BASIC SCIENCE

7 (2011) 480–488

h Article nanomedjournal.com

Researc

Nanomedicine: Nanotechnology, Biology, and Medicine

Cetuximab-labeled liposomes containing near-infrared probe forin vivo imaging

Emma Portnoy, BSca, Shimon Lecht, MScb, Philip Lazarovici, PhDb,Dganit Danino, PhDc, Shlomo Magdassi, PhDa,⁎

aCasali Institute of Applied Chemistry, Institute of Chemistry and Center for Nanoscience and Nanotechnology, Jerusalem, IsraelbSchool of Pharmacy, Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

cDepartment of Biotechnology and Food Engineering, and The Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa, Israel

Received 23 August 2010; accepted 10 January 2011

Abstract

A new liposome-based near-infrared probe that combines both imaging and targeting abilities was developed for application in medicalimaging. The near-infrared fluorescent molecule indocyanine green (ICG), and the cetuximab monoclonal antibody for epidermal growthfactor receptor (EGFR) were attached to liposomes by passive adsorption. It was found that ICG molecules adsorbed to the liposomes aremore fluorescent than free ICG and have a larger quantum yield. Cetuximab-adsorbed fluorescent liposomes preserved EGFR recognition, asis evident from internalization and selective binding to A431 colon carcinoma cells overexpressing EGFR. The binding of cetuximab-targeted fluorescent liposomes to A431 compared with IEC-6 cells (normal enterocytes expressing physiological EGFR levels) was greaterby a factor of 3.5, ensuring imaging abilities with available fluorescent equipment. Due to relatively high quantum yield and specific tumorcell-recognizing ability, this technology deserves further in vivo evaluation for imaging and diagnostic purposes.

From the Clinical Editor: A new liposome-based near-infrared probe combining both imaging and targeting abilities is reported. Due torelatively high quantum yield and EGFR-expressing tumor cell specificity, this technology deserves further in vivo evaluation for imagingand diagnostic purposes.© 2011 Elsevier Inc. All rights reserved.

Key words: Indocyanine green; Near infrared; Liposomes; Imaging; Cetuximab

3

Near- infrared (NIR) optical imaging is an emerging noveltechnique for visualization of cells, tissues, and even the wholebody, because in the NIR wavelengths light is minimallyabsorbed by biological molecules, thus increasing tissue pene-tration depth.1 In addition, autofluorescence in this range is alsolimited, thus enabling a high signal-to-noise ratio; the low energyof emission and excitation is biologically safe.2 Indocyaninegreen (ICG) is a water-soluble tricarbocyanine dye that absorbsand emits in the NIR range. It is the only U.S. Food and DrugAdministration (FDA)-approved NIR molecule that has over50 years of clinical experience with an excellent safety record,which makes it very attractive for various in vivo applications.3,4

For example, in the clinic ICG is widely used for determination ofcardiac output, liver function, visualization of retinal and

Some of the results presented in this article were submitted in a recentprovisional patent application. This work was supported by BioMedicalPhotonics Consortium in the frame of MAGNET program, Israel Ministry ofIndustry and Trade. P.L. is affiliated with the David R. Bloom Center forPharmacy and the Dr. Adolf andKlara Brettler Center for Research inMolecularPharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel.

Please cite this article as: E. Portnoy, S. Lecht, P. Lazarovici, D. Danino, S. Mavivo imaging. Nanomedicine: NBM 2011;7:480-488, doi:10.1016/j.nano.2011.

1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2011.01.001

choroidal vessels, pharmacokinetic analysis, and diagnosis ofburn depth.5 ICG also has potential for photodynamic therapy.6

In current clinical setups, ICG is used in aqueous solution whereit has a relatively low quantum yield,7,8 and undergoes rapidchemical degradation and aggregation, which causes decreasedabsorption and fluorescence.9 Recently, cumulative data indicatethat immobilization of ICG onto various surfaces may result inimproved stability and therefore improved fluorescenceproperties.10,11 Such immobilization was obtained by embeddingthe ICG molecule within polymeric nanoparticles12-14 and byinclusion in liposomes15-17 and micelles.15,18

Liposomes are a very attractive delivery form because they arephysically and chemically well-characterized structures that can bedelivered through almost all routes of administration, and arebiocompatible.19 Utilization of ICG-loaded liposomes in biologicalsystemswas recently described by Sandanaraj et al17 and Proulx et al.16

⁎Corresponding author: Casali Institute of Applied Chemistry, Instituteof Chemistry and Center for Nanoscience and Nanotechnology, The HebrewUniversity of Jerusalem, Jerusalem 91904, Israel.

E-mail address: magdassi@cc.huji.ac.il (S. Magdassi).

gdassi, Cetuximab-labeled liposomes containing near-infrared probe for in01.001

481E. Portnoy et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 480–488

Carcinomas are common but difficult-to-treat diseases.20

Surgery, chemotherapy, and irradiation are common treatments,but they do not always cure the disease. Thus, improvement oftumor diagnosis will assist in the follow-up of therapeuticregimens based on defined biomarkers.21

