2.1.

General Material and Reagent

Cy5.5-mono-maleimide was purchased from GE Healthcare (Piscataway, New Jersey). Dichloromethane, triethylamine, N-hydroxybenzotriazole hydrate (HOBT), diisopropylcarbodiimide (DIC), ethyl acetate, and dithiothriotol (DTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri). Dimethylsulfoxide (DMSO) and ethyl acetate were purchased from Fisher Scientific (Pittsburgh, Pennsylvania). All the other standard reagents were purchased from Sigma-Aldrich Chemical Co. All chemicals were used without further purification.

The Affibody analog, Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ , was prepared by a solid phase peptide synthesis method as previously described.²¹ Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a Dionex Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, California, for analysis and preparation of peptides and bioconjugates) equipped with a photodiode array detector. Five UV wavelengths (218, 254, 280, 500, and $675\mathrm{nm}$ ) were monitored for all the experiments. Analytical RP-HPLC column from Sorbent Tech (Atlanta, Georgia) (Grace Vydac C4, $4.6\times 250\mathrm{mm}$ ) was used for analysis of the labeled small protein. The mobile phase was solvent A, 0.1% trifluoroacetic acid $\left(\mathrm{TFA}\right)∕{\mathrm{H}}_2\mathrm{O}$ , and solvent B, 0.1%TFA/acetonitrile. The flow rate was $1\mathrm{mL}∕\min$ , with the mobile phase starting from 80% solvent A and 20% solvent B $\left(0\text{to}2\min \right)$ to 50% solvent A and 50% solvent B at $32\min$ . Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, model: Perseptive Voyager-DE RP Biospectrometer) (Framingham, Massachusetts) or an electrospray ionization quadrupole mass spectrometer (ESI-MS, model: Micromass ZQ single quadrupole LC-MS) (Milford, Massachusetts) was performed by the Stanford Protein and Nucleic Acid Biotechnology Facility and Stanford Chemistry Department Mass Spectrometry Facility, respectively. The human epithelial carcinoma cancer cell line A431 was obtained from the American Type Tissue Culture Collection (Manassas, Virginia). Female athymic nude mice (nu/nu) were purchased from Charles River Laboratories (Boston, Massachusetts).

2.2.

Synthesis of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$

The general procedure for preparation of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ is as follows. The Affibody molecule Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was dissolved in freshly degassed phosphate buffer ( $0.1\mathrm{M}$ , pH 7.4) at a concentration of approximately $1\mathrm{mg}∕\mathrm{mL}$ . The NIR dye Cy5.5-mono-maleimide $\left(300\mu \mathrm{g}\right)$ in DMSO was then added (1.5 equivalents per equivalent of the Affibody). After vortexing for $2\mathrm{h}$ , the reaction mixture was purified by the HPLC equipped with a Grace Vydac 214TP54-C4 column. Characterization of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was confirmed using MALDI-TOF-MS. The purity of the bioconjugate was confirmed by the analytical HPLC.

Fluorescence emission of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was measured on the Fluomax-3 fluorophotometer (HORIBA Jobin Yvon, Edison, New Jersey), and the spectrum was scanned from $650\text{to}850\mathrm{nm}$ with an increment of $1\mathrm{nm}$ . The wavelength of excitation light is $635\mathrm{nm}$ . The absorption spectrum of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was recorded on an Agilent 8453 UV-visible ChemStation (Agilent Technologies, Wilmington, Delaware). The spectrum was scanned from $500\text{to}850\mathrm{nm}$ with an increment of $1\mathrm{nm}$ .

2.3.

Fluorescence Microscopy Study

The A431 and MCF7 cells were cultured in high-glucose Dulbecco modified eagle medium (DMEM) and modified eagle medium (MEM) respectively supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad, California). The cell line was maintained in a humidified atmosphere of 5% $\mathrm{C}{\mathrm{O}}_2$ at $37°\mathrm{C}$ , with the medium changed every other day. A confluent monolayer was detached with trypsin and dissociated into a single cell suspension for further cell culture.

