Nanographene oxide-based radioimmunoconstructs for in vivo targeting and SPECT imaging of HER2-positive tumors

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Biomaterials xxx (2012) 1e9

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Biomaterials

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Nanographene oxide-based radioimmunoconstructs for in vivo targeting andSPECT imaging of HER2-positive tumors

Bart Cornelissen a, Sarah Able a, Veerle Kersemans a, Philip A. Waghorn a, Sverre Myhra b,Kerstin Jurkshat b, Alison Crossley b, Katherine A. Vallis a,*

aCR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford OX3 7LE, UKbDepartment of Materials, Oxford University, Begbroke Science Park, Oxford OX5 1PF, UK

a r t i c l e i n f o

Article history:Received 1 October 2012Accepted 22 October 2012Available online xxx

Keywords:Nanographene oxide111IndiumMolecular imagingTrastuzumabSPECT

* Corresponding author. Tel.: þ44 (0)1865 255850;E-mail address: [email protected]

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.10.054

Please cite this article in press as: Cornelissimaging of HER2-positive tumors, Biomater

a b s t r a c t

Nanographene oxide (NGO) is a novel nano-wall material that tracks to tumors in vivo, and which, asa consequence of its large surface area, has the capacity to carry a large payload. This study explores theuse of anti-HER2 antibody (trastuzumab)-conjugated NGO, radiolabeled with 111In-benzyl-diethylene-triaminepentaacetic acid (BnDTPA) via pp-stacking, for functional imaging. In two HER2-overexpressingmurine models of human breast cancer, high tumor-to-muscle ratio was achieved, resulting in clearvisualization of tumor using single-photon emission computed tomography (SPECT). In the BALB/neuTmodel and in BALB/c nu/nu mice bearing 231/H2N xenografts, tumor accumulation amounted to12.7 � 0.67 and 15.0 � 3.7% of the injected dose/g (%ID/g) of tumor tissue at 72 h, with tumor-to-muscleratios of 35:1 and 7:1, respectively. Radiolabeled NGO-trastuzumab conjugates demonstrated superiorpharmacokinetics compared to radiolabeled trastuzumab without NGO, with more rapid clearance fromthe circulation. The use of NGO as a scaffold to build radiolabeled nano-immunoconstructs holds promisefor molecular imaging of tumors.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Graphene, a single sheet of sp2-bonded carbon atoms, hasstimulated much interest in the past few years as it has uniqueelectrical, structural and chemical properties [1]. One of the mainadvantages of graphene for life science applications is its largesurface area (2630 m2/g), conferring the ability to adsorb or bindlarge amounts of cargo material [2]. Nanographene, nanometer tomicrometer-sized particles of graphene, is insoluble in water andthis limits its use for in vivo applications. However, its oxidizedform, nanographene oxide (NGO), is water soluble and is easy tosynthesize and functionalize. Its large surface area, together withthe observation that NGO tracks to tumors through the enhancedpermeability and retention (EPR) effect, renders it an attractivedrug delivery system in oncology [3]. NGO has been investigated asa carrier of drugs [4e7], photosensitizers [8], aptamers [9], plasmidDNA [7], iron oxide nanoparticles for cellular MRI [10], as a scaffoldfor photothermal therapy [11,12] and, in two recent reports, asa probe for positron emission tomography (PET) imaging [13,14].

fax: þ44 (0)1865 857533.(K. A. Vallis).

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en B, et al., Nanographene oials (2012), http://dx.doi.org/

Yang et al. used 6-arm PEG-amine conjugated NGO as a photo-sensitizer for photothermal antitumor therapy and used 125I-labeling to track its biodistribution for up to 60 days in mice [11,15].PEGylated NGOdid not cause hematological or biochemical adverseeffects [15]. Zhang et al. described the use of PEG-amine conjugatedNGO nanoparticles for efficient delivery of doxorubicin to tumors,resulting in enhanced cytotoxicity [4]. Dong et al. showed how zincphthalocyanine, pp-stacked onto PEGylated graphene oxide (GO),could be used for photodynamic therapy [8] and Kim et al. usedbranched polyethyleneimine conjugated NGO for introduction ofa plasmid containing luciferase DNA into HeLa and PC-3 cells [16].Recently, Hong et al. reported the use of 64Cu- or 66Ga-labeledPEGylated NGO constructs conjugated to anti-CD105 antibody forPET imaging of tumor vasculature [13,14].