The tyrosine kinases represent a family of about 60physiological proteins and their oncogenic forms. They arecharacterized by an extracellular ligand-binding domain, atransmembrane region, and a tyrosine kinase catalytic motif inthe intracellular part.22 A typical family of these receptors isrepresented by the epidermal growth factor receptor (EGFR)family, which is overexpressed in many carcinomas, such asthose in breast, prostate, and gastrointestinal organs. Preclinicaland clinical studies have shown that targeting the EGFR family isa valid strategy for diagnosis and anticancer therapy.23,24 Inpreclinical studies, for diagnostic purposes, EGFR-expressingtumors both in vitro and in vivo were imaged using a variety ofapproaches such as EGFR-targeted immunoliposomes with theaid of selective antireceptor monoclonal antibodies and frag-ments; fluorescent labeling of FDA-approved monoclonalantibodies such as cetuximab, trastuzumab, and daklizumab;quantum dots conjugated with either EGF or monoclonalantibodies to the EGFR family; and 111In-labeled humanEGF.25-29 Recently, NIR-labeled EGF was prepared anddemonstrated to be a useful tool to study EGFR-overexpressingtumors.30 This approach utilizes the natural ligand of EGFR,which may cause receptor activation and further amplify themutagenic activity of the tumor; therefore, to circumvent thispossibility the FDA-approved monoclonal antibody for EGFRmay be used as a targeting moiety, lacking the receptor activationability. In the present study we use this approach by generatingliposomes loaded with ICG with cetuximab as the targetingmolecule. We describe the preparation of ICG-containingliposomes that are capable of specific targeting in vitro, whilehaving improved fluorescence properties. It is expected that themethod for obtaining these liposomes, which is based onnoncovalent binding of both ICG and antibodies, would meetfuture regulatory requirements.

Methods

Materials

The following materials were used with no furtherpurification: ICG, supplied by Holland Moran (Yehud, Israel),Phospholipon 50 by Lipoid (Ludwigshafen, Germany), fluo-rescein isothiocyanate (FITC)-human IgG by Sigma-Aldrich(Rehovot, Israel). Cetuximab was provided by the PharmacyUnit of the Hadassah-University Medical Center. Human IgGenzyme-linked immunosorbent assay (ELISA) kit E-80G waspurchased from ENCO (Petach Tikvah, Israel), Fluoresceindilaurate was from Fluka (Exeter, UK). Human recombinantEGF was purchased from Peprotech-Asia (Rehovot, Israel).

Experimental methods

Liposome preparationPhospholipon 50 5% (wt/wt) was dispersed in deionized

water and stirred using a magnetic stirrer at 40°C for 40 minutes,

followed by sonication for 10 minutes (Hielscher UP200S;Teltow, Germany; 60 Hz, cycle 0.5). The resulting dispersionwas diluted in phosphate buffer to a final concentration of 1%.All preparations were performed under nitrogen. All experimentswere carried out with 5 mM phosphate buffer (pH 7) unlessotherwise stated.

Binding of ICG to the particlesTypically, the binding of ICG to the liposomes was performed

by addition of various quantities of ICG stock solutions(3.2 mM) to 700 μL of 1% (wt/wt) liposomal dispersion; afinal volume of 3.2 mL was achieved with buffer. The dispersionwas incubated under mild agitation at 5°C for 24 hours.

The quantity of ICG that is bound onto the liposomes wascalculated by determination of the free ICG as follows. The ICGdispersion was filtered through a 300-kDa filtration tube(VS0241 VIVA SPIN; Beit Haemek, Israel) for 10 minutes at40 rpm using a Centrifuge CN-2200 (MRC, Holon, Israel) tocollect the filtrate, which contains the free ICG. The free ICGwas determined by absorbance spectra, using a Cary 100 Biospectrophotometer (Varian, Palo Alto, California) in a 1-cmpolystyrene cell at a scan rate of 600 nm/min.

Binding of IgG-FITC to the ICG-particlesThe ICG was incubated with 1% liposome dispersion under

mild agitation at 5°C for 24 hours; the final ICG concentrationwas 3.2 × 10-2 mM. One hundred microliters of FITC-IgG (1 mg/mL) were added to 1.5 mL of the liposome dispersion, followedby mild agitation at 5°C for 24 hours. The dispersion was thenfiltered (filtration tube of 300 kDa for 10 minutes at 40 rpm) withwashing three times with 1 mL of buffer to remove the free IgG.

ELISA experimentsELISA experiments were performed with the human IgG

ELISA Kit (E-80G; ENCO) according to the manufacturer'srecommendations. The detailed description of ELISA procedureis given in the Supplementary Information, which is available inthe online version of this article.

Fluorescence measurementsFluorescence spectra of the samples were determined with

a Cary Eclipse Fluorimeter (Varian) using a 1-cm quartz cell,at a scan rate of 600 nm/min. For ICG spectra analysis,measurements were performed at λex 720 nm; slits for bothemission and excitation were 10 nm. For relative quantumyield λex and λabs were 720 nm; slit for excitation was 5 nmand for emission 20 nm.

Size measurementSize measurements were performed with a Zetasizer Nano-S

(Malvern Instruments, Worcestershire, United Kingdom). Theaqueous dispersions were measured without dilution. Theliposome aggregates were visualized using an MEE-643 lightmicroscope (Lieder, Germany).

Zeta potentialZeta potential measurements were performed with a Zetasizer

Nano-S (Malvern Instruments). All samples were measured afterdilution (200 μL in 4 mL of buffer).

