeting vectors allowing for a target-specific accumulation(Figures 6(d) and 6(e)) An alternative to the approachof chemical modification of the coating of a nanoparticlewith NIR dyes in order to obtain a fluorescent agent isthe encapsulation of the fluorophore within the particlecoating (Figure 6(b)) which has been shown to result in amuch higher fluorescence signal and photostability of thefluorescent dye than a surficial dye conjugation (Figure 6(c))[8 27]

An important factor in the design of particles intendedfor in vivo imaging purposes is their sufficient stability overthe duration of the examination Thus also the radiolabelhas to be stably introduced by covalent conjugation (in caseof nonmetallic isotopes such as 18F or iodine isotopes) orstable complex formation (in case of radiometal ions) Asthe development of hybrid agents for combined PET and OIis still in the beginning nanoparticles which do not exhibita stable radionuclide introduction have also been reportedIn these cases the particles were only incubated with theradionuclide ldquotrappingrdquo the respective radioisotope by pro-teins used for coating of the particle surface [61] functionalgroups such as primary amines [64] ionic interactions for18F-labeling [68 69] or the use an suboptimal chelator forthe applied radiometal [65] In these cases liberation ofthe radionuclide was inevitable resulting in the expectableunfavorable biodistribution characteristics of the radiolabeland thus low image quality In other cases the potential ofthe labeled nanoparticles was not demonstrated as the agentswere applied via intratumoral injection [62] or incubatedwith tumor cells that in the following could be visualized inanimals directly after implantation of the labeled cells [66]

In contrast also well-designed hybrid nanoparticleprobes were described showing highly promising results andgiving directions for further developments

As alreadymentioned the particle coating allows not onlyfor the conjugation of fluorescent dyes and radiolabels butalso for modification with a targeting vector such as peptidesor proteins for enabling a tumor-specific accumulation andimaging but only few examples of such targeted particlescan be found Two of them describe the surface-modificationof 64Cu-labeled QDs with VEGF and c(RGDyK) for

in vivo imaging of angiogenesis and in both cases a VEGFR2

and 120572v1205733 receptor-specific binding could be demonstratedin vitro and in vivo [70 71] Although the major fractionof the particles was shown to rapidly accumulate in thereticuloendothelial system (which is attributed to theirsize of about 20 nm) both particles allow a visualizationof the tumor entity in the respective tumor xenograftmouse models Furthermore they show a targeting-vectordependent accumulation as the respective particles withouta VEGF or c(RGDyK) derivatization show only a back-ground level tumor accumulation Another even morefavorable example of a hybrid nanoparticle was describedvery recently Small silica particles of 6-7 nm in diametercomprising encapsulated NIR dye Cy55 were PEG-coatedin order to achieve a higher biocompatibility and lowerliver accumulation These particles were further derivatizedon their surface with c(RGDyK) and radiolabeled with124I on the tyrosine moiety of the peptide These particleswere successfully used for whole-body PET imaging fortumor and multiple metastases visualization (showing a veryfavorable biodistribution without accumulation the RES) aswell as intraoperative imaging guidance in a spontaneousmelanoma miniswine model [8] The intraoperative imagingwas performed using a hand-held fluorescence imagingcamera allowing for real-time fluorescence imaging andsurgical guidance By this it was possible to identify sentinellymph nodes and to discriminate between metastatic tumorinfiltration and inflammatory processes during surgery Thisexample of presurgical whole-body PET imaging togetherwith subsequent intraoperative optical image-guided surgeryin a larger animal shows the very high clinical potential ofthis approach

In order to overcome the short circulation half-life andrapid RES accumulation of nanoparticles resulting in a verylow interaction probability of the imaging agent with the tis-sue to be visualized the use of smaller particles (lt12 nm) hasbeen proposed [71 73] Furthermore PEGs can be attachedto the particle surface This modification slows the particleresorption by liver and spleen [72 73] but can in return resultin a higher bone marrow uptake of the compounds in vivo[71 73] Particles comprising no targeting vector showing arapid accumulation in the RES can however also be usefulespecially for sentinel lymph node (SLN) mapping (Figure 1)[27 60]

3 Conclusion

So far the obtained results for hybrid PETOI agents arevariable Nevertheless some examples already show the highpotential of these substances for target visualizationwith bothimaging modalities Future developments of dually labeledhybrid imaging agents for PETOI exhibiting a favorable invivo biodistribution and being applicable in a multimodalclinical setting face the challenge to introduce a radiolabelas well as a fluorescent reporter probe by at the sametime preserving the favorable pharmacokinetic propertiesenabling a successful and specific target visualization


Radiolabel

Fluorescent dye

Targeting vector

Silica particle

Coated iron oxide particle

Coated quantum dot

(a) (b) (c)

(d) (e)

Figure 6 Schematic representation of different kinds of labeled nanoparticles that were already used as hybrid compounds for PET andOI (a)coated quantum dot loaded with chelating agent for radiometal introduction (b) coated iron oxide particle with encapsulated fluorescent dyeand derivatized on its surface with a chelator for radiolabeling (c) coated iron oxide particle derivatized on the surface with fluorescent dyeand chelator for radiometal labeling (d) silica nanoparticles with encapsulated fluorescent dye and surficial derivatization with radionuclideand targeting vector and (e) coated quantum dot loaded with chelating agent for radiometal introduction and a targeting vector