HER2 receptor overexpression occurs in a large sub-group ofbreast cancers and is associated with tumor aggressiveness andmetastatic potential [17]. Molecular imaging of HER2 receptors hasbeen explored extensively [18]. Imaging probes consisting ofa radiolabeled HER2 antibody, trastuzumab, have been tested.However, a disadvantage of using trastuzumab is its long half-life inthe circulation resulting in low tumor-to-background tissue ratios.Herein, we report a strategy for radiolabeling NGO via pp-stackingof the metal ion chelator, 2-(4-aminobenzyl)-diethylene-triaminepentaacetic acid (peNH2eBnDTPA), and subsequent

xide-based radioimmunoconstructs for in vivo targeting and SPECT10.1016/j.biomaterials.2012.10.054

B. Cornelissen et al. / Biomaterials xxx (2012) 1e92

incorporation of indium-111 (111In) for single-photon emissioncomputed tomographic (SPECT) imaging. We have tested thehypothesis that 111In-BnDTPA-NGO-immunoconjugates designedto bind specifically to the HER2 receptor through the incorporationof trastuzumab, could provide favorable pharmacokinetics and beused for functional imaging of HER2-overexpressing tumors.

2. Materials and methods

2.1. Cell lines

The human breast cancer cell line, MDA-MB-231, was acquired from Americantype culture collection (ATCC). MDA-MB-231 breast carcinoma cells, stably trans-fected with the HER2 gene (231/H2N), were a gift from Dr. R. Kerbel (SunnybrookHealth Sciences Centre, Toronto, ON) [19]. Cells were cultured in 5% CO2 usingDMEM cell culture medium (SigmaeAldrich, Dorset, UK) supplemented with 10%fetal calf serum (FCS; Invitrogen, Paisley, UK), and penicillin/streptomycin,100 units/mL (Invitrogen, Paisley, UK). Cells were authenticated by the supplier and used fora maximum of 20 weeks after recovery from liquid nitrogen storage.

2.2. NGO production and quality control

GO was produced from graphite using a modified Hummers method [20].Briefly, 0.25 g of synthetic graphite powder (99.9995% pure, metal basis, conductinggrade, 325 mesh; Alpha Aesar, Ward Hill, MA, USA) was added to a round-bottomflask containing a 30:3.3 mL mixture of H2PO4(co.ac):H3PO4(50%[w/v]) and stir-red. During continuous stirring on ice, KMnO4 (1.5 g) was gradually added. Care wastaken not to increase the temperature of the mixture above 20 �C. The mixturechanged from pink to green as a result of Mn2O7 production. After refluxing over-night at 50 �C themixturewas brown. It was then cooled to room temperature. Afteraddition of 3:0.25 mL H2O:H2O2 (35%w/v H2O2) themixture became yellow andwasstirred for 30 min. Graphite oxide was purified by repeated centrifugation(15,000 � G, 10 min) and washed with water (50 mL). After vacuum-assisted drying,this method yielded 0.35 g of GO. Before analysis, GO was sonicated, washed withNaOH to remove oxidative debris [21], filtered through a 0.2 mm filter, and washedrepeatedly to remove large particles. Dispersal and stability of filtered GO wasconfirmed by storage at 4 �C [22]. Under these conditions GO was stable in solutionfor at least 3 months. Fourier transform infrared spectroscopy (FTIR) was used toconfirm the identity of GO. NGO was obtained by exfoliation through sonication ofGO in water, followed by filtration through a 0.2 mm filter. UV/Vis spectra of NGOwere acquired in water using a nanodrop spectrophotometer (Thermo Scientific,Wilmington, DE). NGOwas deposited on glass cover slips and fluorescence emissionspectrawere acquired under various excitationwavelengths using the lambda modeof a Zeiss 710 fluorescence microscope equipped with a p-mode detector (Zeiss,Germany). NGO in ethanol was deposited on a siliconwafer and knockingmode AFMused to determine particle size. The hydrodynamic radius of NGO was measuredusing dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd,Malvern, UK).

