Cancer is clearly one of the major scourges of the lastcentury. Great improvements have been achieved interms of both diagnosis and treatment procedures;nevertheless, new challenges appear constantly in alllevels. Molecular imaging has promoted the visual-ization, characterization, and measurement of abnor-mal biological processes at the molecular and cellularlevels in living organisms, using appropriate probes.Imaging techniques such as single-photon emissioncomputed tomography (SPECT), positron emission to-mography (PET), magnetic resonance imaging (MRI),and optical fluorescence imaging have been used tomonitor such processes.1 Advances in many basicsciences including chemistry, biology, physics, andengineering have crucially promoted molecular imag-ing into an autonomous scientific discipline, reshap-
ing the philosophy of anticancer drug discovery intomore cost-effective molds. During the last 20 years,an extensive clinical impact of molecular imaging hastaken place, allowing diagnosis, treatment, and ef-ficacy monitoring of a treatment in a noninvasivemanner.
The use of nuclear medicine techniques (SPECTand PET) in oncology had gained great importanceas a rapidly developing diagnostic and therapeuticmultimodality.2 Over the past decades, biospecificimaging agents (e.g., large antibodies, antibody frag-ments, biologically active peptides, nanostructures,etc.) have been labeled with a variety of radionu-clides. The size and general biochemical character-istics of these radiomolecules define their targetingand pharmacokinetic properties, making them favor-able for imaging.
RADIOMETALS IN SPECT
Radiometals are very important for the diagnosticand therapeutic use of nuclear medicine as they
JOURNAL OF PHARMACEUTICAL SCIENCES 1
2 PSIMADAS ET AL.
predominantly possess the most suitable propertiesfor SPECT and PET applications. The type of emis-sion that is produced by a radiometal during its de-cay includes gamma rays, positrons, alpha particlesand Auger, low-energy conversion, and beta electrons.SPECT was the first molecular imaging modality usedclinically. SPECT requires the use of an agent labeledwith a gamma-emitting radionuclide, which shouldhave an ideal gamma energy of 100–250 keV. Ra-dionuclides that decay with gamma energies lowerthan this range produce too much scatter, whereasgamma energies greater than 250 keV are difficultto collimate, resulting in poor imaging quality. Thesegamma rays are recorded by the detectors of a ded-icated gamma camera or SPECT instrument, track-ing the movement of the radiolabeled agent through-out the body.3 Examples of clinically used gamma-emitting radiometals in nuclear medicine are 99mTc,indium-111 (111In), 67Ga, etc.2–5 The major advan-tages of radionuclide-based molecular imaging tech-niques over other modalities (e.g., optical and MRI)are that they are very sensitive (down to the picomolarlevel), quantitative, and there is no tissue penetrationlimit.4 SPECT has also the potentiality to permit si-multaneous imaging of multiple radiometals, as longas they emit gamma rays of different energy.5
Radiometal-labeled agents are the tracers that findgreater application in SPECT. Radiopharmaceuticalslabeled with radiometals are injected into patientsfor the diagnosis of various diseases (e.g., cancer, in-fection, thrombosis, etc.) and are also used to monitorthe efficacy of various cancer treatments and to deter-mine dosimetry in targeted radiotherapy.6 When de-signing radiometal-based tracers, important factorsto consider include the half-life of the radiometal,the mode of decay, as well as its cost and availabil-ity. Generally, the characteristics of a radiometal thatmake it advantageous for use in the production of aradiopharmaceutical include a stable oxidation state,a well-defined coordination chemistry in aqueous en-vironment, and relatively fast complexation kineticsat physiological pH. Furthermore, each radiometalhas additional varied requirements (e.g., ligand selec-tion, complex formation and stability, etc.) that makeits incorporation in a radiopharmaceutical more com-plex than for radioorganic compounds incorporatingradionuclides such as 11C, 18F, and 123/124I.7,8
NANOPARTICLES IN CANCER
Nanotechnology is an applied sciences field, whichinvolves creation and utilization of materials, de-vices, or systems in the nanometer scale. Nanotech-nology is currently undergoing explosive develop-ment in the medicine territory (nanomedicine) andmore specifically on molecular imaging/diagnosis anddrug delivery with the use of nanoparticles (NPs).9–11
NPs are typically characterized as nanometer sizestructures, synthetically produced from a range ofmaterials including polymers, metals, phospholipidbilayers, and so on. The physical and chemical prop-erties of these nanostructures can vary widely de-pending on their structure, affecting their overallin vivo behavior. The variety of NPs that have beendeveloped up to now is attributed on the effort toachieve a high bioavailability within cancer cells, de-crease the reticuloendothelial system (RES) uptake,improve in vivo targeting capabilities, and minimizetoxicity.9–13
The advantageous features of NPs, such as mul-tifunctionality, multivalency, and the ability to carrylarge payloads, have made them the subject of intenseresearch in the cancer field. Numerous investigationshave shown that incorporating anticancer drugs inNPs provide a useful means for controlling their tissu-lar and cellular distribution profiles. The combinationof a controlled drug release with a targeted delivery isconsidered to withdraw the limitations that conven-tional chemotherapy often presents.12,13In addition,hyperthermia and thermal ablation therapy in cancerusing magnetic NPs has significant advantages com-pared with conventional hyperthermia treatment.14
Recently, much attention has been given to inorganicNPs such as gold and magnetic NPs incorporated intopolymer- and lipid-based formations, possessing bothimaging and therapeutic capabilities for improvedoutcome.15,16 Nevertheless, there are several impor-tant issues to be considered for in vivo applications ofNPs in preclinical animal models, such as the biocom-patibility, in vivo kinetics, ability to escape the RES,targeting efficacy, acute and chronic toxicity, and cost-effectiveness.