Promising probes developed so far comprise duallylabeled nanoparticles antibodies and peptides although theagents based on each of these substance classes require acareful optimization regarding the overall biodistribution ofthe hybrid agents Thus one focus of future developmentscould be the design of small molecules comprising theradionuclide as well as the NIR dye enabling the ideallysite-specific one-step dual-labeling of biomolecules exertingonly a minor structural alteration to the targeting vectorand thus only a minor effect on its bioactivity resulting inhighly potent compounds for hybrid imaging In case ofnanoparticles the pharmacokinetic properties need to beoptimized in order to minimize their uptake by the RESand to maximize their target-specific accumulation Suchdevelopments could then result in hybrid imaging agentshaving a significant impact on whole-body in vivo targetdetection by PET as well as subsequent optical imaging-guided intraoperative curative surgery

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support by DeutscheForschungsgemeinschaft and Ruprecht-Karls-UniversitatHeidelberg within the funding programme Open AccessPublishing

References

[1] S R Cherry ldquoMultimodality in vivo imaging systems twicethe power or double the troublerdquo Annual Review of BiomedicalEngineering vol 8 pp 35ndash62 2006

[2] S R Cherry ldquoMultimodality imaging beyond PETCT andSPECTCTrdquo Seminars in Nuclear Medicine vol 39 no 5 pp348ndash353 2009

[3] J Cheon and J H Lee ldquoSynergistically integrated nanoparticlesas multimodal probes for nanobiotechnologyrdquo Accounts ofChemical Research vol 41 no 12 pp 1630ndash1640 2008

[4] T Buckle P T K Chin and F W B van Leeuwen ldquo(Non-targeted) radioactivefluorescent nanoparticles and their poten-tial in combined pre-and intraoperative imaging during sen-tinel lymph node resectionrdquoNanotechnology vol 21 no 48 pp482001ndash482010 2010

[5] M Nahrendorf E Keliher B Marinelli et al ldquoHybrid PET-optical imaging using targeted probesrdquo Proceedings of the


National Academy of Sciences of the United States of Americavol 107 no 17 pp 7910ndash7915 2010

[6] G M van Dam G Themelis L M A Crane et al ldquoIntraop-erative tumor-specific fluorescence imaging in ovarian cancerby folate receptor-120572 targeting first in-human resultsrdquo NatureMedicine vol 17 no 10 pp 1315ndash1319 2011

[7] P T Chin C A Beekman T Buckle L Josephson and F Wvan Leeuwen ldquoMultispectral visualization of surgical safety-margins using fluorescent marker seedsrdquoThe American Journalof Nuclear Medicine and Molecular Imaging vol 2 pp 151ndash1622012

[8] M S Bradbury E Phillips P H Montero et al ldquoClinically-translated silica nanoparticles as dual-modality cancer-targetedprobes for image-guided surgery and interventionsrdquo IntegrativeBiology vol 5 pp 74ndash86 2013

[9] E J T Rutgers ldquoSentinel node biopsy interpretation andmanagement of patients with immunohistochemistry-positivesentinel nodes and those with micrometastasesrdquo Journal ofClinical Oncology vol 26 no 5 pp 698ndash702 2008

[10] C M Balch S-J Soong J E Gershenwald et al ldquoPrognosticfactors analysis of 17600 melanoma patients validation ofthe American Joint Committee on Cancer melanoma stagingsystemrdquo Journal of Clinical Oncology vol 19 no 16 pp 3622ndash3634 2001

[11] LVermeeren RAValdesOlmosWMeinhardt et al ldquoValue ofSPECTCT for detection and anatomic localization of sentinellymph nodes before laparoscopic sentinel node lymphadenec-tomy in prostate carcinomardquo Journal of Nuclear Medicine vol50 no 6 pp 865ndash870 2009

[12] J Kuil T Buckle and F W van Leeuwen ldquoImaging agents forthe chemokine receptor 4 (CXCR4)rdquo Chemical Society Reviewsvol 41 pp 5239ndash5261 2012

[13] J C Knight and F RWuest ldquoNuclear (PETSPECT) and opticalimaging probes targeting the CXCR4 chemokine receptorrdquoMedicinal Chemistry Communications vol 3 pp 1039ndash10532012

[14] K Yan P Li H Zhu et al ldquoRecent advances in multifunctionalmagnetic nanoparticles and applications to biomedical diagno-sis and treatmentrdquo RSC Advances vol 3 pp 10598ndash10618 2013

[15] A Louie ldquoMultimodality imaging probes design and chal-lengesrdquo Chemical Reviews vol 110 no 5 pp 3146ndash3195 2010

[16] S Liu D Li C W Huang et al ldquoEfficient construction of PETfluorescence probe based on sarcophagine cage an opportunityto integrate diagnosis with treatmentrdquo Molecular Imaging andBiology vol 14 pp 718ndash724 2012

[17] R H Kimura Z Miao Z Cheng S S Gambhir and J RCochran ldquoA dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in livingsubjectsrdquo Bioconjugate Chemistry vol 21 no 3 pp 436ndash4442010