2.3. Radiolabeling of NGO with 111In using pp-stacking

For 111In-labeling of NGO, peNH2eBnDTPA (Macrocyclics, Dallas, TX, USA) (5 mg)was added to filtered NGO (35 mg), dissolved in H2O (50 mL), sonicated for 1 h, andthen purified by SEC using a polyacrylamide P4 mini-column and eluted withsodium citrate (0.1 M, pH 5.0). Fractions containing BnDTPA-pp-NGO were selectedby UV/Vis absorbance. BnDTPA-pp-NGO was incubated with indium chloride(111InCl3; Perkin Elmer, Boston, MA, USA) for 1 h at room temperature, yielding 111In-BnDTPA-pp-NGO (referred to as 111In-NGO). Quality control was performed by SECusing a P4 mini-column. The effect of increasing the amount of peNH2eBnDTPA (5e160 mg) and 111In on the yield of the reaction was evaluated. Stability of the 111In-labeling was determined by incubation of 111In-NGO for 0, 4 or 24 h in cell culturemedium containing FCS, and analysis using G50 or P4 size exclusion chromatog-raphy to test for transchelation of 111In to large serum proteins or the formation ofsmaller 111In-labeled fragments, respectively.

2.4. Synthesis and characterization of NGO-immunoconjugates

NGO-immunoconjugates were synthesized using a zero-length linker. NGO-sNHS (1 mg) was incubated with trastuzumab (Tz; 100 mg) or, as a control probe,immunoglobulin from rabbit serum (rIgG), to yield NGO-Tz and NGO-IgG, respec-tively. Purification was by SEC using a G50 mini-column. The efficiency of NGOconjugation to Tz was determined using 123I-NGO. 123I-NGO itself was synthesizedusing the Iodogen method [23]. peNH2eBnDTPA was pp-stacked onto NGO-Tz orNGO-IgG by sonication of peNH2eBnDTPA (5 mg) with NGO-Tz or NGO-IgG in PBSfor 1 h. 111In-labeling was achieved by incubation with 111InCl3 for 1 h at roomtemperature, resulting in 111In-BnDTPA-pp-NGO-Tz and 111In-BnDTPA-pp-NGO-IgG(hereafter referred to as 111In-NGO-Tz and 111In-NGO-IgG respectively). QC was

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performed using a G50 mini-column for SEC. 111In-labeled Tz (111In-Tz) wassynthesized using peSCNeBnDTPA as a chelator, as previously described [24].

2.5. Binding assays to confirm retention of affinity of NGO constructs for HER2receptor

To confirm whether the attachment of NGO to Tz altered affinity of Tz for theHER2 receptor, competition binding assays were carried out. Aliquots of 2� 105 231/H2N cells were seeded in 24-well plates and allowed to adhere overnight. Tz(100 mg) was radioiodinated using the Iodogen method [23], to yield 123I-Tz, andpurified by SEC using a G50 mini-column. Cells were exposed to increasing amountsof cold, unlabeled Tz or BnDTPA-NGO-Tz plus 123I-Tz (1 nM). Cells were incubated at4 �C for 1 h, washed with PBS, lysed using 0.1 M NaOH and counted for radioactivityusing an automated gamma counter (Wizard gamma counter, Perkin Elmer, Wal-tham, MA, USA). Concentration response curves were generated and IC50 valuescalculated. In similar experiments, 231/H2N cells (2 � 105) were exposed to 111In-NGO-Tz or 111In-NGO-IgG (20 nM, 200 mL) for 1 h at 4 �C, with or without the additionof a 100-fold excess of unmodified Tz. Cells were washed with PBS, and solubilizedusing NaOH as described above, and counted for radioactivity.