There are two pathways in order to target NPsto malignant cells: the active targeting pathway,incorporating tumor-specific ligands to the NPs,and the passive targeting pathway, that relies onthe enhanced permeability and retention (EPR)effect.11,17,18 The development of NPs can be sub-divided into three generations. The first-generationNPs are rapidly cleared from the blood stream anduptaken by the macrophages in the RES organs (liverand spleen).19 In order to overcome the RES cap-ture, prolong their blood circulation time and reducetheir toxicity, the NPs surface properties have beenmodified by covalently binding hydrophilic polyethy-lene glycol (PEG) comprising the second-generationNPs.20–22 The above NPs act through passive target-ing and have limitations in their applications (e.g.,suitability only for liver tumors imaging/treatment).A further development on NPs structure was per-formed by binding specific recognition ligands (e.g.,antibodies and peptides) to their surface in orderto actively target specific tumor or tissues throughmolecular interaction or affinity (Fig. 1).23–30
JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps
111In-LABELED NANOPARTICLES 3
Figure 1. PEGylated iron oxide dextran NPs linked with an 111In-labeled antibody.
RADIOLABELING OF NPs
The easiest and most convenient way for an accu-rate estimation of the NPs’ biodistribution profile isits effective radiolabeling with a gamma-emitting ra-diometal. The selection of the appropriate radiometalas well as the methodologies that are needed to be de-veloped in order to perform the radiolabeling of NPsdiffer and have to do with the NP type itself. The syn-thesis of a radiolabeled NP moiety offers the advan-tage of accurate quantification of the amount of thelabeled nanocarrier in each organ and visualization ofits route in vivo at the same time, by performing dy-namic gamma imaging and SPECT.31,32 Studying the
NPs’ in vivo behavior widens the field of application tonovel imaging and therapeutic approaches in nuclearmedicine. Liposomes are already studied for imag-ing of infection33 and targeted diagnosis/therapy ofcancer34,35 and as diagnostic markers in the develop-ment of drugs, based on liposomal or polymeric formu-lations, for both systemic and local administrations.36
There are multiple approaches in order to radiola-bel a NP moiety (Fig. 2). Radiolabeling of many differ-ent types of NPs with radiometals such as 99mTc, 111In,67Ga, 177Lu, and so on, following various radiolabelingmechanisms has already been applied.31,37–56 First ofall, the radiometal or a radiolabeled compound can be
Figure 2. Radiolabeling approaches of NPs: (a) Direct labeling on the NP surface, (b) labelingof chelator-functionalized NPs, (c) encapsulation of radionuclides or lipophilic radiocompounds,and (d) radionuclide incorporation into the lipid bilayer.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES
4 PSIMADAS ET AL.
encapsulated to the nanocarrier during synthesis.This methodology has been used in order to encap-sulate the lipophilic radiopharmaceutical 99mTc-(D,L-hexamethylpropyleneamine oxime), which can beeasily produced by a commercially available kit.57 An-other very common approach especially for 99mTc is af-ter synthesis, by complexing the radionuclide directlyto the surface of the NP after one-step reduction.32,43
A more frequent approach when it comes to 111In isthrough the labeling of a chelator, which is alreadyattached to the surface of the NP or through the label-ing of a chelator–biomolecule conjugate, which is ulti-mately attached to the NP surface.50,52After prepara-tion of the NPs, there are also two other radiolabelingmethods, mostly used for liposomes: the radiometalincorporation into the lipid bilayer or after loading ofthe aqueous phase of the NP.31
The yield of NPs radiolabeling complex reactionsis assessed with instant thin layer chromatographyin silica gel (ITLC-SG) using different mobile phases.In some cases (e.g., gold NPs), the reaction mixturecan pass through a size-exclusion high-performanceliquid chromatography (SE-HPLC) column in orderto evaluate the radiolabeling yield and perform pu-rification if necessary. In most cases, the radiolabeledNPs must be separated from radioactivity, which isnot associated with NPs such as free radiometal orany other radio species. The combination of ultrafil-tration/centrifugation and size-exclusion chromatog-raphy (SEC) is widely applied for this purpose. Fi-nally, before in vivo applications, the radiolabeled NPsmust be tested in order to evaluate the in vitro stabil-ity of the complex in the presence of strong radiometalcompetitors [e.g., diethylenetriaminepentaacetic acid(DTPA), His, and Cys] as well as in the presence ofserum or plasma.32,37,43
111In-LABELED NPs
Indium-111 is a readily available radionuclide which,after 99mTc, is the most widely used in clinical nu-clear medicine. It is mainly produced by two meth-ods, either from the enriched isotopes of cadmium (ir-radiation with protons or deuterons) or from silver(irradiation with a-particles or 3He ions), althoughin lower yields in the latter case. It decays by elec-tron capture and Auger electrons emission to 111Cd,which de-excites via the emission of two low-energyphotons, which are only slightly internally converted,so it is practically stable. The mean energies of itsdecay photons, which are useful for detection andimaging, are 171.3 and 245.4 keV and lie within therange of skills of the detector devices.58 A significantadvantage of 111In-labeled radiotracers is that theyare often used as the imaging surrogates for biodis-tribution and dosimetry determination of their corre-sponding 90Y analogs due to their similar coordination
Figure 3. Chemical structures of (a) DTPA and (b) DOTAmacrocyclic chelators.
chemistry.59 Especially for NPs studying, 111In is veryconvenient because of its appropriate half-life of 2.8days, which offers the opportunity to evaluate treat-ment schemes and assess biodistribution for a longperiod after the radiopharmaceutical administration.In addition, the parallel use of 111In in order to imageas well as to obtain a therapeutic effect via the Augerelectrons emitted is a substantial advantage of thisradiometal, which has not yet been widely exploredwhen it comes to NPs.
For the radiolabeling of a chemical moleculewith any radiometal, a bifunctional chelating agent(BFCA) is essential most of the times, in or-der to facilitate the radiocomplex formation.60
DTPA is the BFCA of choice for radiolabeling ofmolecules with 111In. Another BFCA, which hasgained a lot of attention and tends to replaceDTPA, is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA can form highly stablecomplexes with metals of various ionic radiuses andit has become a universal chelator for the integrationof a variety of radiometals (e.g., 111In, 177Lu, 86/90Y,67/68Ga, 64Cu, etc.) into biomolecules.60 The chemicalform of both BFCAs is presented in Figure 3.
The NP categories that have been radiolabeled with111In so far, their overall characteristics, as well astheir application in biomedical nanoresearch are pre-sented in Table 1.