[18] P Paudyal B Paudyal Y Iida et al ldquoDual functional molecularimaging probe targeting CD20 with PET and optical imagingrdquoOncology Reports vol 22 no 1 pp 115ndash119 2009

[19] R Weissleder and U Mahmood ldquoMolecular imagingrdquo Radiol-ogy vol 219 no 2 pp 316ndash333 2001

[20] K Shah and R Weissleder ldquoMolecular optical imaging appli-cations leading to the development of present day therapeuticsrdquoNeuroRx vol 2 no 2 pp 215ndash225 2005

[21] J V Frangioni ldquoIn vivo near-infrared fluorescence imagingrdquoCurrent Opinion in Chemical Biology vol 7 no 5 pp 626ndash6342003

[22] J M Park and S S Gambhir ldquoMultimodality radionuclidefluorescence and bioluminescence small-animal imagingrdquo Pro-ceedings of the IEEE vol 93 no 4 pp 771ndash782 2005

[23] J P Holland G Normand A Ruggiero J S Lewis and JGrimm ldquoIntraoperative imaging of positron emission tomo-graphic radiotracers using cerenkov luminescence emissionsrdquoMolecular Imaging vol 10 no 3 pp 177ndash186 2011

[24] J C Park M K Yu G I An et al ldquoFacile preparationof a hybrid nanoprobe for triple-modality opticalPETMRimagingrdquo Small vol 6 no 24 pp 2863ndash2868 2010

[25] A Azhdarinia NWilganowski H Robinson et al ldquoCharacter-ization of chemical radiochemical and optical properties of adual-labeledMMP-9 targeting peptiderdquoBioorganic ampMedicinalChemistry vol 19 no 12 pp 3769ndash3776 2011

[26] W B Edwards B Xu W Akers et al ldquoAgonistmdashantagonistdilemma inmolecular imaging evaluation of a monomolecularmultimodal imaging agent for the somatostatin receptorrdquo Bio-conjugate Chemistry vol 19 no 1 pp 192ndash200 2008

[27] J S Kim Y-H Kim J H Kim et al ldquoDevelopment and invivo imaging of a PETMRI nanoprobe with enhanced NIRfluorescence by dye encapsulationrdquo Nanomedicine vol 7 no 2pp 219ndash229 2012

[28] L Sampath S Kwon S Ke et al ldquoDual-labeled trastuzumab-based imaging agent for the detection of human epidermalgrowth factor receptor 2 overexpression in breast cancerrdquoJournal of Nuclear Medicine vol 48 no 9 pp 1501ndash1510 2007

[29] L SampathWWang and EM Sevick-Muraca ldquoNear infraredfluorescent optical imaging for nodal stagingrdquo Journal ofBiomedical Optics vol 13 no 4 Article ID 041312 2008

[30] J Kuil T Buckle J Oldenburg et al ldquoHybrid peptide den-drimers for imaging of chemokine receptor 4 (CXCR4) expres-sionrdquo Molecular Pharmaceutics vol 8 no 6 pp 2444ndash24532011

[31] J Kuil T Buckle H Yuan et al ldquoSynthesis and evaluation of abimodal CXCR4 antagonistic peptiderdquo Bioconjugate Chemistryvol 22 no 5 pp 859ndash864 2011

[32] WWang S Ke S Kwon et al ldquoA new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chainrdquo Bioconjugate Chemistry vol 18 no 2 pp 397ndash402 2007

[33] J P Houston S Ke W Wang C Li and E M Sevick-Muraca ldquoQuality analysis of in vivo near-infrared fluorescenceand conventional gamma images acquired using a dual-labeledtumor-targeting proberdquo Journal of Biomedical Optics vol 10 no5 Article ID 054010 2005

[34] W B Edwards W J Akers Y Ye et al ldquoMultimodal imag-ing of integrin receptor-positive tumors by bioluminescencefluorescence gamma scintigraphy and single-photon emissioncomputed tomography using a cyclic RGD peptide labeled witha near-infrared fluorescent dye and a radionucliderdquo MolecularImaging vol 8 no 2 pp 101ndash110 2009

[35] C Li WWang QWu et al ldquoDual optical and nuclear imagingin humanmelanoma xenografts using a single targeted imagingproberdquoNuclearMedicine and Biology vol 33 no 3 pp 349ndash3582006

[36] Z Zhang K Liang S Bloch M Berezin and S AchilefuldquoMonomolecular multimodal fluorescence-radioisotope imag-ing agentsrdquo Bioconjugate Chemistry vol 16 no 5 pp 1232ndash12392005

[37] C A Boswell P K Eck C A S Regino et al ldquoSynthesis cha-racterization and biological evaluation of integrin 120572v1205733-targeted PAMAM dendrimersrdquo Molecular Pharmaceutics vol5 no 4 pp 527ndash539 2008


[38] D R Vera D J Hall C K Hoh P Gallant L MMcIntosh andR F Mattrey ldquoCy55-DTPA-galactosyl-dextran a fluorescentprobe for in vivo measurement of receptor biochemistryrdquoNuclear Medicine and Biology vol 32 no 7 pp 687ndash693 2005