For fluorescence microscopy study, A431 or MCF7 cells $(1\times {10}^4)$ were cultured on the $35\text{-}\mathrm{mm}$ MatTek glass-bottom culture dishes (Ashland, Massachusetts). After $24\mathrm{h}$ , the cells were washed with phosphate-buffered saline (PBS) and then incubated at $37°\mathrm{C}$ with Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(100\mathrm{nM}\right)$ for $1\mathrm{h}$ in dark. The EGFR-binding specificity of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ in cell culture was verified by incubating the A431 and MCF7 cells with or without large amounts of (blocking dose) nonfluorescent Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ peptide $\left(10\mu \mathrm{M}\right)$ . After the incubation period, cells were washed three times with ice-cold PBS. The fluorescence signal of the cells was recorded using an Axiovert 200M fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, New Jersey) equipped with a Cy5.5 filter set (Exciter, HQ $650∕20\mathrm{nm}$ ; Emitter, HQ $695∕35\mathrm{nm}$ ). An AttoArc HBO $100\text{-}\mathrm{W}$ microscopic illuminator was used as a light source for fluorescence excitation. Images were taken using a thermoelectrically cooled charge-coupled device (CCD) (Micromax, model RTE/CCD-576, Princeton Instruments Inc., Trenton, New Jersey) and analyzed using MetaMorph Software version 6.2r4 (Molecular Devices Corporation, Downingtown, Pennsylvania).

2.4.

Binding Affinity Measurement with Flow Cytometry

The flow cytometric analysis was performed as described in the literature with minor modification.²³ Samples were illuminated with a sapphire laser at $488\mathrm{nm}$ and a red laser at $635\mathrm{nm}$ on a BD FACSCalibur. The fluorescence and the forward-scattered and side-scattered light from 10,000 cells was detected at a rate of approximately $150\text{events}∕\mathrm{s}$ . Flow cytometric data were analyzed with FACSDiva Software (BD Biosciences, San Jose, California). Prior to the flow cytometric analysis, A431 cells seeded in $75\text{-}{\mathrm{cm}}^2$ flasks approximately $3\text{days}$ before the experiment were trypsinized [0.05% trypsin to 0.02% ethylenediaminetetraacetic acid (EDTA), $2.5\mathrm{mL}$ , $\sim 10\min$ , $37°\mathrm{C}$ ; Lonza Verviers S.P.R.L.] followed by a centrifugation ( $1000\mathrm{g}$ , $3\min$ ). The pellet was subsequently resuspended in the binding buffer [PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide (NaAz)]. A431 cells ( $0.2\mathrm{M}$ each, expressing $\sim 2\times {10}^6$ receptors/cell) were then distributed in Falcon tubes $\left(15\mathrm{mL}\right)$ . After centrifugation, the cells were incubated with $5\mathrm{mL}$ of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ at different concentrations (ranging from $0.1\text{to}100\mathrm{nM}$ ) for $1\mathrm{h}$ at room temperature in the dark, with continuous rocking. The cells were then centrifuged ( $178\mathrm{g}$ , $4\min$ , $4°\mathrm{C}$ ), washed with the binding buffer twice, resuspended in $500\mu \mathrm{L}$ of binding buffer, and subjected to flow cytometric analysis. All procedures after the incubation with the probe were performed on ice with ice-cold buffers. A triplicate of the flow cytometric data was analyzed with GraphPad Prism 5 (GraphPad Software, San Diego, California) and a nonlinear regression one-site-specific model was used to calculate the $K_D$ of the probe.

2.5.

Optical Imaging of A431 Tumors in Mice

All animal studies were carried out according to a protocol approved by the Stanford University Administrative Panels on Laboratory Animal Care (APLAC). Female athymic nude mice (nu/nu) at $4\text{to}6\text{weeks}$ of age were injected subcutaneously in the shoulder with $5\times {10}^6$ A431 or MCF7 cells suspended in $100\mu \mathrm{L}$ of PBS. For the MCF7 mice model, $4.7\mathrm{mg}$ of estrogen was planted in the back $2\text{days}$ before inoculation. The tumor-bearing mice were subjected to in vivo optical imaging studies $2\text{to}3\text{weeks}$ after implantation.