Liposomes
Liposomes were the first class of nanostructures de-veloped and they have undergone the most extensivestudy during the last 30 years.61 One of the greatadvantages of liposomes is that they can be easilyprepared and their composition can vary simply byusing different derivatized phospholipids in the for-mulation process, for example, PEG-modified phos-pholipid building blocks, in order to impart signifi-cant advantageous characteristics such as prolonged
JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps
111In-LABELED NANOPARTICLES 5
Table 1. 111In-Labeled NP Types in Biomedical Research
NPs 111In Labeling Application References
Liposomes 111InCl3 in pH 5 at RT Biodistribution and imaging properties evaluation 37,45,46111In-oxine entrapment at RT Biodistribution and therapeutic evaluation of
anticancer drug delivery.Therapy via Auger e−
Polymeric micelles 111InCl3 in pH 5.2 at RT Biodistribution and imaging properties evaluation 47–50Imaging of chemotherapy-induced apoptosisIn vivo cellular uptake evaluation
Gelatin NPs Sodium citrate intermediate at RT Biodistribution evaluation 51Iron oxide NPs Conjugation of 111In-labeled Ab Hyperthermia treatment evaluation 52Gold NPs 111InCl3 in pH 5.5 at RT Biodistribution evaluation 53Carbon NPs Sodium citrate intermediate at RT or 40◦C Biodistribution and imaging properties evaluation 54, 55Antisense carrying NPs 111InCl3 in pH 5.5 at RT Antisense-mediated cytotoxicity evaluation 56
Therapy via Auger e−
circulation, integrity, etc.22 Radiolabeling of lipo-somes, mainly with radiometals, can be performeddirectly, using lipid-soluble chelating agents62 orby synthesizing chelator-derivatized phospholipidmonomers, which can bind the radiometal onto theliposome.63 Such potential chelator is DTPA, whichcan be used for 111In, 90Y, and 177Lu radiolabeling forSPECT, but also for Gd loading for MRI.64
Helbok et al.37 have recently evaluated DTPA-derivatized liposomes and micelles with respect toradiolabeling with trivalent radiometals, including111In and 177Lu. PEGylated liposomes and cholesterolliposomes as well as PEGylated micelles were cou-pled with different amounts of DTPA and were radio-labeled with 111In by the addition of 111InCl3 solutionin slightly acidic environment (pH 5) at room tem-perature (RT). The quality control of the radiolabelednanostructures was performed by ITLC-SG and SECon a PD-10 column and verified high labeling effi-ciencies for 111In (approximately 98% for 50:g of li-posome). All radiolabeled species were stable againsttranschelation after serum, DTPA, and His challengestudies. Although PEGylated formations were chosenin order to avoid the RES uptake, the in vivo biodis-tribution in healthy Lewis rats showed high radioac-tivity concentration in the liver and spleen. Neverthe-less, the 111In-labeled liposomes remained in the bloodpool for quite a long time, not showing high intestineexcretion before 12 h after injection. The amount ofDTPA loading, the lipid composition and final size ofliposomes, as well as the amount of injected radio-labeled liposomes showed no detectable influence onbiodistribution. Furthermore, no release of the metalsfrom the complexes was found, as no significant boneuptake was observed. However, it is well known thatDTPA is not an ideal chelator for trivalent radiomet-als compared with its derivatives or cyclic chelators(e.g., DOTA).58 Therefore, when 177Lu or 90Y are usedfor radionuclide therapy or Gd ions are loaded to the
NPs for MRI, these alternative ligands should be ap-plied, as radiometal release in vivo is not acceptabledue to severe side effects.
Two other studies evaluated the therapeutic effi-cacy of small unilamellar PEGylated liposomal vesi-cles (size <100 nm), in which an antineoplastic agent[vinorelbine (VNB)] was encapsulated to produceNanoVNB.45,46 These studies were performed in micebearing human colorectal adenocarcinoma (HT-29)xenografts, transfected with the luciferase gene forbioluminescence imaging (BLI). Biodistribution andimaging properties of NanoVNB were assessed by111In labeling of the nanostructure via an after-loading radiolabeling procedure by which radiola-beled liposomal preparations can be obtained withhigh in vivo stability. NanoVNB was labeled with111In-oxine at RT and purified after passage througha Sephadex G50 column (111In entrapment: >90%).Biodistribution of 111In–NanoVNB indicated that at1 h after injection, radioactivity accumulation wasfound mainly in the spleen and blood, whereas at 48 hafter injection, almost all radioactivity was clearedfrom the blood pool. However, liver and spleen werethe two main organs still with the maximum accumu-lation at that timepoint. The tumor–to-blood ratios of111In–NanoVNB were 0.1, 2.7, 20.6, and 24.3, at 4,24, 48, and 72 h after injection, respectively. Gammascinitigraphy was performed 9 days after injectionfor therapeutic efficiency evaluation. Results showedthat the tumor–muscle ratios were 2, 2.3, and 4.4, at0, 5, and 10 mg/kg doses, respectively, and that signif-icant tumor reduction was observed in 5 and 10 mg/kgdoses, but not in the control group. The results are inagreement with autoradiography and BLI and indi-cate that the higher the treatment dose, the smallerthe tumor size. An interesting finding was that theadministration of 111In–liposome only (without VNB)was also effective in tumor reduction, suggesting thatAuger electrons may also act therapeutically.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES
6 PSIMADAS ET AL.
Polymeric Micelles
Peptides in general have a short blood half-life in vivo,which could lead to limited tumor exposure time andthus lower tumor uptake. Long-circulating NPs canmake peptides available for longer time in order tobind to their receptors. PEGylated core-cross-linkedpolymeric micelles (CCPMs) have been used for op-timizing peptide pharmacokinetics. More specifically,a 14-peptide with high affinity for the EphB4 recep-tor (EphB4r-peptide), which is overexpressed in nu-merous tumors, was attached on the surface of PEG–CCPM. This structure (average diameter 23 nm) wasdually labeled with 111In and near-infrared fluo-rescence fluorophores (Cy7), for both SPECT andoptical imaging.47 Cy7 dye was entrapped in thecore, whereas 111In was complexed to DTPA, whichwas conjugated on the surface of CCPM. Radio-labeling was performed under acidic environmentat RT and was assessed by ITLC-SG. Biodistribu-tion studies showed high liver and spleen uptakebut also indicated that this NP formulation hadsignificantly reduced clearance, higher blood con-centration, and consequently better tumor target-ing than the peptide alone. Histopathologically, 111In–DTPA–CCPM–(EphB4r-peptide) can bind specifi-cally to the region of EphB4 expression in tumorxenografts, which is consistent with autoradiographicand fluorescent findings in tumor sections, and con-sequently it possess the potentiality to image tumorsthat overexpress this receptor, as micro SPECT/CTimages revealed.