[39] N Mitchell T L Kalber M S Cooper et al ldquoIncorporation ofparamagnetic fluorescent and PETSPECT contrast agents intoliposomes for multimodal imagingrdquo Biomaterials vol 34 pp1179ndash1192 2013

[40] Z Yang S Zheng W J Harrison et al ldquoLong-circulating near-infrared fluorescence core-cross-linked polymeric micellessynthesis characterization and dual nuclearoptical imagingrdquoBiomacromolecules vol 8 no 11 pp 3422ndash3428 2007

[41] S K Pandey A L Gryshuk M Sajjad et al ldquoMultimodalityagents for tumor imaging (PET fluorescence) and photody-namic therapy A possible ldquosee and treatrdquo approachrdquo Journal ofMedicinal Chemistry vol 48 no 20 pp 6286ndash6295 2005

[42] E R Ranyuk N Cauchon H Ali R Lecomte B Guerin and JE Van Lier ldquoPET imaging using64Cu-labeled sulfophthalocya-nines synthesis and biodistributionrdquo Bioorganic amp MedicinalChemistry Letters vol 21 no 24 pp 7470ndash7473 2011

[43] R Lebel N Zarifyussefian M Letendre-Jauniaux et al ldquoUltra-high sensitivity detection of bimodal probes at ultra-low noisefor combined fluorescence and positron emission tomographyimagingrdquo vol 8574 of Proceedings of SPIE pp 8574ndash8580 2013

[44] E Ranyuk R Lebel Y Berube-Lauziere et al ldquo(68)GaDOTA-and (64)CuNOTA-phthalocyanine conjugates as fluorescentPET bimodal imaging probesrdquo Bioconjugate Chemistry vol 24pp 1624ndash1633 2013

[45] T Priem C Bouteiller D Camporese et al ldquoA novel sulfonatedprosthetic group for [F-18]-radiolabelling and imparting watersolubility of biomolecules and cyanine fluorophoresrdquo Organicamp Biomolecular Chemistry vol 11 pp 469ndash479 2013

[46] J A Hendricks E J Keliher D P Wan S A HilderbrandR Weissleder and R Mazitschek ldquoSynthesis of [F-18]BODIPYbifunctional reporter for hybrid opticalpositron emissiontomography imagingrdquo Angewandte Chemie International Edi-tion vol 51 pp 4603ndash4606 2012

[47] H Xu K Baidoo A J Gunn et al ldquoDesign synthesis andcharacterization of a dual modality positron emission tomog-raphy and fluorescence imaging agent for monoclonal antibodytumor-targeted imagingrdquo Journal of Medicinal Chemistry vol50 no 19 pp 4759ndash4765 2007

[48] C Bernhard M Moreau D Lhenry et al ldquoDOTAGA-anhyd-ride a valuable building block for the preparation of DOTA-like chelating agentsrdquo ChemistrymdashA European Journal vol 18pp 7834ndash7841 2012

[49] S C Ghosh P Ghosh N Wilganowski et al ldquoMultimodal che-lation platform for near-infrared fluorescencenuclear imag-ingrdquo Journal of Medicinal Chemistry vol 56 pp 406ndash416 2013

[50] B Wangler R Schirrmacher P Bartenstein and C WanglerldquoChelating agents and their use in radiopharmaceutical sci-encesrdquo Mini Reviews in Medicinal Chemistry vol 11 pp 968ndash983 2011

[51] T W Liu J M Stewart T D MacDonald et al ldquoBiologically-targeted detection of primary and micro-metastatic ovariancancerrdquoTheranostics vol 3 pp 420ndash427 2013

[52] G Ren G Blum M Verdoes et al ldquoNon-invasive imagingof cysteine cathepsin activity in solid tumors using a64Cu-labeled activity-based proberdquo PLoS ONE vol 6 no 11 ArticleID e28029 2011

[53] B Behnam Azad C-F Cho J D Lewis and L G Luyt ldquoSyn-thesis radiometal labeling and in vitro evaluation of a targeted

PPIX derivativerdquo Applied Radiation and Isotopes vol 70 no 3pp 505ndash511 2012

[54] Y Zhang H Hong G W Severin et al ldquoImmunoPET andnear-infrared fluorescence imaging of CD105 expression usinga monoclonal antibody dual-labeled with 89Zr and IRDye800CWrdquo The American Journal of Translational Research vol4 pp 333ndash346 2012

[55] R Cohen M A Stammes I H de Roos M Stigter-vanWalsum G W Visser and G A van Dongen ldquoInert couplingof IRDye800CW to monoclonal antibodies for clinical opticalimaging of tumor targetsrdquoEuropean Journal of NuclearMedicineand Molecular Imaging Research vol 1 no 31 2011

[56] H Hong Y Zhang G W Severin et al ldquoMultimodalityimaging of breast cancer experimental lung metastasis withbioluminescence and a monoclonal antibody dual-labeled withZr-89 and IRDye 800CWrdquo Molecular Pharmacology vol 9 pp2339ndash2349 2012

[57] Y Zhang H Hong JW Engle et al ldquoPositron emission tomog-raphy and optical imaging of tumor CD105 expression with adual-labeled monoclonal antibodyrdquo Molecular Pharmaceuticsvol 9 no 3 pp 645ndash653 2012