2.6. Mouse models and SPECT/CT imaging

All animal procedures were carried out in accordance with the UK Animals(Scientific Procedures) Act 1986 and with local ethical committee approval. Bio-distribution and SPECT/CT imaging studies were performed in two mouse models:spontaneous tumors in female BALB/neuT mice and BALB/c nu/numice (Harlan, UK)bearing 231/H2N xenografts. BALB/neuT mice are a genetically engineered strain inwhich the NeuT-gene is overexpressed and under regulation of an MMTV promotor,resulting in the development of trastuzumab-avid tumors in mammary fat padswhen mice are approximately 120 days old [25]. The presence of Tz-avid NeuTreceptors in spontaneously developing tumors in BALB/neuT mice was confirmedusing immunohistochemistry. Tz was Cy3-conjugated using a Cy3-succinimide ester(Amersham Health, Amersham, UK). Frozen sections (10 mm-thick) from BALB/neuTtumors were fixed (4% formaldehyde, 10 min, room temperature), blocked (1%bovine serum albumin in PBS, 1 h, 37 �C), stained with Cy3-Tz (1 mg/mL, 1 h, 37 �C),and mounted with Vectashield mounting medium containing DAPI to stain nuclei.231/H2N xenografts were established in female BALB/c nu/nu mice (Harlan, UK) bysubcutaneous (s.c.) injection in the right flank of 5 �106 cells in DMEM:matrigel 1:1(matrigel, BD, Oxford, UK). Imaging was performed when xenografts or spontaneoustumors reached a volume of approximately 500 mL. For the 231/H2N xenograftmodel, groups of 3 mice received 10 mg (5 MBq) of 111In-Tz, 111In-NGO-Tz or 111In-NGO-IgG by intravenous (i.v.) injection. In some cases, a 100-fold excess of cold,unlabeled Tz was co-injected to block the HER2 receptor. SPECT/CT images wereacquired at 1, 24, 48 and 72 h post-injection (p.i). For SPECT/CT imaging, BALB/neuTmice received 10 mg (5 MBq) of 111In-Tz, 111In-NGO-Tz or 111In-NGO-IgG i.v. andimages were acquired at 1, 24, 48 and 72 h post injection (p.i.). Volume renderedimages of SPECT/CT images were generated using segmented images producedusing ITK-snap [26].

2.7. Statistical analysis

All curve fitting and statistical analyses were performed using Graphpad Prism(Graphpad software Inc, LaJolla, CA, USA). 1-way or 2-way ANOVA was used formultiple comparisons. Tukey post-tests were used after 1-way ANOVA. F-tests wereused to compare fitted curves.

3. Results

3.1. Preparation of NGO

GO nanoparticles were synthesized by a modified Hummersmethod, using a prolonged oxidation step to obtain small GOfragments [6,27]. Color and solubility of NGO before and after base-washing with NaOH, and FTIR spectra were consistent withprevious reports (Fig. 1A, B) [6]. FTIR also confirmed oxidation ofgraphene (Fig. 1B) [6]. AFM analysis showed that particles had anaverage diameter of 64 nm and heights of 6, 12 or 18 nm, equivalentto 1, 2 and 3 layers of NGO, indicating incomplete exfoliation(Fig. 1C,D). DLS corroborated these results (Fig. 1E). UV/Vis andfluorescence spectrophotometry, resulted in spectra similar tothose previously reported (Fig. 1F,G) [6].

xide-based radioimmunoconstructs for in vivo targeting and SPECT10.1016/j.biomaterials.2012.10.054

Fig. 1. Quality control of NGO produced using a modified Hummers method. (A) Photograph of GO starting material before and after base wash with NaOH (10 M). (B) FTIR of driedNGO. (C) AFM image of filtered NGO. Inset: detailed image of one NGO flake. (D) Quantification of size (surface area) and height of dried, filtered NGO, by AFM image analysis. (E)Hydrodynamic radii of unfiltered NGO, NGO after filtration, IgG, Tz, and NGO-Tz, obtained by dynamic light scattering. (F) UV/Vis spectra of NGO inwater. (G) Fluorescence spectra ofgraphite and NGO.