The same NP formulation was used in order toperform dual imaging of chemotherapy-induced apop-tosis in tumor models.48 Annexin A5 is an endoge-nous human protein (319 amino acids) that recog-nizes phosphatidylserine residue. Because of its shortblood half-life, the peak apoptotic activity can only beassessed by multiple scans after multiple injectionsof 99mTc-labeled annexin A5. Conjugation of annexinA5 with CCPM (average diameter 25 nm) was per-formed in order to prolong the time that the target-ing moiety has to reach and bind to therapy-inducedapoptotic cells. 111In–DTPA–CCPM–A5 was stronglybound in a specific manner to apoptotic tumor cellsin vitro. in vivo studies in tumor-bearing mice afterchemotherapy (in order to induce apoptosis) showed amean retention half-life of 12.5 h, whereas mean ini-tial blood concentration was 22.4% injected dose (ID)/mL. Apoptosis was efficiently visualized dually andimaging data showed that the tumor-to-muscle andtumor-to-blood ratios in treated mice were almost twotimes higher than those in untreated mice bearingEL4 lymphoma. In addition, 111In–DTPA–CCPM–A5exhibited significantly higher tumor uptake than the99mTc-labeled annnexin A5 and the plain CCPM con-trol in the treated mice tumors.
Many studies so far have focused on the whole bodyclearance and distribution of a variety of NPs as far astheir in vivo fate is concerned, whereas their in vivocellular uptake and subcellular distribution as a func-tion of size and/or targeting remains unexplored. Asdiscussed previously, large particles with size lessthan 200 nm generally tend to have prolonged cir-culation half lives and can accumulate in a passiveway to tumors; nevertheless the optimal NP size formaximum tumor penetration and retention have notyet been clearly delineated.50,65 Lee et al.49 have stud-ied the ability of PEGylated-block-poly-g-caprolactoneblock copolymer micelles (BCMs) to concentrate insolid tumors in vivo passively, as well as after target-ing with epidermal growth factor (EGF). NP formu-lations with diameter of 60 nm (BCM-60) were con-jugated with DTPA and effectively labeled with 111Inat 37◦C (ITLC-SG: ≥94%). Targeting of the BCMs didnot alter their in vivo distribution and eliminationprofile. SPECT/CT images were in accordance withbiodistribution data showing highest BCM accumula-tion in the liver and spleen (9%–30% ID/g), whereastumor uptake was limited, except from the targetedformulation tested in an EGF receptor positive tu-mor model (∼5% ID/g at 48 h after injection). Tumortissue fractionation indicated that most of the tumor-accumulated radioactivity was found in the extracel-lular area (70%–90%). Autoradiography and CD-31staining showed that the intratumoral distribution ofBCMs is in correlation with the blood vessel densities.
A later study by the same group50 showed that thesame NP formulations with a reduced mean diam-eter of 25 nm (BCM-25) cleared more rapidly fromthe plasma, leading to an almost twofold decreasein their total tumor accumulation. At 48 h after injec-tion, no statistical difference was detected in the accu-mulation of BCMs in organs responsible for clearance(liver, spleen, and kidneys). BCM-25, which was addi-tionally functionalized with EGF (T-BCM-25) showeddelay in the tumoral clearance, which resulted in acomparable level of total tumor deposition as the non-targeted BCM-60 (NT-BCM-60).
Unfortunately, autoradiography failed to detectany differences between targeted and nontargetedNPs of 25 and 60 nm in tumor penetration, whereasconfocal fluorescence microscopy revealed that par-ticle size and molecular targeting has an effect inthe intratumoral transport of polymeric NPs. EGFintroduction retards the tumor penetration ability ofT-BCM-25, whereas a significant enhancement in cy-toplasmic and nuclear uptake due to molecular tar-geting was detected. Therefore, the design parame-ters of polymeric micelle drug-encapsulated deliverysystems must be carefully optimized concerning theirin vivo behavior, in order to become useful tools forspecific therapeutic applications.
JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps
111In-LABELED NANOPARTICLES 7
Gelatin NPs
The biodistribution and tumor-targeting potentialof gelatin NPs, which were PEGylated (PEG–Gel)and/or thiolated (PEG–SHGel) were also investi-gated in vivo, in mice bearing estrogen-negative hu-man breast adenocarcinoma.51 NPs were derivatizedwith DTPA and radiolabeled with 111In at RT usingsodium citrate as intermediate. These NP formula-tions showed quick uptake by the RES, localizing inliver as early as 1 h after injection However, PEG mod-ification of the NPs (mean size 290–350 nm) resultedin longer circulation and availability in blood as wellas greater accumulation and retention in the tumor,when compared with their unmodified counterparts.More specifically, about 13% of the recovered dose ofPEG–SHGel and PEG–Gel NPs was retained in thetumor at 12 h after injection, in contrast to Gel andSHGel NPs that had 7% and 9% retention, respec-tively. Besides the plasma, tumor, and liver, these NPswere predominantly accumulated in the kidneys andspleen and to a lesser extent in the lungs and heart.The PEG-modified thiolated gelatin NPs, which havelonger circulation times, higher sensitivity towardhighly reducing environment, and enhanced tumorextravasation, could potentially be used in passivedrug and gene targeting of solid tumors in vivo.