[58] L Sampath S Kwon M A Hall R E Price and E M Sevick-Muraca ldquoDetection of cancer metastases with a dual-labelednear- infraredpositron emission tomography imaging agentrdquoTranslational Oncology vol 3 no 5 pp 307ndash318 2010

[59] M A Hall M B Aldrich A Azhdarinia et al ldquoQuantify-ing multimodal contrast agent biological activity using near-infrared flow cytometryrdquo Contrast Media amp Molecular Imagingvol 7 no 3 pp 338ndash345 2012

[60] R Ting T A Aguilera J L Crisp et al ldquoFast18F labeling of anear-infrared fluorophore enables positron emission tomogra-phy and optical imaging of sentinel lymph nodesrdquo BioconjugateChemistry vol 21 no 10 pp 1811ndash1819 2010

[61] X Lin J Xie G Niu et al ldquoChimeric ferritin nanocagesfor multiple function loading and multimodal imagingrdquo NanoLetters vol 11 no 2 pp 814ndash819 2011

[62] S H Li B Goins L J Zhang and A D Bao ldquoNovel multifunc-tional theranostic liposome drug delivery system constructioncharacterization andmultimodalityMR near-infrared fluores-cent and nuclear imagingrdquo Bioconjugate Chemistry vol 23 pp1322ndash1332 2012

[63] A Ruggiero C H Villa E Bander et al ldquoParadoxical glomeru-lar filtration of carbon nanotubesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no27 pp 12369ndash12374 2010

[64] L Stelter J G Pinkernelle R Michel et al ldquoModification ofaminosilanized superparamagnetic nanoparticles feasibility ofmultimodal detection using 3T MRI small animal PET andfluorescence imagingrdquo Molecular Imaging and Biology vol 12no 1 pp 25ndash34 2010

[65] M Nahrendorf H Zhang S Hembrador et al ldquoNanoparticlePET-CT imaging of macrophages in inflammatory atheroscle-rosisrdquo Circulation vol 117 no 3 pp 379ndash387 2008

[66] D W Hwang H Y Ko S-K Kim D Kim D S Lee and SKim ldquoDevelopment of a quadruple imaging modality by usingnanoparticlesrdquo ChemistrymdashA European Journal vol 15 no 37pp 9387ndash9393 2009

[67] J Xie K Chen J Huang et al ldquoPETNIRFMRI triple func-tional iron oxide nanoparticlesrdquo Biomaterials vol 31 no 11 pp3016ndash3022 2010


[68] Q Liu M Chen Y Sun et al ldquoMultifunctional rare-earth self-assembled nanosystem for tri-modal upconversion lumines-cence fluorescence positron emission tomography imagingrdquoBiomaterials vol 32 pp 8243ndash8253 2011

[69] J Zhou M Yu Y Sun et al ldquoFluorine-18-labeled Gd3+Yb3+Er3+ co-doped NaYF4 nanophosphors for multimodalityPETMRUCL imagingrdquo Biomaterials vol 32 no 4 pp 1148ndash1156 2011

[70] K Chen Z-B Li H Wang W Cai and X Chen ldquoDual-moda-lity optical and positron emission tomography imaging of vas-cular endothelial growth factor receptor on tumor vasculatureusing quantum dotsrdquo European Journal of NuclearMedicine andMolecular Imaging vol 35 no 12 pp 2235ndash2244 2008

[71] W Cai K Chen Z-B Li S S Gambhir and X Chen ldquoDual-function probe for PET and near-infrared fluorescence imagingof tumor vasculaturerdquo Journal of Nuclear Medicine vol 48 no11 pp 1862ndash1870 2007

[72] F Duconge T Pons C Pestourie et al ldquoFluorine-18-labeledphospholipid quantum dot micelles for in vivo multimodalimaging from whole body to cellular scalesrdquo BioconjugateChemistry vol 19 no 9 pp 1921ndash1926 2008

[73] M L Schipper Z Cheng S-W Lee et al ldquoMicroPET-basedbiodistribution of quantum dots in living micerdquo Journal ofNuclear Medicine vol 48 no 9 pp 1511ndash1518 2007

[74] X Michalet F F Pinaud L A Bentolila et al ldquoQuantum dotsfor live cells in vivo imaging and diagnosticsrdquo Science vol 307no 5709 pp 538ndash544 2005


Despite these favorable properties of PETCT and alsoPETMRI systems in whole-body imaging for the identifi-cation of target structures these modalities exhibit certainlimitations after having specifically identified and localized atumor target tissue the resection of the tumormass is difficultdue to the intricate intraoperative identification of tumormargins and smallmetastases Additionally the identificationof the sentinel lymph node (SLN) which is often resected forhistology is not trivial For this purpose another combinationof imagingmodalities could be of special interest namely thecombination of PET with optical imaging (OI)