In vivo fluorescence imaging was performed with an IVIS 200 small animal imaging system (Xenogen, Alameda, California). A Cy5.5 filter set (excitation $615\text{to}655\mathrm{nm}$ ; emission $695\text{to}770\mathrm{nm}$ ) was used for acquiring Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ fluorescence in vivo. Identical illumination settings (lamp voltage, filters, $f$ /stop, fields of view, binning) were used to acquire all images, and fluorescence emission was normalized to photons per second per centimeter squared per steradian $(\mathrm{p}∕\mathrm{s}∕{\mathrm{cm}}^2∕\mathrm{sr})$ . Images were acquired and analyzed using Living Image 3.0 software (Xenogen, Alameda, California). For the positive control experiment, mice $(n=3)$ were injected via tail vein with $0.5\mathrm{nmol}$ of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $(20\mathrm{nmol}∕\mathrm{kg})$ and subjected to optical imaging at various time points p.i. For the blocking experiment, mice ( $n=3$ for each probe also) were injected with the mixture of $300\mu \mathrm{g}$ of Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ and $0.5\mathrm{nmol}$ of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ . IVIS-200 NIR fluorescent images were acquired using a $1\text{-}\mathrm{s}$ exposure time ( $f∕\text{stop}=4$ , $\text{binning}=4$ ).

To subtract the autofluorescence, optical imaging using an IVIS spectrum small animal imaging system was also performed on mice for ex vivo imaging. The mice injected with Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ were euthanized at $24\mathrm{h}$ p.i. and used for ex vivo fluorescence imaging. The tumor and other major tissues were dissected and placed on black papers. A slightly blue shifted excitation filters at $585\text{to}620\mathrm{nm}$ was used to measure the tissue autofluorescence. The background filter image was then subtracted from the primary filter image (excitation filter $620\text{to}655\mathrm{nm}$ ) of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ fluorescence by image-math. IVIS-spectrum NIR fluorescent images were acquired using a $5\text{-}\mathrm{s}$ exposure time ( $f∕\text{stop}=4$ , $\text{binning}=4$ ) because of the much narrower emission filter range $\left(710\text{to}730\mathrm{nm}\right)$ . The fluorescence images were acquired, and mean fluorescence flux $(\mathrm{p}∕\mathrm{s}∕{\mathrm{cm}}^2∕\mathrm{sr})$ for each sample was obtained.

2.6.

Statistical Analysis

All the data are given as $\text{means}\pm \mathrm{SD}$ (standard deviation) of $n$ independent measurements. Statistical analysis was performed using a Student’s $t$ test. Statistical significance was assigned for $P$ values $<0.05$ . To determine tumor contrast, mean fluorescence intensities of the tumor area $\left(T\right)$ at the shoulder of the animal and of the area $\left(N\right)$ at the right flank (normal tissue) were calculated by the region-of-interest (ROI) function of Living Image software integrated with Igor (Wavemetrics, Lake Oswego, Oregon). Dividing $T$ by $N$ yielded the contrast between tumor tissue and normal tissue.

3.1.

Synthesis of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$

The Affibody small protein Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ with a cysteine at the $N$ terminal was successfully synthesized using a peptide synthesizer and purified by semipreparative HPLC. The peptide was generally obtained in 10% yield with over 95% purity [Fig. 1 ]. The purified Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was characterized by MALDI-TOF-MS. The measured molecular weight (MW) was consistent with the expected MW: $m∕z=6690.0$ for ${[\mathrm{M}+\mathrm{H}]}^{+}$ (Calculated $\mathrm{MW}=6688.6$ ). Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ was then conjugated with Cy5.5-mono-maleimide in the PBS buffer and purified by HPLC. MS analysis verified the success preparation of the final product Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ (the observed MW: $m∕z=7727.2$ for ${[\mathrm{M}+\mathrm{H}]}^{+}$ , calculated ${\mathrm{MW}}_{[\mathrm{M}+\mathrm{Na}]}^{+}=7727.4$ ). The purity for the final product was over 95% (retention time: $25\min$ ) [Fig. 1], and the recovery yield of the probe was 47%. Spectrum study showed that Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ had similar absorption and fluorescence emission characteristics of free Cy5.5 dye, with ${\lambda }_{\mathrm{ab}}=680\mathrm{nm}$ and ${\lambda }_{\mathrm{em}}=696\mathrm{nm}$ [Fig. 1].

Fig. 1

(a) HPLC chromatogram of purified Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ and (b) Absorption and emission fluorescence spectra of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ .

3.2.

Binding Specificity and Affinity of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$

To demonstrate the EGFR specificity and subcellular localization of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ , the probe was incubated with A431 tumor cells and fluorescent microscopic imaging was performed. With an $1\mathrm{h}$ incubation at $37°\mathrm{C}$ , intensive fluorescent signal was observed from the A431 cells membrane, and some of probe was also found to be inside of the cells (Fig. 2 ). Furthermore, the fluorescent signal from the cells could be significantly reduced by incubation of the cells with large excess of the Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(10\mu \mathrm{M}\right)$ , demonstrating the binding specificity of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ (Fig. 2). The staining of low EGFR expression MCF7 is barely visible. The flow cytometry study was performed for measurement of the binding affinity of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ . A representative flow cytometric analysis was shown in Fig. 3 . The equilibrium dissociation constant $K_D$ was determined to be $43.6\pm 8.4\mathrm{nM}$ using a nonlinear regression one-site-specific model (Fig. 3).

Fig. 2

Cell uptake of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(100\mathrm{nM}\right)$ in A431 and MCF7 cells at $37°\mathrm{C}$ incubated with (blocking) or without the presence of the Affibody molecule Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(10\mu \mathrm{M}\right)$ . NIR fluorescent (NIR) and light images (LI) are shown at the top and bottom, respectively.

Fig. 3

(a) Flow cytometric analysis of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ binding to A431 cells at different concentrations $\left(0.1\text{to}100\mathrm{nM}\right)$ and (b) cell population gated against a control blank, where triplicate data at each concentration were fitted with a nonlinear regression one-site- specific model.

3.3.

In Vivo and Ex Vivo Optical Imaging

Figure 4 shows typical NIR fluorescent images of nude mice bearing subcutaneous A431 and MCF7 tumor after intravenous injection of $0.5\mathrm{nmol}$ of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ . The A431 tumor could be clearly visualized from the surrounding background tissue from $0.5\text{to}4\mathrm{h}$ p.i.. Quantification analysis of ROIs was performed and the tumor to normal tissue ratio $(T∕N)$ as a function of time is depicted in Fig. 4. Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ exhibited a fast tumor targeting property in vivo. The tumor-to-normal tissue ratio reached a peak at $2\mathrm{h}$ p.i. The receptor specificity of the probe was verified by the blocking experiment. Unlabeled Ac-Cys- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ significantly reduced tumor uptake and tumor contrast at all the time points [Figure 4]. Tumor contrast as quantified by ROI analysis of images indicated that the $T∕N$ value at $4\mathrm{h}$ p.i. was reduced from $2.9\pm 0.1$ to $1.8\pm 0.4$ $(P<0.05)$ . At $4\mathrm{h}$ p.i., MCF7 tumor showed much lower $T∕N$ value $(1.54\pm 0.19)$ .

Fig. 4

(a) In vivo fluorescence IVIS-200 imaging of subcutaneous A431 and MCF7 tumor-bearing nude mice at $10\min$ , $30\min$ , $1\mathrm{h}$ , $2\mathrm{h}$ , and $4\mathrm{h}$ . Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(0.5\mathrm{nmol}\right)$ with (bottom) or without (top) coinjection of unlabeled Affibody ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ $\left(300\mu \mathrm{g}\right)$ were injected. Arrows indicate the location of tumors. (b) ROI analysis of tumor-to-normal tissue ratios of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ in mice bearing A431 tumor at $10\min \text{to}4\mathrm{h}$ p.i. $(n=3)$ .

The result for ex vivo tumor mice imaging at $24\mathrm{h}$ p.i., as shown in Fig. 5 . Quantitative analysis showed that there is also large amount of probe trapped inside the kidney without clearance [Fig. 5]. The tumor-to-normal organ (except kidney) ratios are also high [Fig. 5]. For example, the ratios for tumor-to-lung and tumor-to-muscle are $5.6\pm 1.8$ and $6.5\pm 0.4$ , respectively.

Fig. 5

(a) Ex vivo imaging of tumor and normal tissues of Cy5.5- ${\mathrm{Z}}_{\mathrm{EGFR}:1907}$ after sacrificed the mice at $24\mathrm{h}$ p.i.: Tm, tumor; H, heart; L, lung; Lv, liver; Kn, kidney; Sp, spleen; St, stomach; B, blood; Sk, skin; Ms, Muscle. (B) ROI analysis of fluorescent signal from tumor and normal tissues. (b) Fluorescence intensity ratios of tumor-to-normal tissues based on the ROI analysis. Error bar was calculated as the standard deviation $(n=3)$ .