Iron Oxide NPs
Magnetic iron oxide NPs can be used in thermal can-cer therapy applications, when they are inductivelyheated by an externally applied alternating magneticfield (AMF). If these NPs are linked to a cancer-specific antibody or peptide, tumor-specific hyperther-mia treatment can be developed. In order to evaluatethe potential of this therapy, in vivo tumor targetingefficacy and radionuclide-based heat dosimetry wasstudied using 111In-labeled NPs in a human breastcancer xenograft model.52 111In–DOTA–ChL6 [ChL6,monoclonal antibody (mAb) with affinity for an inte-gral membrane glycoprotein highly expressed in var-ious cancers] was conjugated via amide linkage tothe carboxyl-terminated PEG coating of the iron ox-ide–impregnated dextran NPs (20 nm) using the car-bodiimide method and purified as 111In–ChL6–NPs.Quality control with cellulose acetate electrophore-sis verified monomeric final product formation inhigh yield (<91%). Pharmacokinetic data were ob-tained at 1, 2, 3, and 5 days after injection in tumor-bearing mice. Mean 111In–ChL6–NPs concentrations(%ID/g) in most organs were similar to those of111In–ChL6, whereas liver and spleen concentrationswere twice that of 111In–ChL6. The mean tumoruptake of 111In–ChL6–NPs remained constant (be-tween 9.7% and 13.7% ID/g) over the 5-day study.AMF was delivered 72 h after injection for 20 min andthe treated and control animals were monitored for
90 days. Tumor total heat dose from activated tumor111In–ChL6–NPs was calculated for each heat-dosegroup using 111In–ChL6–NPs tumor uptake and pre-measured particle heat response to AMF amplitudes.All groups presented tumor growth delay correlatedwith heat dose and, except for the lowest heat-dosegroup, this was statistically significant when com-pared with the untreated group.
Gold NPs
Gold NPs can facilitate transfer of thermal energiesto cancer tissue, reducing the laser energy neces-sary for tumor cell destruction in laser-induced pho-totherapy, and enhancing photothermal ablation effi-ciency. However, the targeted delivery of the currentlyused gold NPs to tumor cells is limited. Melanconet al.53 synthesized gold nanoshells (HAuNS; size:30 nm) covalently attached to C225, an mAb directedat EGF receptor (EGFR).53 HAuNS were composedonly of a thin gold wall with a hollow interior andC225 was functionalized with DTPA for 111In la-beling, prior to NP attachment. Radiolabeling wasperformed at RT in slightly acidic conditions (pH5.5) by adding 111InCl3 to a HAuNS–C225–DTPAsolution. The labeled compound was purified bycentrifugation and radiochemical control was per-formed by ITLC-SG (>95%). Biodistribution studiesof 111In-labeled HAuNS–C225 in nude mice-bearingEGFR-positive A431 human squamous carcinomaxenografts indicated that 24 h after injection, theorgans with the higher radioactivity concentrationwere liver (33.1 ID/g), spleen (17.5 ID/g), and kidney(13.6 ID/g). The biodistribution results of the controlmolecule (111In-labeled HAuNS–IgG) were compara-ble except from the lower liver (13.2 ID/g) and tu-mor (4.6 ID/g) uptake observed, a finding that was at-tributed to no specific interaction with EGFR, which ispresent in both tissues. Additional radiotracer count-ing study indicated significant increase in the num-ber of nanoshells per field observed under dark-fieldmicroscope in the perivascular tumor area in miceinjected with C225–HAuNS than in mice injectedwith IgG–HAuNS. Thus, combining the facts that111In-labeled anti-EGFR mAb-conjugated HAuNSverified enhanced EGFR-positive tumors deliveryin vivo and showed increased extravasation efficiencyfrom tumor vessels to interstitial space, we may con-clude that their applications could potentially be ex-tended to in vivo molecular therapy.
Carbon NPs
Carbon nanotubes are carbon allotropes with acylindrical nanostrusture. They possess extraordi-nary properties, including high electrical and ther-mal conductivity, great strength, and rigidity, andhave been developed lastly for biomedicine applica-tions. Water-soluble, single-walled carbon nanotubes
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES
8 PSIMADAS ET AL.
(SWNTs) have been functionalized with DTPA andlabeled with 111In in order to evaluate their biodis-tribution profile.54 Compounds with a surface com-pletely saturated with DTPA molecules as well aswith only 60% of DTPA functionalization and 40%of free amine groups were synthesized. Radiola-beling was performed by cationic exchange from asolution of 111In–citrate at RT. Surface charge den-sity differences of 111In–DTPA–SWNT did not al-ter significantly the biodistribution profile, showingrapid clearance from the blood pool through the re-nal excretion route without any toxic side effects. The111In–DTPA–SWNT compound, which carries free-NH2 groups showed even greater kidney excre-tion (almost two times higher than the fully DTPA-saturated compound) and it can be used for systemicgene transfer in vivo.
Another study in the field of carbon NPs investi-gated the suitability of PEGylated perfluorocarbonNPs conjugated with a vitronectin antagonist, forin vivo tumor angiogenesis targeting.50 These NPswere linked with a DOTA-derivatized chelator and la-beled with 111In. Radiolabeling was performed at 40◦Cat an overnight shaker, using citrate as an intermedi-ate shuttle, and coupling was assessed by ITLC-SG.Biodistribution results showed higher uptake in thespleen at all dosages applied (0.25, 0.5, and 1.0 mL/kg). The 111In–"<$3–NPs administration provided ahigh sensitivity, low-resolution signal from the tu-mor angiogenesis site that was well recognized within15–30 min after injection and remained persistent forhours. The tumor signal obtained was higher for theformulations with 10 radionuclides per NP versus 1.The in vivo blocking of "<$3 integrin resulted in lowertarget uptake, confirming the affinity of the modi-fied NPs with the receptors. The tumor accumula-tion in this study was a result of both specific bindingand passive entrapment within the tumor neovascu-lature, suggesting that "<$3-targeted 111In perfluoro-carbon NPs may provide a clinically useful tool forsensitively detecting angiogenesis in cancer, poten-tially combined with other imaging modalities, suchas MRI.