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Cryo-transmission electron microscopy (cryo-TEM)The nanoparticles were imaged using cryo-TEM. Samples

were prepared in the controlled-environment vitrificationsystem31 at 25°C and at water saturation to avoid evaporation.Specimens were studied in a Philips CM120 transmissionelectron microscope (Philips, Eindhoven, The Netherlands)operating at 120 kV with an Oxford CT3500 (OxfordInstruments, Abingdon, Oxfordshire, United Kingdom) cryo-holder, maintained below −172°C. Digital images were recordedon a cooled Gatan MultiScan 791 charge-coupled device camera(Gatan, Abingdon, Oxfordshire, United Kingdom) using theDigital Micrograph 3.1 software (Gatan). Imaging was done inthe low-dose mode to minimize beam exposure and electron-beam radiation damage.32

Synthesis and purification of EGF labeled with FITCThe covalent attachment of human recombinant EGF with

FITC was performed according to published protocol.33 Theexact preparation procedure and validation of the conjugateactivity are described in the Supplementary Information,available in the online version of this article.

Confocal microscopyFor visualization of liposomes binding to EGFR, cetuximab or

IgG solutions (0.01 mg/ml in buffer) were added 1:1 to 1% wt/wtliposomal dispersion, and the unlabeled liposomes weredispersed similarly in buffer. The dispersion was incubatedunder mild agitation at 5°C for 24 hours. Fifty microliters offluorescein dilaurate (1 mg/mL) in pure ethanol were added to0.5% wt/wt liposomal dispersion, mixed for 3 hours at roomtemperature 22–25°C, then filtered through a 300-kDa filtrationtube and washed three times with buffer. For cell labeling, A431cells were placed on cover slides and left overnight to adhere. Thefollowing day the adherent cells were incubated with EGF-FITCor liposome solutions for 30 minutes followed by three washeswith phosphate buffered saline (PBS). Thereafter the cells werefixed with 4% paraformaldehyde (Sigma-Aldrich) and washedthree times with PBS. In the negative control experiments, theEGF-FITC or liposome solution incubation step was omittedwhile the other steps remained the same. The cells were examinedin a FluoView FV300 confocal laser scanning microscope(Olympus, Tokyo, Japan).

In vitro NIR imaging using Odyssey scannerThe ICG with 1% liposome dispersion was incubated under

mild agitation at 5°C for 24 hours; the final concentration of ICGwas 3.2 × 10-2 mM. Cetuximab solution (0.01 mg/mL) wasadded 1:1 to 1% wt/wt liposomal dispersion; in parallel, theunlabeled liposomes and IgG-bound liposomes were similarlydiluted in buffer. The dispersion was incubated under mildagitation at 5°C for 24 hours. To start the binding, cultures wereincubated with liposome solutions for 30 minutes. At the end ofincubation the cultures were washed three times with PBS andimaging was performed. (The focal cell model is described inSupplementary Information, which can be found in the onlineversion of the article). The tissue culture plates were placedinside an Odyssey NIR laser scanner (Li-Cor; Lincoln,Nebraska). The plates were scanned with fixed intensity atexcitation 785 nm and emission 800 nm. The intensity of

scanning was calibrated with free liposome solution (data notshown) and was kept constant during all experiments to avoidoverflow values. The obtained images were quantified using theLi-Cor imaging program provided with the scanner. The resultspresented as the mean ± SD of at least three independentexperiments (n = 9–15) were evaluated using the InStat3statistics program (GraphPad, La Jolla, California). Statisticallysignificant differences between experimental groups weredetermined by analysis of variance with Bonferroni post hoctest, with P b 0.05 considered significant.

Results

ICG binding

The liposomes composed of Phospholipon 50 (Lipoid), which ispermitted for oral delivery, were prepared by high-energysonication, which typically leads to formation of small unilamellarliposomes. Dynamic light scattering measurements showed that themean size (by volume) of the liposomes is 29.8 ± 0.6 nm with apolydispersity index of 0.235–0.250, and zeta potential of −30.5 ±1 mV (at pH 7). Cryo-TEM imaging (Figure 1) confirms that theliposomes are unilamellar in the size range of 20–40 nm, inagreement with dynamic light scattering results.

The ICG binding experiments were performed by incubationof the liposomes with solutions of ICG at various concentrations.The adsorbed quantity of ICG was calculated by measuring theoptical density of the solution obtained after filtration of thesamples by a 300-kDa filter, which is expected to remove allthe liposomes. The retained particles had a green color, giving avisual indication that the ICG is bound to the liposomes.

As seen in the adsorption isotherm (Figure 2, A), the quantityof adsorbed ICG increased with the increase in ICG concentra-tion in solution. The liposome number was calculated accordingto Jones.34 A bilayer thickness was taken as 4 nm,35 and a cross-sectional area of phospholipid as 0.71 nm2.35 It should be notedthat high ICG concentrations caused aggregation of liposomes,and the maximal concentration of ICG that does not causeaggregation was 46 molecules per liposome. For the greatest ICGconcentration in the adsorption isotherm (1519 molecules perliposome), the aggregate size, as seen by optical microscope, is1.4–4.2 μm (data not shown).

In addition to the size increase, it was found that binding ICGcaused a decrease in the absolute value of zeta potential, from−30.5 ± 1 mV to −15.35 ± 0.35 mV (Figure 2, B).

Because the nanoprobes are intended for colon imaging andwill be delivered by capsules suitable for colon delivery, weperformed an experiment aimed at evaluating the stability of ICGadsorption to liposomes in physiological conditions. Therefore,nanoprobes were incubated in human colon fluids for 7 hours(procedure described in the Supplementary Information, avail-able in the online version of this article).