Although OI is a modality with restricted applicabilityfor whole-body in vivo imaging due to the limited tissuepenetration of the light emitted by the fluorescent probeit is a valuable methodology for surface imaging applica-tions such as intraoperative image-guided surgery due toits favorable spatial resolution and sensitivity [3ndash5] Thusa combined bimodal imaging consisting of an initial PETscan using 120574-radiation with a high tissue penetration rangeto identify and localize tumor lesions throughout the bodyand a subsequent intraoperativeOI in order to identify tumormargins and infected lymph nodes can result in a significantclinical improvement [6ndash8] Especially in breast and prostatecarcinomas as well as melanomas the prognosis stronglydepends on the presence of lymph node metastases [9ndash11]However the secure intraoperative identification of sentineland infected lymph nodes is crucial for efficient diagnosisand treatment but is difficult if the surgeon can only rely onabnormal visual appearance and palpation to discriminatebetween lymph nodes and surrounding tissues or to identifyinfected nodes The use of a specific tumor-accumulatingagent which can be visualized during surgery by opticalimaging techniques emitting light which can penetrate tissuein a reasonable range (so that a target node can be detectedeven if not already fully surgically exposed) would mean asignificant improvement for surgery (Figure 1)

The development of new combined imaging techniqueshowever also requires the development of the respectivehybrid imaging agents that are suitable for all involvedimaging modalities Thus considerable research has beenconducted in this field of hybrid contrast agents over the lastyears [3 4 12ndash15]

For combined PET and optical imaging in principle theuse of two separate molecular markers one for PET andone for OI (instead of using a hybrid imaging agent) wouldalso be possible However this is no optimal approach asboth agents are likely to exhibit differing biodistribution andpharmacokinetic properties (especially in cases of relativelysmall specifically accumulating biomolecules such as pep-tides) Hence to achieve reliable results that are comparablebetween both imaging modalities a hybrid marker has to beapplied

It has to be kept in mind that optical imaging is not fullyquantifiable as it is surface-weighted due to absorbance andscattering of the photons by tissue penetration (especiallywhen imaging deep tissues exhibiting a low imaging agentaccumulation) and thus cannot be fully correlated to PETimaging data [16ndash19] As PET however is fully quantifiableand used for whole-body imaging whereas OI is used for

intraoperative imaging purposes only in order to identifytumor tissues (tumormargins small metastases and infectedlymph nodes) the quantification of optical signals is nocritical criterion

For OI different classes of reporter probes detectableby optical imaging techniques can in principle be used ina hybrid PETOI agent (i) fluorescent proteins that can bedetected by bioluminescence imaging (BLI) (ii) 120574-emittingradionuclides that can be visualized by Cherenkov lumi-nescence imaging (CLI luminescence that can be observedwhen a particle travels faster than light in the examinedmedium) (iii) fluorescent small dye molecules that canideally emit near infrared light and (iv) quantum dots whichare semiconductor nanocrystals consisting of CdTe or CdSematerials and whose emission characteristics can be tailoredby particle size For all these probes that can be used in thedevelopment of a hybrid PETOI agent substances emittinglight of the near-infrared and infrared spectrum (700ndash900 nm) aremost useful as light of thesewavelengths exhibitsthe highest tissue permeability of several mm to cm in vivo[20 21]

Large proteins such as GFP (green fluorescent protein) orRFP (red fluorescent protein) are in principle applicable inthe synthesis of a hybrid compound However they are struc-turally demanding and would most possibly have a severeimpact on the pharmacokinetic properties of the resultingimaging agent Thus it is only conceivable to use these com-pounds in combination with particle carriers Furthermorethe quantum yield of these proteins is rather limited and theydo not enable near-infrared photon emissions [22] furtherrestricting the use of fluorescent proteins in hybrid opticalimaging agents

In contrast CLI using different positron-emitting radio-nuclides has been proposed as a favorable optical imagingtechnique for imaging-guided surgery [23] This techniquedoes not require the conjugation of an additional fluorescentcompound in order to obtain a bimodal imaging agent Thisis favorable as an additionally conjugated fluorescent dyecanmdashif susceptible to the radiolabeling conditions appliedmdashinterfere with the radiosynthesis or result in a significantalteration of the pharmacokinetic properties of the result-ing hybrid compound Unfortunately using the Cherenkovluminescence imaging approach one of the most valuableproperties of combined PETOI probes to be applied in intra-operative imaging namely the consecutive detection via PETand the subsequent later resection of the tumor cannot beutilized Using a hybrid compound consisting of a fluorescentdye in addition to a radionuclide the optical intraoperativeimaging can be performed delayed in time after identifyingand localizing the tumorous tissue by a whole-body PETscan By this procedure the radionuclide at least partiallydecayed before surgery resulting in no or only low radiationburden to the surgeon during intraoperative imaging andresection In contrast using CLI for intraoperative imagingcan result in a significant radiation burden as is indicatedby a recent study systematically investigating the potential ofCLI in a preclinical setting In this workmdashwhen imaging an124I activity depot located subcutaneously in 4mmdepthmdashan