Other NPs
Alongside with imaging and biodistribution evalua-tion, 111In-labeled compounds can be applied in orderto induce cytotoxicity via the emission of Auger elec-trons in the nucleus of tumor cells. NP formulationsthat consist of streptavidin linked with three biotiny-lated components (antiHer2 Ab trastuzumab, the tatpeptide, and a DTPA-derivatized antisense or sensemorpholino oligomer: DTPA–MORF/tat/trastuzumabNP) were synthesized and radiolabeled with 111In.56
The in vitro cytotoxicity of cells incubated with thesense NPs showed no dose-dependent increase, in-
dicating that when incubated as the sense NP, the111In is not reaching the nucleus effectively. The de-creasing survival efficiency for the antisense NPsis strong evidence that in this case the 111In isreaching the nucleus. The 111In–DTPA–MORF/tat/trastuzumab NP performed efficient tumor targeting,intracellular transport, 111In nuclear migration, andspecific radiation cytotoxicity.
PRESPECTIVES AND LIMITATIONS
Nanoparticles are of great scientific interest be-cause they represent a bridge between bulk materi-als and molecules and structures at an atomic level.Nanoparticle research is currently very active in thebiomedicine field against cancer, covering areas suchas molecular imaging, drug delivery, and hyperther-mia treatment. Molecular imaging and therapeuticapplications of radiolabeled NPs have been exploredwith encouraging results. The most unique feature ofradionulide-based biodistribution and imaging tech-niques is that they are highly sensitive as they arequantitative. Multimodal imaging techniques can ad-ditionally allow simultaneous detection of a radiola-beled NP with SPECT and MRI (e.g., magnetic NPs)providing more accurate results. When it comes totherapy, labeling with a therapeutic radionuclide canprovide therapeutic outcome as well as informationabout pharmacokinetics and tumor-targeting efficacyat the same time, whereas the obstacle of controlledanticancer drug release is overcome.
Most research in tumor targeting with radiola-beled NPs has been performed based on the EPR ef-fect, which has, as a prerequisite, a prolonged bloodcirculation.11,51 This can indeed act therapeuticallyin some cases, but in many other it results prolongedexposure of the normal organs to the radionuclidecausing undesired toxicity. That is why the next stepin applying radiolabeled NPs for cancer diagnosis/treatment is the radiolabeling of targeted NPs.49,50,53
Advances in chemistry, biology, and physics mustcombine in order to produce stable and well-definednanostructures with specific ligands/biomolecules at-tached on their surface.
Another obstacle to overcome when it comes to NPsis the appearance of possible side effects derived fromtheir in vivo use, due to their toxicity. Adverse biolog-ical reactions and immunotoxic effects caused fromthe material itself or the prolonged time the NPs stayin the body can be limited by thoroughly modifyingtheir structure through robust synthetic chemistry.
Finally, when it comes to commercialization of NPs,many problems arise due to their complexity and mul-ticomponent nature. Furthermore, the use of radionu-clides adds an additional level of difficulty, which isthe radiation safety.
JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps
111In-LABELED NANOPARTICLES 9
CONCLUSION
The use of 111In for radiolabeling a wide range of NPsin order to visualize their route into the body as wellas the efficacy of a treatment can offer substantialhelp for potential application of NPs in clinical prac-tice. 111In is a radiometal with a sufficient half-lifefor performing in vivo evaluation of an injectable ra-diopharmaceutical even several days after adminis-tration and additionally has a potential therapeuticeffect, due to the emission of Auger electrons. The ul-timate goal of nanomedicine in the cancer field is thecreation of nanoagents appropriate for simultaneoustargeted diagnosis and therapy, which is critical to ad-dressing the challenges of cancer heterogeneity andadaptation.
ACKNOWLEDGMENTS
The authors would like to acknowledge financial sup-port provided by the European Commission throughthe Seventh Framework Program (FP7) for Researchand Development (CP-IP 213631-2 NANOTHER).
REFERENCES
1. Reubi JC, Maecke HR. 2008. Peptide-based probes for cancerimaging. J Nucl Med 49:1735–1738.
2. Imam SK. 2005. Molecular nuclear imaging: The radiophar-maceuticals. Cancer Biother Radiopharm 20:163–172.
3. Hawkins RA. 2000. Radionuclide imaging in cancer medicine.In Holland-Frei Cancer Medicine; Bast RC, Kufe DW, PollockRE, Weichselbaum RR, Holland JF, Frei E, Eds. 5th ed. Hamil-ton, Ontario: BC Decker Inc: Chapter 30H.
4. Massoud TF, Gambhir SS. 2002. Molecular imaging in livingsubjects: Seeing fundamental biological processes in a newlight. Genes Dev 17:545–580.
5. Berman DS, Kiat H, van Train K, Friedman JD, Wang FP,Germano G. 1994. Dual-isotope myocardial perfusion SPECTwith rest thallium-201 and stress Tc-99m sestamibi. CardiolClin 12:261–270.
6. Cutler CS, Lewis JC, Anderson CJ. 1999. Utilization ofmetabolic, transport and receptor-mediated processes to de-liver agents for cancer diagnosis. Adv Drug Deliv Rev37:189–211.
7. Anderson CJ, Welch MJ. 1999. Radiometal-labeled agents(non-technetium) for diagnostic imaging. Chem Rev99:2219–2234.
8. Jurisson S, Cutler C, Smith CV. 2008. Radiometal complexes:Characterization and relevant in vitro studies. Q J Nucl Med52:222–234.
9. Wang MD, Shin DM, Simons JW, Nie S. 2007. Nanotechnol-ogy for targeted cancer therapy. Exp Rev Anticancer Ther7:833–837.
10. Hoet PH, Bruske-Hohlfeld I, Salata OV. 2004. Nanoparticles—Known and unknown health risks. J Nanobiotechnology2(1):12.
11. Choi HS, Frangioni JV. 2010. Nanoparticles for biomedicalimaging: Fundamentals of clinical translation. Mol Imaging9:291–310.
12. Gu FX, Karnik R, Wang AZ, Alexis F, Levy-Nissenbaum E,Hong S, Langer RS, Farokhzad RS. 2007. Targeted nanoparti-cles for cancer therapy. NanoToday 2:14–21.
13. Lian T, Ho RJ. 2001. Trends and developments in liposomedrug delivery systems. J Pharm Sci 90:667–80.
14. Kumar CS, Mohammad F. 2011. Magnetic nanomaterials forhyperthermia-based therapy and controlled drug delivery. AdvDrug Deliv Rev 63:789–808.