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B. Cornelissen et al. / Biomaterials xxx (2012) 1e94

3.2. Synthesis and characterization of radiolabeled NGO constructs

An overview of the synthetic strategy is shown in Fig. 2.Following sonication for 1 h the metal ion chelator, 2-(4-aminobenzyl)-diethylenetriaminepentaacetic acid (peNH2e

BnDTPA), was adsorbed onto the surface of NGO particles, by pp-stacking. Addition of 111InCl3 resulted in the formation of 111In-NGOwith a radiolabeling yield of >99%, as demonstrated by SEC usinga P4 mini-column (Fig. 3A). At least 30 MBq 111In associated withNGO via 20 mg peNH2eBnDTPA (Fig. 3A). When BnDTPA wasomitted, <2% of 111In transferred onto NGO suggesting that there isno direct interaction between In3þ and the NGO. At least 160 mg peNH2eBnDTPA could be stacked onto 35 mg of NGO without reduc-tion of 111In-labeling yield (Fig. 3B). To verify the stability of 111In-NGO, SEC with both P4 and G50 mini-columns was used. Theproduct was stable in serum at 37 �C for up to 24 hwith less than 5%transchelation to serum proteins as shown by G50 SEC, and nodiscernible smaller fragments as shown by P4 SEC (Fig. 3C,D). Incontrast, similar studies with 111InCl3 show immediate trans-chelation to large proteins (Fig. 3E). Conjugation to trastuzumab, ornon-specific rIgGs was performed using EDC/sNHS activation ofNGO, yielding NGO-Tz and NGO-IgG, respectively (Fig. 2). Theconjugation efficiency of NGO to Tz was approximately 86%, asdetermined using 123I-NGO. peNH2eBnDTPA was adsorbed ontothe surface of NGO-IgG particles by pp-stacking. Addition of111InCl3 resulted in the formation of 111In-NGO-Tz or 111In-NGO-IgGwith good radiochemical yield (>95%) as shown by SEC using a G50mini-column (Fig. 3F). The stability of 111In-NGO-Tz over 24 hincubation in cell growth medium containing FCS, as measured byG50 SEC chromatography, was comparable to that of 111In-NGO(data not shown). To investigate whether Tz retains affinity forHER2 when linked to NGO, the ability of BnDTPA-NGO-Tz todisplace 123I-Tz from HER2 receptors in 231/H2N cells wasmeasured. The IC50 for BnDTPA-NGO-Tz was 18-fold greater thanthat of unmodified Tz (37.8 vs. 2.5 nM, respectively) (Fig. 3G). Fromthese results, the affinity of BnDTPA-NGO-Tz (expressed as the

Fig. 2. Synthesis of 111In-NGO-IgG or 111In-NGO-Tz (Note: th

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dissociation constant, kD) for the HER2 receptor was calculated tobe 23 nM. An excess of unmodified Tz was found to reduce bindingof 111In-NGO-Tz to 231/H2N cells by 24% (P < 0.01) (Fig. 3H). Todetermine the influence of HER2 receptor expression level onuptake, the HER2-negative cell line, MDA-MB-231, was used. Theabsence of HER2 receptors in MDA-MB-231 resulted in moderatereduction of cellular uptake of 111In-NGO-Tz compared to 231/H2N(35% reduction [P < 0.001]).

3.3. SPECT imaging using 111In-labeled NGO-IgG constructs

Two trastuzumab-avid tumor models (BALB/c nu/nu micebearing 231/H2N xenografts and spontaneous tumors in BALB/neuTmice) were used for SPECT imaging with 111In-labeled NGO-IgGconstructs (Figs. 4 and 5). Tumor accumulation of 111In-NGO-Tzwas visualized clearly using SPECT imaging in 231/H2N xenografts(Fig. 4, Fig. S1). Tumor uptake of 111In-NGO-Tz at 72 h p.i. was15.0 � 3.7% injected dose/g (%ID/g) of tumor tissue. Tumor accu-mulation of 111In-NGO-Tz was slower than that of 111In-Tz (at 24 hp.i. uptake was 9.1 � 2.3 %ID/g vs. 16.9 � 3.7 %ID/g, respectively[P ¼ 0.026]), but at 72 h p.i. uptake of both compounds wascomparable (15.0 � 3.7 vs. 18.5 � 4.3 %ID/g, respectively [P > 0.05])(Fig. S1A). The slightly lower tumor uptake of 111In-NGO-Tz wasoffset by lower non-target organ uptake compared to 111In-Tz. Forexample, muscle uptake at 48 h was 1.97 � 0.7 and 4.13 � 0.5 %ID/gfor 111In-NGO-Tz and 111In-Tz, respectively (P ¼ 0.0018) resulting intumor:muscle ratios of 7.15 vs. 3.92 at 48 h, respectively (Fig. 4B).Tumor uptake of the non-specific control radioimmunoconstruct(111In-NGO-IgG) was significantly lower than that of 111In-NGO-Tz(at 72 h p.i.: 0.11�0.04 vs.15.0� 3.7 %ID/g). Also, when an excess ofunmodified Tz was co-injected with 111In-NGO-Tz to block HER2receptors, tumor uptake was significantly reduced (at 72 h p.i.uptake was 1.45 � 0.04 vs. 15.0 � 3.7 %ID/g), an indication thattumor uptake of 111In-NGO-Tz is Tz- and HER2-mediated. Theexpression of NeuT in tumors that developed in BALB/neuT micewas confirmed using immunohistochemistry (Fig. S2A). Marked