It was found by fluorescence measurements that although theICG is not bound covalently to the liposome, during at least7 hours the ICG remained bound to the liposomes (data notshown), meaning that the nanoprobe did not disintegrate in thecolon fluids.

Figure 1. Cryo-TEM image of liposomes labeled by 3.2 × 10-5 M ICG.

Figure 2. (A) Adsorption isotherm of ICG bound to liposomes as a functionof free ICG at equilibrium (Ceq). (B) Zeta potential of liposome dispersion asa function of ICG concentration.

Figure 3. (A) Fluorescence of ICG in buffer (black diamonds) and ICG-liposome dispersion at various ICG concentrations (black triangles).Excitation at 720 nm; the emission intensities are presented at thewavelengths in which the peak was observed. (B) Effect of ICGconcentration in liposomal dispersion on fluorescence shift (black squares)and fluorescence intensity (black circles).

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Fluorescence of the ICG-liposomes

The fluorescence spectra of the ICG-liposomes wereevaluated in comparison to those of ICG in aqueous solution.It should be noted that because of a red shift that occurs in thesystems containing the liposomes, as discussed below, theemission intensities will be presented at the wavelengths inwhich the maximum of the peak was observed. Figure 3, Apresents the fluorescence intensities of ICG solutions and forICG bound to liposomes. As shown, the fluorescence intensityincreases with the increase of ICG concentration, reaches amaximum at 6.4 × 10-3 mM, and is followed by a gradualdecrease. The concentration at which the maximum is observedis close to that reported by Saxena et al9 (2.6 × 10-3 mM) for ICGin aqueous solution. It is clearly seen that the fluorescenceintensity is greater for liposomal ICG throughout nearly theentire concentration range studied.

The decrease in fluorescence intensity at high ICG concentra-tions, both in solution and in liposomal dispersion, was explained byquenching due to formation of aggregates of dye molecules.9 TheICG emission peak in water was observed at 805 nm atconcentrations less than 6.4 × 10−3 mM; above that there is ashift up to 814 nm. Those results correlate well with data reportedby Saxena et al.9 The dependence of fluorescence in dispersions ofliposomes with bound ICG is different: up to 3.2 × 10-3 mM ICGthe emission peak occurs at about 820 nm, whereas above thisconcentration a red shift that is dependent on ICG concentration isobserved. As presented in Figure 3, B, increasing the ICGconcentration to 9.6 × 10-2 mM causes a shift to about 30 nm.

The red shift is accompanied with quenching of fluorescence—in other words, the larger the red shift the lower thefluorescence intensity. Because the fluorescence intensity ofICG-liposomes was greater than that of unbound ICG molecules(aqueous solution), the quantum yield ratio between the twostates in which ICG is present was determined, for aconcentration of 1.28 × 10-3 mM.

The relative quantum yield was calculated according to thefollowing equation36:

/x

/s=

As

Ax

� �×

Fx

Fs

� �×

nxns

� �2

where ϕ is the quantum yield, F is the area under the emissionpeak (expressed in number of photons), A is absorbance at theexcitation wavelength, and n is the refractive index of thesolvents. The subscript x denotes the respective values of the

Figure 4. IgG binding presented as TMB (3,3′,5,5′-tetramethylbenzidine)absorbance (HRP substrate) as a function of liposome concentration. The IgGconcentration was kept constant throughout this experiment.

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sample (liposome-ICG), and s denotes standard (free ICGin buffer).

It was found that the quantum yield of liposomal ICG is morethan three times greater than that of free ICG in buffer solution.

IgG binding to ICG liposomes

Active targeting can be achieved by conjugation of targetingmolecules to liposomes, which specifically bind to an antigen orreceptor that is overexpressed on the tumor cell surface. In thepresent study we used human IgG as a model recognition ligandfor evaluating its binding to the ICG-liposomes.

As discussed by Torchilin et al,37 binding of proteins onto theliposome surface can be achieved by simple adsorption. Using thisapproach, we incubated preformed liposomes with solutions ofIgG, followed by removal of the unbound IgG by ultrafiltration.

The evaluation of IgG binding to the liposomes wasperformed by an immunoassay (ELISA). As presented inFigure 4, the immunoassay results show an increase in thehorseradish peroxidase (HRP) related signal with an increase inliposome-to-IgG ratio. Nonspecific binding to the ELISA platedid not occur in this experiment, because there was no HRPsignal in the samples that did not contain IgG.

Specific binding of cetuximab-labeled liposomes evaluated byconfocal microscopy

To evaluate whether antibody adsorbed to a liposomepreserves its specific recognition ability, we performed bindingexperiments between the cetuximab (monoclonal antibody toEGFR) adsorbed to liposomes and A431 carcinoma cellsoverexpressing EGFR (see Supplementary Information, avail-able in the online version of this article). The specificity ofcetuximab-labeled liposomes in comparison to nonspecific IgGantibody-labeled liposomes, liposomes only, and fluorescentEGF, was investigated by confocal microscopy (Figure 5). Thenonspecific labeling of the cultures by nonlabeled liposomes wasvery low, as evident in Figure 5, A, reflecting mainly theautofluorescence of the cells. The binding of IgG-labeledliposomes was eightfold greater than that of nonlabeledliposomes to the cell culture (Figure 5, C and data not shown).