15. Schladt TD, Schneider K, Schild H, Tremel W. 2011. Synthesisand bio-functionalization of magnetic nanoparticles for medi-cal diagnosis and treatment. Dalton Trans 40:6315–6343.
17. Maeda H, Matsumura Y. 1989. Tumoritropic and lymphotropicprinciples of macromolecular drugs. Crit Rev Ther Drug Car-rier Syst 6:193–210.
18. Gil PR, Parak WJ. 2008. Composite nanoparticles take aim atcancer. ACS Nano 2:2200–2205.
19. Boerman OC, Laverman P, Oyen WJG, Corstens FHM, StormG. 2000. Radiolabeled liposomes for scintigraphic imaging.Progr Lipid Res 39:461–475.
20. Gref R, Minamitake Y, Peracchia V, Trubetskoy V, TorchillinV, Langer R. 1994. Biodegradable long-circulating polymericmicrospheres. Science 263:1600–1603.
21. Blume G, Cevc G, Crommelin MD, Bakker-Woudenberg IA,Kluft C, Storm G. 1993. Specific targeting with poly(ethyleneglycol)-modified liposomes: Coupling of homing devices tothe ends of the polymeric chains combines effective targetbinding with long circulation times. Biochim Biophys Acta1149:180–184.
22. Immordino ML, Dosio F, Cattel L. 2006. Stealth liposomes:Review of the basic science, rationale, and clinical applications,existing and potential. Int J Nanomedicine 1:297–315.
23. Kocbek P, Obermajer N, Cegnar M, Kos J, Kristl J. 2007. Tar-geting cancer cells using PLGA nanoparticles surface modifiedwith monoclonal antibody. J Control Release 120:18–26.
24. Liu Y, Miyoshi H, Nakamura M. 2007. Nanomedicine for drugdelivery and imaging: A promising avenue for cancer therapyand diagnosis using targeted functional nanoparticles. Int JCancer 120:2527–2537.
25. Lee HY, Li Z, Chen K, Hsu AR, Xu C, Xie J, Sun S,Chen X. 2008. PET/MRI dual-modality tumor imaging usingarginine–glycine–aspartic (RGD)-conjugated radiolabeled ironoxide nanoparticles. J Nucl Med 49:1371–1379.
27. Saad M, Garbuzenko OB, Ber E, Chandna P, Khandare JJ,Pozharov VP, Minko T. 2008. Receptor targeted polymers, den-drimers, liposomes: Which nanocarrier is the most efficientfor tumor-specific treatment and imaging? J Control Release130:107–114.
28. Shokeen M, Fettig NM, Rossin R. 2008. Synthesis, in vitro andin vivo evaluation of radiolabeled nanoparticles. Q J Nucl MedMol Imaging 52:267–277.
29. Lucignani G. 2009. Nanoparticles for concurrent multimodal-ity imaging and therapy: The dawn of new theragnostic syn-ergies. Eur J Nucl Med Mol Imaging 36:869–874.
30. Elbayoumi TA, Pabba S, Roby A, Torchilin VP. 2007. Antinu-cleosome antibody-modified liposomes and lipidcore micellesfor tumor-targeted delivery of therapeutic and diagnosticagents. J Liposome Res 17:1–14.
31. Ting G, Chang CH, Wang HE, Lee TW. 2010. Nanotargetedradionuclides for cancer nuclear imaging and internal radio-therapy. J Biomed Biotechnol 2010:953537.
32. Arulsudar N, Subramanian N, Mishra P, Sharma RK, MurthyRS. 2003. Preparation, characterisation and biodistributionof 99mTc-labeled liposome encapsulated cyclosporine. J DrugTarget 11:187–196.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES
10 PSIMADAS ET AL.
33. Laverman P, Boerman OC, Oyen WJ, Dams ET, Storm G,Corstens FH. 1999. Liposomes for scintigraphic detection ofinfection and inflammation. Adv Drug Deliv Rev 37:225–235.
34. Elbayoumi TA, Torchilin VP. 2006. Enhanced accumulationof long-circulating liposomes modified with thenucleosome-specific monoclonal antibody 2C5 in various tumours inmice: Gamma-imaging studies. Eur J Nucl Med Mol Imaging33:1196–205.
35. Li L, Wartchow CA, Danthi SN, Shen Z, Dechene N, Pease J,Choi HS, Doede T, Chu P, Ning S, Lee DY, Bednarski MD,Knox SJ. 2004. A novel antiangiogenesis therapy using anintegrin antagonist or anti-Flk-1 antibody coated 90Y-labelednanoparticles. Int J Radiat Oncol Biol Phys 58:1215–1227.
36. Torchilin VP, Lukyanov AN. 2003. Peptide and protein drugdelivery to and into tumors: Callenges and solutions. DrugDiscov Today 8:259–266.
37. Helbok A, Decristoforo C, Dobrozemsky G, Rangger C,Diederen E, Stark B, Prassl R, von Guggenberg E. 2010. Radio-labeling of lipid-based nanoparticles for diagnostics and thera-peutic applications: A comparison using different radiometals.J Liposome Res 20:219–227.
38. Hwang do W, Ko HY, Lee JH, Kang H, Ryu SH, Song IC, LeeDS, Kim S. 2010. A nucleolin-targeted multimodal nanoparti-cle imaging probe for tracking cancer cells using an aptamer.J Nucl Med 51:98–105.
39. Chakraborty S, Das T, Sarma HD, Venkatesh M, Banerjee S.2008. Preparation and preliminary studies on 177Lu-labeledhydroxyapatite particles for possible use in the therapy of livercancer. Nucl Med Biol 35:589–97.
40. Hamoudeh M, Kamleh MA, Diab R, Fessi H. 2008. Radionu-clides delivery systems for nuclear imaging and radiotherapyof cancer. Adv Drug Deliv Rev 60:1329–1346.
41. McCarthy JR, Weissleder R. 2008. Multifunctional magneticnanoparticles for targeted imaging and therapy. Adv Drug De-liv Rev 60:1241–1251.
42. Hong H, Zhang Y, Sun J, Cai W. 2009. Molecular imaging andtherapy of cancer with radiolabeled nanoparticles. Nano Today4:399–413.
43. Psimadas D, Baldi G, Ravagli C, Bouziotis P, Xanthopoulos S,Franchini MC, Georgoulias P, Loudos G.Preliminary evalua-tion of a 99mTc labeled hybrid nanoparticle bearing a cobaltferrite core: in vivo biodistribution. J Biomed Nanotechnol (inpress).
44. Loudos G, Kagadis GC, Psimadas D. 2011. Current status andfuture perspectives of in vivo small animal imaging using ra-diolabeled nanoparticles. Eur J Radiol 78:287–295.
45. Chow TH, Lin YY, Hwang JJ, Wang HE, Tseng YL, Pang VF,Wang SJ, Whang-Peng J, Ting G. 2008. Diagnostic and ther-apeutic evaluation of 111In-vinorelbine-liposomes in a humancolorectal carcinoma HT-29/luc-bearing animal model. NuclMed Biol 35:623–634.
46. Lee WC, Hwang JJ, Tseng YL, Wang HE, Chang YF, Lu YC,Ting G, Whang-Peng J, Wang SJ. 2006. Therapeutic efficacyevaluation of 111in-VNB-liposome on human colorectal adeno-carcinoma HT-29/luc mouse xenografts. Nucl Instr MethodsPhys Res 569:497–504.
47. Zhang R, Xiong C, Huang M, Zhou M, Huang Q, Wen X, LiangD, Li C. 2011. Peptide-conjugated polymeric micellar nanopar-ticles for Dual SPECT and optical imaging of EphB4 receptorsin prostate cancer xenografts. Biomaterials 32:5872–5879.
48. Zhang R, Lu W, Wen X, Huang M, Zhou M, Liang D, Li C. 2011.Annexin A5-conjugated polymeric micelles for dual SPECTand optical detection of apoptosis. J Nucl Med 52:958–964.
49. Lee H, Hoang B, Fonge H, Reilly RM, Allen C. 2010. in vivo dis-tribution of polymeric nanoparticles at the whole-body, tumor,and cellular levels. Pharm Res 27:2343–2355.
50. Lee H, Fonge H, Hoang B, Reilly RM, Allen C. 2010. The effectsof particle size and molecular targeting on the intratumoraland subcellular distribution of polymeric nanoparticles. MolPharm 7:1195–1208.
51. Kommareddy S, Amiji M. 2007. Biodistribution andpharmacokinetic analysis of long-circulating thiolatedgelatin nanoparticles following systemic administra-tion in breast cancer-bearing mice. J Pharm Sci 96:397–407.
52. DeNardo SJ, DeNardo GL, Natarajan A, Miers LA, Fore-man AR, Gruettner C, Adamson GN, Ivkov R. 2007. Ther-mal dosimetry predictive of efficacy of 111In-ChL6 nanopar-ticle AMF-induced thermoablative therapy for human breastcancer in mice. J Nucl Med 48:437–444.
53. Melancon MP, Lu W, Yang Z, Zhang R, Cheng Z, Elliot AM,Stafford J, Olson T, Zhang JZ, Li C. 2008. in vitro and in vivotargeting of hollow gold nanoshells directed at epidermalgrowth factor receptor for photothermal ablation therapy. MolCancer Ther 7:1730–1739.
54. Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C,Prato M, Bianco A, Kostarelos K. 2006. Tissue biodistribu-tion and blood clearance rates of intravenously administeredcarbon nanotube radiotracers. Proc Natl Acad Sci U S A103:3357–3362.
55. Hu G, Lijowski M, Zhang H, Partlow KC, Caruthers SD, KieferG, Gulyas G, Athey P, Scott MJ, Wickline SA, Lanza GM.2007. Imaging of Vx-2 rabbit tumors with alpha(nu)beta3-integrin-targeted 111In nanoparticles. Int J Cancer 120:1951–1957.
56. Liu X, Wang Y, Nakamura K, Kawauchi S, Akalin A, Cheng D,Chen L, Rusckowski M, Hnatowich DJ. 2009. Auger radiation-induced, antisense-mediated cytotoxicity of tumor cells usinga 3-component streptavidin-delivery nanoparticle with 111In.J Nucl Med 50:582–590.
57. Pereira MA, Mosqueira VC, Carmo VA, Ferrari CS, ReisEC, Ramaldes GA, Cardoso VN. 2009. Biodistribution studyand identification of inflammatory sites using nanocapsuleslabeled with (99m)Tc-HMPAO. Nucl Med Commun 9:749–755.
58. Kocher DC. 1981. Radioactive Decay Data Tables-A Handbookof Decay Data for Application to Radiation Dosimetry and Ra-diological Assessments, DOE/TIC-11026; U.S. Department ofEnergy, Oak Ridge, Tennessee: 115.
59. Virgolini I, Traub T, Novotny C, Leimer M, Fuger B, Li SR,Patri P, Pangerl T, Angelberger P, Raderer M, Burggasser G,Andreae F, Kurtaran A, Dudczak R. 2002. Experience withindium-111 and yttrium-90-labeled somatostatin analogs.Curr Pharm Des 8:1781–1807.
60. Meares CF, Moi MK, Diril H, Kukis DL, McCall MJ,Deshpande SV, DeNardo SJ, Snook D, Epenetos AA. 1990.Macrocyclic chelates of radiometals for diagnosis and therapy.Br J Cancer Suppl 10:21–26.
61. Lasic DD, Papahadjopoulos D. 1995. Liposomes revisited. Sci-ence 267:1275–1276.
62. McAfee JG, Thakur ML. 1976. Survey of radioactive agents forin vitro labeling of phagocytic leukocytes. I. Soluble agents. JNucl Med 17:480–487.
63. Rolland A, Collet B, Le Verge R, Toujas L. 1989. Bloodclearance and organ distribution of intravenously adminis-tered polymethacrylic nanoparticles in mice. J Pharm Sci78:481–484.
![99mTc-HYNIC-[Tyr3]-octreotide for imaging somatostatin-receptor-positive tumors: preclinical evaluation and comparison with 111In-octreotide](https://static.documents.pub/doc/80x56/634f330b48cb0bd7320f576e/99mtc-hynic-tyr3-octreotide-for-imaging-somatostatin-receptor-positive-tumors.jpg)