e graphene oxide sheets extend further than depicted).

xide-based radioimmunoconstructs for in vivo targeting and SPECT10.1016/j.biomaterials.2012.10.054

Fig. 3. (A) Labeling of NGO with 111In, returned good radiolabeling yields, without evidence of saturation over the specific activity range tested. (B) Increasing amounts of BnDTPAwere pp-stacked onto a fixed amount of NGO. The resulting BnDTPA-NGO was then radiolabeled with a fixed amount of 111In, and the radiolabeling yield was determined using SECwith P4 mini-columns. (C) G50 SEC of 111In-NGO after 0, 4, or 24 h after incubation in cell culture medium containing fetal calf serum. (D) As in C, but using 111InCl3 (E) as in C, butusing P4 SEC (F) G50 SEC of 111In-NGO-Tz. (G) Increasing amounts of Tz or BnDTPA-NGO-Tz were incubated with 231/H2N cells and 1 nM

123I-Tz. Results are expressed as themean � SD of three independent experiments. (H) HER2-negative MDA-MB-231 cells, or HER2-positive 231/H2N cells were exposed for 1 h at 4 �C to 1 nM

111In-NGO-Tz, with orwithout a 100-fold excess of unmodified Tz and the amount of 111In associated with cells then measured. Results are expressed as the mean � SD of three independent experiments,*indicates significant differences (P < 0.05).

B. Cornelissen et al. / Biomaterials xxx (2012) 1e9 5

tumor uptake of 111In-NGO-Tz was observed in NeuT-positivespontaneous tumors arising in BALB/neuT mice (Fig. 5, Fig. S2).Tumor accumulation amounted to 15.7 � 0.67 %ID/g, with tumor-to-muscle ratio of 39:1 at 24 h p.i. Specificity of tumor accumula-tion of 111In-NGO-Tz was demonstrated through comparison to thenon-specific control probe, 111In-NGO-IgG, which resulted ina significantly lower tumor-to-muscle ratio (7:1 vs. 39:1 at 24 h p.i;P < 0.05). In the BALB/neuT model, tumor uptake of 111In-NGO-Tzwas similar to 111In-Tz at 24 h (15.7 � 0.67 vs 11.0 � 1.7 %ID/g,[P> 0.05]), but tumor-to-muscle ratio was much higher i.e. 7.8-foldhigher (P < 0.01) (Fig. 5B, Fig. S2).

4. Discussion

NGO has been used for a range of biomedical applications. Thecharacteristics of NGO that render it suitable as a scaffold formolecular imaging probe construction are its large surfacearea which can be loaded via pp-stacking, its long edge lengthwhich allows covalent conjugation and its versatile chemistry[28,29]. These features can be exploited for radiopharmaceutical

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development. The large surface area of NGO makes radiolabelingto high specific activity possible and functionalization withtumor targeting moieties can be readily achieved [13,14]. Multi-functionalization for optical, photoacoustic and magnetic reso-nance imaging is also possible, as shown by Yang et al. [30]. The useof GO-based agents for molecular imaging was recently reviewedby Zhang et al. [31]. Here we report the synthesis, in vivo bio-distribution and tumor uptake of an 111In-labeled NGO constructthat was conjugated to the anti-HER2 antibody, trastuzumab, forSPECT imaging. The probe design differs from that reported byHong et al. who used PEG as a spacer between NGO and the anti-CD105 antibody, TRC105 [13,14]. In addition, radiolabeling wasachieved via pp-stacking of peNH2eBnDTPA onto the surface ofNGO rather than via covalent chemistry or via a PEG linker as re-ported previously by others [13e15].