Most probably, IgG-labeled liposome binding reflects non-EGFR-mediated cell association. The binding of cetuximab-labeled liposomes to the cell culture is about 10-fold greater thanthat of nonlabeled liposomes (Figure 5, D and data notshown). Interestingly, careful visualization of the micrographsat high magnification indicates that whereas IgG-labeledliposomes are mainly associated with cell plasma membrane(Figure 5, C'), Cetuximab-labeled liposomes are both associatedwith cell plasma membrane and internalized into the cells(Figure 5,D’). Quantization of liposome distribution between theplasma membrane and intracellular compartment (Figure 5, E)clearly indicates that a greater fraction of cetuximab-labeled,compared with IgG-labeled, liposomes is found in intracellularcompartments. We point out that A431 and IEC-6 cultures treatedwith these liposomes were alive for several days, indicating lackof toxicity of these preparations (data not shown).

Specific binding of cetuximab-labeled liposomes evaluated byNIR imaging of living cells

In another approach to evaluating the specific binding ofcetuximab-labeled liposomes containing ICG, we took advan-tage of a focal in vitro model in which the EGFR overexpressorA431 cells are plated in the center and normal IEC-6 colon cellssurround the tumor cells (Figure 6). Exposure of this tumorlike invitro model system to cetuximab-labeled liposomes resulted in aspecific NIR imaging signal detected with the tumor cells but notwith the surrounding normal colorectal cells (Figure 6). Thebinding of cetuximab-targeted fluorescent liposomes to A431compared with IEC-6 cells (normal enterocytes expressingphysiological EGFR levels) was larger by a factor of 3.5(Figure 6), and it was larger than nonspecific binding ofliposomes without the targeting moiety (cetuximab) by 40%,whereas IgG-labeled liposomes also caused relatively highbackground labeling. As expected, the control untreated culturesemitted very low NIR autofluorescence signal.

Discussion

Colorectal cancer is one of the most common cancers and hasa relatively poor prognosis38; early diagnosis is the best way toimprove overall survival.39 Although conventional colonoscopyis the acknowledged gold standard for detecting colonneoplasms, it is not perfect40; for example, the detection rateof carcinomas was 95%41 by endoscope and 74% by imagingcapsule.42 Obviously, with specific marking of the lesion byoptical probes we can significantly improve the detection andtherefore reduce morbidity and mortality from cancer.

The approach described here refers to preparation of amultimodal nanoprobe capable of binding specifically to cancercells and detected by an NIR fluorescence probe that is already inclinical use, ICG. Both the fluorescence probe and targetingmolecules are noncovalently bound to the liposomes.

The nanoprobe is composed of liposomeswith an average size of30 nm (Figure 1) and zeta potential of −30 mV (Figure 2, B). Itseems that ICG was passively adsorbed to the liposomes throughelectrostatic interactions, because the zeta potential decreased inabsolute value to about −15 mV (Figure 2, B). The electrostatic

Figure 5. Binding of fluorescein-containing liposomes to A431 carcinoma cell culture visualized by confocal microscopy. The cultures were incubated for 40minutes at 37°C with 0.5 mg/mL of (A) nonlabeled liposomes, (B) EGF-FITC, (C) IgG-labeled liposomes (Lipo-FITC-IgG), (C’) IgG-labeled liposomes, (D)cetuximab-labeled liposomes (Lipo-FITC-Cetuximab), and (D’) cetuximab-labeled liposomes. Scale bar, 25 μm. (E) Fluorescence intensities at cell membrane(white bars) or cytosol (black bars) were quantified and are presented in arbitrary units (a.u.). #P b 0.05 vs. membrane IgG-FITC-labeled liposomes; ⁎P b 0.05vs. cytosol IgG-FITC-labeled liposomes.

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mechanismof adsorptionwas also reported for other cyanine dyes.43

However, the hydrophobic moiety of the ICG molecule permits theICG also to be embedded fully or partially within the liposomebilayer, as suggested by Devoiselle et al.15 Liposomal ICG wasmore fluorescent than the free ICG in solution (Figure 3, A),probably as a result of the higher monomeric fraction of ICG in theliposomal form. A similar phenomenonwas reported by Sidorowiczet al, for another cyanine dye, 3,3-disulfobutyl-5,5-diphenyl-9-ethyl-oxacarbocyanine, in the presence of phosphatidylserine.44

As shown in the binding isotherm (Figure 2, A) and thedecrease in zeta potential (Figure 2, B), increasing the concentra-tion of ICG leads to a greater concentration of ICG moleculesbound to the liposome. This is expected to enable closer packingof the ICG molecules, and thus a greater probability foraggregation of the dye molecules, resulting in a red shift andquenching, which is seen in Figure 3, B. The shift in fluorescence

might be explained by the less polar environment43,44 andformation of ICG aggregates that emit at a higher wavelength.9,45

In addition, we found that liposomal ICG had an approx-imately threefold higher quantum yield than that of a simplesolution of ICG, and this finding should show a significantimprovement in detection capabilities. Similarly, an increase inquantum yield ratio was also observed in a micellar system byKirchherr et al.18 As reported for other cyanine dyes by DeRossiet al,46 the greater quantum yield can be explained by the higherorder of the molecules in the liposomes and by the more rigidstructure, which prevents free molecular motion and preferableadsorption in monomeric form.7

Another important issue is the stability of the nanoprobe inthe colon; because the ICG was bound to the liposomes by asimple adsorption process, without covalent bonds, and is stillstable, it opens a way for accelerated clinical use, because all the

Figure 6. NIR imaging of living cells: binding of ICG-containing liposomesto A431 carcinoma cell culture in the center and IEC-6 normal colon cells inthe surroundings. The cultures were incubated for 30 minutes at 37°C with0.5 ng/mL of various liposome preparations. Lipo, liposomes only; Lipo-IgG,control liposomes containing IgG as targeting moiety; Lipo-Cetux, liposomescontaining cetuximab as targeting agent. Upper panel: representative imagesof the in vitro focal colon cancer model. ⁎P b 0.05 vs. Lipo.

486 E. Portnoy et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 480–488

molecules from which the nanoprobe is made are alreadyapproved for clinical applications.

The IgG molecule was used as a model targeting agent that canbe passively adsorbed to the liposomal surface. The ELISA results(Figure 4) indicated passive absorbance of IgG molecules. Tosupport the conclusions from the ELISA measurements, and also toevaluate whether ICG-liposome binding antibodies retain their NIRfluorescence, the fluorescence of both ICG and FITC was measured(data not shown). It was found that the fluorescence of both ICG andFITC was detected in the fraction of the solution that was retainedafter filtration, indicating that both ICG and IgG are bound to theliposomes. The physical proximity of ICG and IgG as obtained inliposomes resulted in conservation of ICG fluorescence, in contrastto the disappearance of its fluorescence while it is covalently boundto IgG.47 The retention of ICG fluorescence in presence of IgG andincreasing its quantum yield in liposomes indicates the advantage ofthe binding through simple adsorption methodology.

Cetuximab adsorbed on liposomes kept its EGFR-specificrecognition ability as was shown by 10-fold higher fluorescentsignal relative to control, untargeted liposomes (Figure 5, D anddata not shown). Interestingly, that analysis revealed that a largerfraction of cetuximab-labeled liposomes was incorporated withinthe intracellular compartment (Figure 5, E) relative to IgG-labeled liposomes, which were mainly membrane associated(Figure 5, C’). This difference in the membrane-to-cytosol ratioindicates an EGFR-mediated specific process associated withcetuximab-labeled liposomes. Internalization of cetuximab-labeled liposomes has the added benefit of amplifying thefluorescent signal, because the fluorescent probe will accumulatein the tumor target cell. Therefore, adsorption of ICG andcetuximab to liposomes provides a practical approach forgenerating a tool for imaging of carcinomas overexpressingEGFR. Another approach for imaging is based on covalentattachment of ICG molecules directly to an antibody.47,48

However, the covalent attachment approach has several major

drawbacks: (i) It was found that the ICG significantly loses itsfluorescence activity as a result of the chemical binding47; (ii)The number of ICG molecules per antibody was limited to onlyfive47 or one,48 whereas liposomes may bear potentially largerICG quantities; (iii) The ICG-antibody conjugate is obtained bycovalent attachment, and therefore the resulting molecule is anew moiety that would require an additional approval processbefore it can be utilized in clinics. Our approach of physicaladsorption of ICG and cetuximab onto the liposome overcomesthese drawbacks and provides a new and simple paradigm forEGFR-overexpressing tumor imaging.

Nonspecific binding is a critical issue for noninvasive tumorimaging. Therefore, NIR imaging of living cells was used forevaluation of the specificity of the nanoprobes (Figure 6). Thesignal ratio of A431/IEC6 was 3.5 for cetuximab-labeledliposomes, whereas for unlabeled liposomes it was 40% lower,thus indicating more pronounced nonspecific binding. Cetuximab-labeled liposomes enhanced the signal-to-background ratio bydecreasing the level of the background labeling of the cells asopposed to nonspecific IgG-labeled liposomes. The variation insignal strength between the binding of cetuximab and that of IgG-labeled liposomes, measured with carcinoma cultures in vitro, hasthe potential to predict the usefulness of this approach in future invivo experiments. Indeed, liposomes conjugated with EGFRligand peptides49 or cetuximab50 were able to bind to EGFR in invivo experiments, thus further supporting the usefulness of ICG-loaded cetuximab-labeled liposomes for in vivo targeting.Liposomes hold tremendous potential as diagnostic imagingpharmaceutical tools and promise for the development ofmultimodality agents with both imaging and therapeutic capabil-ities. The creation of biocompatible liposomes will provide anadvanced enabling technology for diagnosis and therapy ofcarcinomas based on EGFR and its oncogenes.

In conclusion, the presented system may serve as a new probefor visualization of EGFR overexpression in colon carcinomas.The ICG-liposomes have improved fluorescence propertiescompared with those of ICG in aqueous solutions, are stable incolon fluids, and are capable of binding monoclonal antibodiesby noncovalent bonds. We found that the cetuximab-labeledliposomes are safe in vitro, have specific recognition ability, bindselectively to carcinoma cells overexpressing EGFR, and areinternalized by the cells. Altogether, these properties propose apromising tool for EGFR-overexpressing tumors. It is anticipa-ted that this technology can be further adopted for clinical use.

Supplementary materials related to this article can be foundonline at doi:10.1016/j.nano.2011.01.001.

Acknowledgments

The authors appreciate the technical assistance of Dr. EllinaKesselman.

References

1. Zako T, Hyodo H, Tsuji K, Tokuzen K, Kishimoto H, Ito M, et al.Development of near infrared-fluorescent nanophosphors and

487E. Portnoy et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 480–488

applications for cancer diagnosis and therapy. Journal of Nanomaterials2010;2010:1-7.