Tumor

SLN

Lymphaticflow


agent




and SLN

120574

120574

120582em

120582ex











N HN

NNH

O

OOO

124I

124I-PS41

NN

NN

NN

NN

NaO3S

NaO3S

SO3Na

SO3Na

64 Cu

64 Cu-PcS42

N

N

NNN

N N

N

Zn

HN

O

N

NN

OO

O

O

NaO3S

NaO3S

SO3Na

64 Cu


O

NN

NH

OO

18FSO3

NH2

18F-Cy554

N N

F

O O

N O

B

O

18F


N NB

F F

OHN

HN

HN

O

N

N N

N

O

OO

HO

HO

OH

OH

O

O


NN

NN

O

N

O

O O O

O

O

NO

N

O

O

HN

HO3S

HO3SSO3H

SO3H

SO3

64 Cu


O

NN

N

N

N

O

O

HO

HO

HO

O

O OH

OH

O

NH

NH

NH

OO

N OO

HO3S

O3S

SO3H

SO3H


A998400998400-DTPA

S=C=N





NH

O HN

O

O

O

OO

NN

NN

O

N

O

O

O O

O

O

N

R 9

10

N N

NN

O

GDEVDGSGKO HN

NH

NN

NNH

NH

O

OO

COOH

R =

[64 Cu Cu] [64 ]GB173GB170 and 9 and 10


11

SO3H

64 Cu64 Cu

NH2

NH2

O3S

HN

NH











O

NN

ON

NN

N

OO

O

O

OO

NH

HN N

H

HN

NH


O

O

O

O

HN

O

OO

O

O

HOOC

O

O

NH

OO

O

OO

HOOC

O

NN

NN

N N

O

N

N

N N

NN

OHO NHO

O O

HN

O

NH

HN

NH

NH

NHNH

NH

NH

NH

NH

HN

HNHN

HN

HNHN

HN HN

HNHN

HN

HN

HN

HN

HN

HN

HN

OH

HN

HN

HN

HNHN

HN

NH

NH

NH

NH

NH

NHNH

NH

NH

OO

O

OH

OH

OH

OH

O

HN

O

OO

O

OO

O

OO

N

O

OHO

HN

O

NH

O

N

N

N NO

O

O

OH

HO

HO

O

ON

O

NH O

O

O

OO

O

O

HN

SS

O

HN

NN

NN

O

OO

O

O

OO

O

N

N

N

O

O

ONH2

NH2

NH2

NH2

NH2

H2N

H2N

64 Cu

64 Cu

64 Cu

68 Ga




12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S


H3C













ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References

















































































Tumor

SLN

Lymphaticflow


agent




and SLN

120574

120574

120582em

120582ex











N HN

NNH

O

OOO

124I

124I-PS41

NN

NN

NN

NN

NaO3S

NaO3S

SO3Na

SO3Na

64 Cu

64 Cu-PcS42

N

N

NNN

N N

N

Zn

HN

O

N

NN

OO

O

O

NaO3S

NaO3S

SO3Na

64 Cu


O

NN

NH

OO

18FSO3

NH2

18F-Cy554

N N

F

O O

N O

B

O

18F


N NB

F F

OHN

HN

HN

O

N

N N

N

O

OO

HO

HO

OH

OH

O

O


NN

NN

O

N

O

O O O

O

O

NO

N

O

O

HN

HO3S

HO3SSO3H

SO3H

SO3

64 Cu


O

NN

N

N

N

O

O

HO

HO

HO

O

O OH

OH

O

NH

NH

NH

OO

N OO

HO3S

O3S

SO3H

SO3H


A998400998400-DTPA

S=C=N





NH

O HN

O

O

O

OO

NN

NN

O

N

O

O

O O

O

O

N

R 9

10

N N

NN

O

GDEVDGSGKO HN

NH

NN

NNH

NH

O

OO

COOH

R =

[64 Cu Cu] [64 ]GB173GB170 and 9 and 10


11

SO3H

64 Cu64 Cu

NH2

NH2

O3S

HN

NH











O

NN

ON

NN

N

OO

O

O

OO

NH

HN N

H

HN

NH


O

O

O

O

HN

O

OO

O

O

HOOC

O

O

NH

OO

O

OO

HOOC

O

NN

NN

N N

O

N

N

N N

NN

OHO NHO

O O

HN

O

NH

HN

NH

NH

NHNH

NH

NH

NH

NH

HN

HNHN

HN

HNHN

HN HN

HNHN

HN

HN

HN

HN

HN

HN

HN

OH

HN

HN

HN

HNHN

HN

NH

NH

NH

NH

NH

NHNH

NH

NH

OO

O

OH

OH

OH

OH

O

HN

O

OO

O

OO

O

OO

N

O

OHO

HN

O

NH

O

N

N

N NO

O

O

OH

HO

HO

O

ON

O

NH O

O

O

OO

O

O

HN

SS

O

HN

NN

NN

O

OO

O

O

OO

O

N

N

N

O

O

ONH2

NH2

NH2

NH2

NH2

H2N

H2N

64 Cu

64 Cu

64 Cu

68 Ga




12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S


H3C













ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References

















































































N HN

NNH

O

OOO

124I

124I-PS41

NN

NN

NN

NN

NaO3S

NaO3S

SO3Na

SO3Na

64 Cu

64 Cu-PcS42

N

N

NNN

N N

N

Zn

HN

O

N

NN

OO

O

O

NaO3S

NaO3S

SO3Na

64 Cu


O

NN

NH

OO

18FSO3

NH2

18F-Cy554

N N

F

O O

N O

B

O

18F


N NB

F F

OHN

HN

HN

O

N

N N

N

O

OO

HO

HO

OH

OH

O

O


NN

NN

O

N

O

O O O

O

O

NO

N

O

O

HN

HO3S

HO3SSO3H

SO3H

SO3

64 Cu


O

NN

N

N

N

O

O

HO

HO

HO

O

O OH

OH

O

NH

NH

NH

OO

N OO

HO3S

O3S

SO3H

SO3H


A998400998400-DTPA

S=C=N





NH

O HN