To test the feasibility of the selected synthetic route, we firstsynthesized 111In-labeled NGO, through pp-stacking of the radio-metal chelator, peNH2eBnDTPA, onto NGO sheets. In the absenceof peNH2eBnDTPA, 111In itself was not adsorbed onto NGO, elimi-nating the possibility that 111In3þ interacts directly with graphene,

xide-based radioimmunoconstructs for in vivo targeting and SPECT10.1016/j.biomaterials.2012.10.054

Fig. 4. (A) Representative whole-body SPECT images (coronal maximum intensity projections) of the biodistribution of 111In-NGO-Tz, 111In-NGO-IgG, or 111In-Tz in 231/H2Nxenograft-bearing BALB/c nu/nu mice, with or without an excess of unlabeled Tz at 1, 24, 48, or 72 h p.i. The location of the tumor is indicated by the dashed white circle. (B)Quantification of the biodistribution of the mice in (A). Results are expressed as the mean � SD (n � 3).

B. Cornelissen et al. / Biomaterials xxx (2012) 1e96

as has been reported for 188Re [32]. There was no evidence ofsignificant transchelation from 111In-NGO to serum proteins or ofdegradation, indicating that NGO labeled with 111In-BnDTPAthrough pp-stacking/charge interaction is very stable. It is possible

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that the DTPA-moiety contributes to the charge interaction of 111In-NGO with the graphene oxide sheet.

The facile radiolabeling and stability of pp-stacking of BnDTPAonto NGO sheets allowed successful 111In-labeling of NGO-Tz

xide-based radioimmunoconstructs for in vivo targeting and SPECT10.1016/j.biomaterials.2012.10.054

Fig. 5. (A) Representative whole-body SPECT images (MIPs) of the biodistribution of 111In-NGO-Tz, 111In-NGO-IgG, or 111In-Tz in spontaneous tumor-bearing BALB/neuT mice at 1, 24,48, or 72 h p.i. Tumors are located inmammary fat pads, withmetastases in the axial and inguinal lymph nodes (white dashed circle in 72 h images). Tumor burden is likely different ineach animal due to the nature of the spontaneous tumor model. (B) Quantification of the biodistribution of the mice in (A). Results are expressed as the mean � SD (n � 3).

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conjugates, which was achieved with radiolabeling efficiencies of>95%. Large amounts of BnDTPA and 111Inwere adsorbed onto NGOand NGO-Tz without difficulty. Radiolabeling Tz to high specificactivity in this way would be expected to increase its ability todetect small tumors with enhanced sensitivity. Further, the samesynthetic method could be used to efficiently radiolabel any anti-body or protein for in vivo imaging. In both a xenograft anda spontaneous breast cancer model, tumor accumulation of 111In-NGO-Tz was shown to be Tz-mediated and HER2-selective. Theefficiency of HER2-targeting was comparable to that reported for111In- or 89Zr-labeled trastuzumab in HER2-overexpressing tumorxenografts [33,34]. In the experiments reported here, 111In-NGO-Tzwas superior to 111In-Tz as it resulted in higher tumor-to-

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background ratios and these were achieved rapidly as a result offast plasma excretion (Fig. 5).

The mechanism of the rapid excretion of the NGO-Tz constructscompared to Tz remains unclear. One possible explanation is thatNGO acts as a cell-penetrating peptide (CPP)-like entity, withcharge interactions between NGO and cell membranes promotingextravasation [35]. Alternatively, it is possible that NGO-basedconstructs are opsonized, triggered perhaps by adsorption ofplasma proteins on their surface, leading to macrophage clearance[36]. The involvement of the reticuloendothelial system in theclearance of NGO was demonstrated previously when dextran-functionalized NGO was observed to accumulate in the liverand spleen [37]. In the experiments reported here, the uptake of

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B. Cornelissen et al. / Biomaterials xxx (2012) 1e98

111In-NGO-Tz in the liver of BALB/neuTmice was higher than that of111In-Tz (39 vs. 22 %ID/g [P< 0.05]; Fig. S2B), however no significantuptake of either was observed in the spleen. In BALB/c nu/nu mice,splenic uptake of 111In-NGO-Tz and 111In-NGO-IgG was lower thanthat of 111In-Tz, and liver uptake was similar for all three (Fig. S1A).Thus, there was no clear evidence that removal via the reticulo-endothelial system is a significant determinant of the rapid phar-macokinetics of 111In-NGO-Tz.

The results presented here suggest a synergy between NGO-and Tz-mediated tumor uptake, as the uptake of 111In-NGO-Tz inHER2-positive tumors is greater than that of 111In-Tz (Fig. 5). Wehypothesize that NGO-Tz immunoconjugates are able to targettumor through the action of both Tz and NGO. The contribution totumor uptake of Tz binding to HER2 seems to be much greaterin vivo than in vitro, as indicated by the difference in resultsobtained when HER2 receptors were blocked with an excess of Tz(33% reduction of binding in vitro, but almost 90% in vivo)(Figs. 3H and 4). This may be explained by differences in thetumor cell microenvironment in 2D culture compared to in vivo.Furthermore, tumor uptake was almost completely absent whenthe non-specific control construct, 111In-NGO-IgG, was used. Theseobservations indicate that tumor uptake of 111In-NGO-Tz is notpredominantly due to the EPR effect. At the most, EPR plus any asyet undetermined mechanism, accounts for 10% of tumor uptake,the remainder being mediated by the interaction between Tz andthe HER2 receptor.

NGO-based immunoconstructs combine the excellent targetspecificity and affinity of antibodies with the rapid pharmacoki-netics and large payload carrying capacity of NGO. Furthermore, thestraightforward synthesis of NGO-based particles means that theycan be directed against different molecular targets by simpleintegration of an appropriate IgG in construct design. The use ofwhole antibodies for molecular imaging has been limited by theirlarge size and consequently slow removal from the circulationwhich can result in low tumor-to-blood ratio and poor imagequality. This has been the driving force behind the development ofimmuno-PET and immuno-SPECT probes that are based on anti-body fragments such as ScFv, diabodies, minibodies and affibodies.However, the rapidity with which NGO-IgGs are cleared from thecirculation as reported here, suggests that NGO, and not theattached antibody, is the major determinant of pharmacokineticbehavior. This opens up the possibility of using whole antibodies inprobe design while avoiding the problem of unfavorable pharma-cokinetics. Since whole antibodies are often readily commerciallyavailable, this approach lends itself to ease of translation. Anotheradvantage of NGO-based immunoconstructs is that they can beeasily adapted for therapeutic applications, since NGO is a capa-cious and, therefore, efficient carrier of anticancer drugs, photo-sensitizers for photodynamic therapy or other therapeutic entities.Similarly, NGO-IgG constructs could be used as carriers of thera-peutic radionuclides for targeted radiotherapy, since their largesurface area allows radiolabeling to very high specific activity.

5. Conclusion

NGO was radiolabeled with 111In through pp-stacking of themetal ion chelator, peNH2eBnDTPA, in a simple two-step process.Trastuzumab was conjugated to NGO for targeting of HER2 recep-tors. 111In-NGO-Tz showedHER2-specific tumor uptake in vivo, withmore favorable kinetics than 111In-Tz. The modular approach toradioimmunoconjugate construction that was used lends itself tostraightforward modification, with addition of other tumor-targeting moieties, tailored to specific applications. NGO holdspromise as a versatile scaffold material for the construction ofmolecular imaging probes.

Please cite this article in press as: Cornelissen B, et al., Nanographene oimaging of HER2-positive tumors, Biomaterials (2012), http://dx.doi.org/

Acknowledgments

This research was supported by the CR-UK/EPSRC/MRC/NIHROxford Cancer Imaging Centre, the NIHR Oxford BiomedicalResearch Centre and Cancer Research-UK.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2012.10.054.

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