2. Chatterjee DK, Rufaihah AJ, Zhang Y. Upconversion fluorescenceimaging of cells and small animals using lanthanide doped nanocrystals.Biomaterials 2008:937-43.

3. Dzurinko VL, Gurwood AS, Price JR. Intravenous and indocyaninegreen angiography. Optometry 2004;75:743-55.

4. Sakka SG. Assessing liver function. Curr Opin Crit Care 2007;13:207-14.

5. Still JM, Law EJ, Klavuhn KG, Island TC, Holtz JZ. Diagnosis of burndepth using laser-induced indocyanine green fluorescence: a preliminaryclinical trial. Burns 2001;27:364-71.

6. Bozkulak O, Yamaci RF, Tabakoglu O, GulsoyM. Photo-toxic effects of809-nm diode laser and indocyanine green on MDA-MB231 breastcancer cells. Photodiagn Photodyn 2009;6:117-21.

7. Philip R, Penzkofer A, Baumler W, Szeimies RM, Abels C. Absorptionand fluorescence spectroscopic investigation of indocyanine green.J Photochem Photobiol A 1996;96:137-48.

8. Sevick-Muraca EM, Lopez G, Reynolds JS, Troy TL, Hutchinson CL.Fluorescence and absorption contrast mechanisms for biomedical opticalimaging using frequency-domain techniques. Photochem Photobiol1997:55-64.

9. Saxena V, Sadoqi M, Shao J. Degradation kinetics of indocyanine greenin aqueous solution. J Pharm Sci 2003;92:2090-7.

10. Desmettre T, Devoisselle JM, Mordon S. Fluorescence properties andmetabolic features of indocyanine green (ICG) as related to angiography.Surv Ophthalmol 2000;45:15-27.

11. Gathje J, Steuer RR, Nicholes KR. Stability studies on IndocyanineGreen dye. J Appl Physiol 1970;29:181-5.

12. Saxena V, Sadoqi M, Shao J. Indocyanine green-loaded biodegradablenanoparticles: preparation, physicochemical characterization and in vitrorelease. Int J Pharm 2004;278:293-301.

13. Yaseen MA, Yu J, Jung BS, Wong MS, Anvari B. Biodistribution ofencapsulated Indocyanine Green in healthy mice. Mol Pharm 2009;6:1321-32.

14. Yu J, Javier D, Yaseen MA, Nitin N, Richards-Kortum R, Anvari B,et al. Self-assembly synthesis, tumor cell targeting, and photothermalcapabilities of antibody-coated indocyanine green nanocapsules. J AmChem Soc 2010;132:1929-38.

15. Devoiselle JM, Soulie-Begue S, Mordon SR, Desmettre T, Maillols H.Fluorescence properties of indocyanine green: I. In-vitro study withmicelles and liposomes. Proc SPIE 1997;2980:530-7.

16. Proulx ST, Luciani P, Derzsi S, Rinderknecht M, Mumprecht V,Leroux JC, et al. Quantitative imaging of lymphatic function withliposomal indocyanine green. Cancer Res 2010;70:7053-62.

17. Sandanaraj BS, Gremlich HU, Kneuer R, Dawson J, Wacha S.Fluorescent nanoprobes as a biomarker for increased vascular perme-ability: implications in diagnosis and treatment of cancer andinflammation. Bioconjug Chem 2010;21:93-101.

18. Kirchherr AK, Briel A, Mader K. Stabilization of Indocyanine Greenby encapsulation within micellar systems. Mol Pharm 2009;6:480-91.

19. Barenholz Y, Crommelin DJA. Liposomes as a pharmaceutical dosageforms. In: Swarbrick B, editor. Encyclopedia of pharmaceuticaltechnology, Vol. 9. New York: Marcel Dekker; 1994. p. 1.

20. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CACancer J Clin 2005;55:74-108.

21. Duffy MJ, van Dalen A, Haglund C, Hansson L, Holinski-Feder E,Klapdor R, et al. Tumour markers in colorectal cancer: European Groupon Tumour Markers (EGTM) guidelines for clinical use. Eur J Cancer2007;43:1348-60.

22. Hubbard SR, Till JH. Protein tyrosine kinase structure and function.Annu Rev Biochem 2000;69:373-98.

23. Bouche O, Beretta GD, Alfonso PG, Geissler M. The role of anti-epidermal growth factor receptor monoclonal antibody monotherapy inthe treatment of metastatic colorectal cancer. Cancer Treat Rev 2010;36(Suppl 1):S1-10.

24. Becker JC, Muller-Tidow C, Serve H, Domschke W, Pohle T. Role ofreceptor tyrosine kinases in gastric cancer: new targets for a selectivetherapy. World J Gastroenterol 2006;12:3297-305.

25. Hu M, Scollard D, Chan C, Chen P, Vallis K, Reilly RM. Effect of theEGFR density of breast cancer cells on nuclear importation, in vitrocytotoxicity, and tumor and normal-tissue uptake of [111In]DTPA-hEGF.Nucl Med Biol 2007;34:887-96.

26. Koyama Y, Barrett T, Hama Y, Ravizzini G, Choyke PL, Kobayashi H.In vivo molecular imaging to diagnose and subtype tumors throughreceptor-targeted optically labeled monoclonal antibodies. Neoplasia2007;9:1021-9.

27. Kriete A, Papazoglou E, Edrissi B, Pais H, Pourrezaei K. Automatedquantification of quantum-dot-labelled epidermal growth factor receptorinternalization via multiscale image segmentation. J Microsc 2006;222:22-7.

28. Mamot C, Ritschard R, Kung W, Park JW, Herrmann R, RochlitzCF. EGFR-targeted immunoliposomes derived from the monoclonalantibody EMD72000 mediate specific and efficient drug deliveryto a variety of colorectal cancer cells. J Drug Target 2006;14:215-23.

29. Tada H, Higuchi H, Wanatabe TM, Ohuchi N. In vivo real-time trackingof single quantum dots conjugated with monoclonal anti-HER2 antibodyin tumors of mice. Cancer Res 2007;67:1138-44.

30. Gong H, Kovar J, Little G, Chen H, Olive DM. In vivo imaging ofxenograft tumors using an epidermal growth factor receptor-specificaffibody molecule labeled with a near-infrared fluorophore. Neoplasia2010;12:139-49.

31. Bellare JR, Davis HT, Scriven LE, Talmon Y. Controlled environ-ment vitrification system: an improved sample preparation technique.J Electron Microsc Tech 1988;10:87-111.

32. Danino D, Bernheim-Grosswasser A, Talmon Y. Digital cryogenictransmission electron microscopy: an advanced tool for direct imaging ofcomplex fluids. Colloid Surf A 2001;183:113-22.

33. Haigler H, Ash JF, Singer SJ, Cohen S. Visualization by fluorescenceof the binding and internalization of epidermal growth factor inhuman carcinoma cells A-431. Proc Natl Acad Sci U S A 1978;75:3317-21.

34. Jones MN. Liposomal antibacterial, antifungal and antiviral agents. In:Nejan D, editor. Methods in enzymology, Vol. 391. San Diego: Elsevier-Academic Press; 2005. p. 218.

35. Torre LG, Carneiro AL, Rosada RS, Silva CL, Santana MHA. Amathematical model describing the kinetic of cationic liposomeproduction from dried lipid films adsorbed in a multitubular system.Braz J Chem Eng 2007;24:477-86.

36. Fery-Forgues S, Lavabre D. Are fluorescence quantum yields so tricky tomeasure? A demonstration using familiar stationery products. J ChemEduc 1999;76:1260-4.

37. Torchilin VP, Klibanov AL. Immobilization of proteins on liposomesurface. Enzyme Microb Tech 1981;3:297-304.

38. Murray GI, Duncan ME, O'Neil P, Melvin WT, Fothergill JE. Matrixmetalloproteinase-1 is associated with poor prognosis in colorectalcancer. Nat Med 1996;2:461-2.

39. Lowenfels AB. Multimodality approaches to diagnosis and managementof colorectal cancer. [cited 2005, May 27]; [about 1 p.]. Available from:http://cme.medscape.com/viewarticle/505554.

40. Ferrucci JT. Virtual colonoscopy for colon cancer screening: furtherreflections on polyps and politics. Am J Roentgenol 2003;181:795-7.

41. Rex DK, Rahmani EY, Haseman JH, Lemmel GOT, Kaster S, BuckleyJS. Relative sensitivity of colonoscopy and barium enema for detectionof colorectal cancer in clinical practice. Gastroenterology 1997;112:17-23.

42. Van Gossum A, Munoz-Navas M, Fernandez-Urien I, Carretero C,Gay G, Delvaux M, et al. Capsule endoscopy versus colonoscopyfor the detection of polyps and cancer. N Engl J Med 2009;361:264-70.

488 E. Portnoy et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 480–488

43. Sidorowicz A, Pola A, Dobryszycki P. Spectral properties of 3,3′-diethyloxadicarbocyanine included in phospholipid liposomes. J Photo-chem Photobiol B 1997;38:94-7.

44. Sidorowicz A, Mora C, Jblonka S, Pola A, Modrzycka T,Mosiądz D, et al.Spectral properties of two betaine-type cyanine dyes in surfactant micellesand in the presence of phospholipids. J Mol Struct 2005;744:711-6.

45. Minjoong Yoon YC. Dongho Kim. Absorption and fluorescencespectroscopic studies on dimerization of chloroaluminum(III) in aqueousalcoholic solutions. Photochem Photobiol 1993;58:31-6.

46. DeRossi U, Daehne S, LindrumM. Increased coupling size in J-aggregatesthrough N-n-alkyl betaine surfactants. Langmuir 1996;12:1159-65.

47. Ogawa M, Kosaka N, Choyke PL, Kobayashi H. In vivo molecularimaging of cancer with a quenching near-infrared fluorescent probe

using conjugates of monoclonal antibodies and Indocyanine Green.Cancer Res 2009;69:1268-72.

48. Withrow KP, Gleysteen JP, Safavy A, Skipper J, Desmond RA, Zinn K,et al. Assessment of indocyanine green-labeled Cetuximab to detectxenografted head and neck cancer cell lines. Otolaryngol Head Neck2007;137:729-34.

49. Song S, Liu D, Peng J, Deng H, Guo Y, Xi LX, et al. Novel peptideligand directs liposomes toward EGF-R high-expressing cancer cells invitro and in vivo. FASEB J 2009;23:1396-404.

50. Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, et al.Epidermal growth factor receptor-targeted immunoliposomes signifi-cantly enhance the efficacy of multiple anticancer drugs in vivo. CancerRes 2005;65:11631-8.