O

O

O

OO

NN

NN

O

N

O

O

O O

O

O

N

R 9

10

N N

NN

O

GDEVDGSGKO HN

NH

NN

NNH

NH

O

OO

COOH

R =

[64 Cu Cu] [64 ]GB173GB170 and 9 and 10


11

SO3H

64 Cu64 Cu

NH2

NH2

O3S

HN

NH











O

NN

ON

NN

N

OO

O

O

OO

NH

HN N

H

HN

NH


O

O

O

O

HN

O

OO

O

O

HOOC

O

O

NH

OO

O

OO

HOOC

O

NN

NN

N N

O

N

N

N N

NN

OHO NHO

O O

HN

O

NH

HN

NH

NH

NHNH

NH

NH

NH

NH

HN

HNHN

HN

HNHN

HN HN

HNHN

HN

HN

HN

HN

HN

HN

HN

OH

HN

HN

HN

HNHN

HN

NH

NH

NH

NH

NH

NHNH

NH

NH

OO

O

OH

OH

OH

OH

O

HN

O

OO

O

OO

O

OO

N

O

OHO

HN

O

NH

O

N

N

N NO

O

O

OH

HO

HO

O

ON

O

NH O

O

O

OO

O

O

HN

SS

O

HN

NN

NN

O

OO

O

O

OO

O

N

N

N

O

O

ONH2

NH2

NH2

NH2

NH2

H2N

H2N

64 Cu

64 Cu

64 Cu

68 Ga




12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S


H3C













ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References

















































































NH

O HN

O

O

O

OO

NN

NN

O

N

O

O

O O

O

O

N

R 9

10

N N

NN

O

GDEVDGSGKO HN

NH

NN

NNH

NH

O

OO

COOH

R =

[64 Cu Cu] [64 ]GB173GB170 and 9 and 10


11

SO3H

64 Cu64 Cu

NH2

NH2

O3S

HN

NH











O

NN

ON

NN

N

OO

O

O

OO

NH

HN N

H

HN

NH


O

O

O

O

HN

O

OO

O

O

HOOC

O

O

NH

OO

O

OO

HOOC

O

NN

NN

N N

O

N

N

N N

NN

OHO NHO

O O

HN

O

NH

HN

NH

NH

NHNH

NH

NH

NH

NH

HN

HNHN

HN

HNHN

HN HN

HNHN

HN

HN

HN

HN

HN

HN

HN

OH

HN

HN

HN

HNHN

HN

NH

NH

NH

NH

NH

NHNH

NH

NH

OO

O

OH

OH

OH

OH

O

HN

O

OO

O

OO

O

OO

N

O

OHO

HN

O

NH

O

N

N

N NO

O

O

OH

HO

HO

O

ON

O

NH O

O

O

OO

O

O

HN

SS

O

HN

NN

NN

O

OO

O

O

OO

O

N

N

N

O

O

ONH2

NH2

NH2

NH2

NH2

H2N

H2N

64 Cu

64 Cu

64 Cu

68 Ga




12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S


H3C













ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References

















































































O

NN

ON

NN

N

OO

O

O

OO

NH

HN N

H

HN

NH


O

O

O

O

HN

O

OO

O

O

HOOC

O

O

NH

OO

O

OO

HOOC

O

NN

NN

N N

O

N

N

N N

NN

OHO NHO

O O

HN

O

NH

HN

NH

NH

NHNH

NH

NH

NH

NH

HN

HNHN

HN

HNHN

HN HN

HNHN

HN

HN

HN

HN

HN

HN

HN

OH

HN

HN

HN

HNHN

HN

NH

NH

NH

NH

NH

NHNH

NH

NH

OO

O

OH

OH

OH

OH

O

HN

O

OO

O

OO

O

OO

N

O

OHO

HN

O

NH

O

N

N

N NO

O

O

OH

HO

HO

O

ON

O

NH O

O

O

OO

O

O

HN

SS

O

HN

NN

NN

O

OO

O

O

OO

O

N

N

N

O

O

ONH2

NH2

NH2

NH2

NH2

H2N

H2N

64 Cu

64 Cu

64 Cu

68 Ga




12

13

15 16

14

68 Ga

SO3H

SO3

SO3

SO3

SO3H

SO3H

SO3H

HO3S

HO3SHO3S

HO3S

HO3S


H3C













ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References



























































































ON

NN

N

OO

O

O

OO

ON

N

O

N

N

NO

O

O

O

O

ONH

S

ON

O

NH

O

NNHO

O

N OHN

OOOO

Alexa fluor 750

64 Cu

89 Zr

64 Cu

SO3H

SO3H

SO3H

SO3


m

n

=

=











2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References


















































































2or other








3 Conclusion



Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References

















































































Radiolabel

Fluorescent dye

Targeting vector

Silica particle


Coated quantum dot

(a) (b) (c)

(d) (e)





Acknowledgments


References
















































































Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid...

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Transcript of Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid...