Key words

1 Introduction

Near-infrared fluorescence (NIRF) imaging is an excellent noninvasive technique for studying diseases at the molecular level in living subjects [1–4]. As an excellent complement to nuclear imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), NIRF imaging does not employ ionizing radiation or radioactive materials, rendering it cost-effective, robust, sensitive, and straightforward over other imaging modalities [3, 5, 6]. Although absorption and scattering from biological tissues may limit penetration of light through the body, photon penetration into and out of tissue can be more efficient in the near-infrared window (650–900 nm) with minimal intra-tissue scattering. Recent research advances have demonstrated that NIRF imaging is playing a valuable role in better understanding of biology, early diagnosis of diseases, and effective assessment of treatment [7–9].

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a fundamental process occurring during tumor progression [10–13]. A sizable body of evidence suggests that the formation of tumor angiogenesis is a complex multi-step process that follows a characteristic sequence of events mediated and controlled by growth factors, cellular receptors, and adhesion molecules [14–16]. Therefore, biomarkers exclusively expressed in tumor angiogenesis can be recognized as potential targets for cancer diagnosis and therapy. Numerous studies have proved that CD13 receptor is an important regulator of endothelial morphogenesis during tumor angiogenesis [17]. CD13 receptor , also known as aminopeptidase N (APN), is a zinc-dependent membrane-bound ectopeptidase that degrades preferentially proteins and peptides with an N-terminal neutral amino acid [18]. Research results suggest that overexpression of CD13 is associated with the progression of many tumors, such as prostate, colon, and pancreatic cancer [19–21]. Through in vivo screening of a phage-displayed peptide library, a tumor vasculature homing phage carrying sequence CNGRCVSGCAGRC was selected by using human breast carcinoma xenografts [22]. Later, the molecular basis behind NGR tumor-homing properties was revealed, and NGR-containing peptide was identified as a specific ligand for CD13 receptor [23]. Consequently, a number of NGR-containing derivatives have been developed for both CD13-targeted tumor imaging and therapy [6, 24–30].

Click chemistry offers chemists a platform for modular and high-yielding synthetic transformations for constructing highly diverse molecules [31]. The Huisgen 1,3-dipolar cycloaddition reaction, which fuses an azide and an alkyne together, and provides access to a variety of five-membered heterocycles, has become of great use in the development of new molecular probes [32]. Based on our previous experience [33], a catalyst-free click chemistry system using the ligation of dibenzocyclooctyne and azide was chosen to prepare peptide-based near-infrared fluorescent probes (Fig. 1) [34, 35]. To this end, the dimeric NGR peptide (NGR2) was conjugated with an azide-terminated Cy5.5 fluorophore (Cy5.5-N3) to rapidly afford Cy5.5-NGR2 in a quantitative yield. The Cy5.5-NGR2 peptide provided highly sensitive and target-specific imaging of CD13 receptor expression in tumors. The excellent tumor-to-normal tissue ratio and fast tumor targeting ability of Cy5.5-NGR2 have proved it a promising molecular probe, not only allowing the NIRF imaging of CD13 overexpressed tumor angiogenesis, but also having the potential to facilitate noninvasive monitoring of CD13-targeted anti-angiogenesis therapy.

Fig. 1
figure 1

Schematic representation of the construction of peptide-based near-infrared fluorescent probe via bioorthogonal click chemistry for in vivo tumor angiogenesis imaging

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2 Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

  1. 1.

    Monomeric NGR peptide [GGGCNGRC; disulfide Cys:Cys = 4 − 8] (C S Bio, Inc., Menlo Park, CA, USA).

  2. 2.

    Dimeric NGR peptide (NGR2) is prepared using the reported procedure [30].

  3. 3.

    Cy5.5 azide (Cy5.5-N3) (Lumiprobe, Hallandale Beach, FL, USA).

  4. 4.

    DBCO-PEG4-NHS ester (Click Chemistry Tools, Inc., Scottsdale, AZ, USA).

  5. 5.

    Sodium borate buffer (pH 8.5).

  6. 6.

    Phosphate-buffered saline (PBS).

  7. 7.

    0.1 % TFA in water (Solution A, which has been rigorously degassed for high-performance liquid chromatography [HPLC] use).

  8. 8.

    0.1 % TFA in acetonitrile (Solution B, which has been rigorously degassed for HPLC use).

  9. 9.

    Luna C18 reversed phase column (5 μm, 250 × 4.6 mm) (Phenomenex, Torrance, CA, USA).

  10. 10.

    Chamber slides (VWR Corporate, Radnor, PA, USA).

  11. 11.

    Dulbecco’s modified Eagle’s medium (DMEM) (USC Cell Culture Core, Los Angeles, CA, USA).

  12. 12.

    HT-1080 human fibrosarcoma cell line (ATCC, Manassas, VA, USA).

  13. 13.

    MCF-7 human breast cancer cell line (ATCC, Manassas, VA, USA).

  14. 14.

    Fetal bovine serum (FBS).

  15. 15.

    Paraformaldehyde (PFA).

  16. 16.

    Matrigel (BD Biosciences, San Jose, CA, USA).

  17. 17.

    Female athymic nude mice (Harlan Laboratories, Livermore, CA, USA).

3 Methods

3.1 Preparation of DBCO-Conjugated NGR2 Peptide

  1. 1.

    Dissolve the dimeric NGR peptide (NGR2) (2.6 μmol) in 200 μL of sodium borate buffer (pH = 8.5).

  2. 2.

    Add DBCO-PEG4-NHS ester (3.1 μmol) dissolved in 20 μL of DMSO.

  3. 3.

    Adjust the mixture to pH 8.5 (see Note 1 ).

  4. 4.

    Sonicate at room temperature for 1 h.

  5. 5.

    Purify the crude peptide by HPLC (see Note 2 ).

  6. 6.

    Collect the peak containing the desired product.

  7. 7.

    Lyophilize to afford a fluffy white powder (see Note 3 ).

3.2 Preparation of Cy5.5-NGR2 Peptide

  1. 1.

    Dissolve the DBCO-conjugated NGR2 peptide (0.48 μmol) in 100 μL of PBS.

  2. 2.

    Add Cy5.5-N3 (0.53 μmol) in 10 μL of DMSO and 100 μL of acetonitrile (see Note 4 ).

  3. 3.

    Shake the mixture in the dark at room temperature for 15 min.

  4. 4.

    Purify the crude peptide by HPLC (see Note 5 ).

  5. 5.

    Collect the peak containing the desired product.

  6. 6.

    Lyophilize to afford Cy5.5-NGR2 peptide (see Note 6 ).

The chemical structure of Cy5.5-NGR2 peptide is illustrated in Fig. 2.

Fig. 2
figure 2

Schematic structure of Cy5.5-NGR2 peptide. Blue: NGR peptide; red: Cy5.5 dye. Reproduced from Ref. 6 with permission from Springer

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3.3 Absorption and Emission Spectra of Cy5.5-NGR2 Peptide

  1. 1.

    The absorption spectrum of Cy5.5-NGR2 peptide is recorded on a Cary 14 UV-Vis spectrometer (Bogart, GA, USA). The spectrum is scanned from 550 to 800 nm with an increment of 1 nm.

  2. 2.

    The fluorescence emission of Cy5.5-NGR2 peptide is measured using a Shimadzu RF-5301PC spectrofluorophotometer (Columbia, MD, USA), and the spectrum is scanned from 550 to 800 nm with an increment of 1 nm. The wavelength of excitation light is set at 650 nm.

The absorption and emission fluorescence spectra of Cy5.5-NGR2 peptide in deionized water are illustrated in Fig. 3.

Fig. 3
figure 3

Absorption and emission fluorescence spectra of Cy5.5-NGR2 peptide. Reproduced from Ref. 6 with permission from Springer

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3.4 Binding Specificity of Cy5.5-NGR2 to CD13 Receptor

  1. 1.

    HT-1080 and MCF-7 cells are grown in DMEM supplemented with 10 % FBS at 37 °C in humidified atmosphere containing 5 % CO2.

  2. 2.

    HT-1080 and MCF-7 cells are grown in chamber slides with a density of 2 × 104/well for 24 h.

  3. 3.

    Wash cells with serum-free DMEM medium for 3 min, fix cells in each well with 2 % PFA for 10 min, and then wash cells with serum-free DMEM medium (three times, 3 min/wash).

  4. 4.

    Add 20 nM of Cy5.5-NGR2 peptide in 200 μL of serum-free DMEM medium to the HT-1080 or MCF-7 cells.

  5. 5.

    Incubate at 37 °C in the dark for 15 min.

  6. 6.

    Wash cells with PBS (three times, 5 min/wash).

  7. 7.

    For the blocking group, the HT-1080 cells are co-incubated with 20 nM of Cy5.5-NGR2 peptide and 50 μM of non-labeled monomeric NGR peptide (see Note 7 ).

  8. 8.

    Mount the chamber slides with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium.

  9. 9.

    Place the slides under a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) with a Cy5 filer and record the images at 633 nm excitation wavelength.

It is critical that the CD13-binding specificity is maintained after chemical modification of NGR peptides (see Note 8 ). A representative result is shown in Fig. 4.

Fig. 4
figure 4

Confocal microscopy results of Cy5.5-NGR2 with HT-1080 cells (CD13 positive) and MCF-7 cells (CD13 negative) (magnification: 100×). The blocking study is achieved by adding unlabeled monomeric NGR peptide. Top: Incubation of Cy5.5-NGR2 (20 nM) with CD13-positive HT-1080 cells; middle: incubation of Cy5.5-NGR2 (20 nM) with CD13-positive HT-1080 cells blocked by a non-labeled monomeric NGR peptide (50 μM); bottom: incubation of Cy5.5-NGR2 (20 nM) with CD13-negative MCF-7 cells. Reproduced from Ref. 6 with permission from Springer

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3.5 In Vivo NIRF Optical Imaging

  1. 1.

    Generate the tumor xenografts by subcutaneous injection of five million HT-1080 cells suspended in 50 μL of cell culture media and 50 μL of BD Matrigel into the right shoulder of female athymic nude mice (Harlan Laboratories, Livermore, CA, USA) (see Note 9 ). The mice are subjected to in vivo NIRF imaging studies when the tumor volume reaches 200–300 mm3 (about 2 weeks after inoculation). Monitor the tumor volume every other day using a caliper following the formula \( V={(witdth)}^2\times length/2 \).

  2. 2.

    Set up the IVIS Imaging System 200 Series (PerkinElmer Inc., Alameda, CA, USA). A Cy5.5 filter set is used to acquire the fluorescent signals.

  3. 3.

    Obtain the fluorescent signals by scanning a tumor-bearing mouse before the injection of Cy5.5-NGR2.

  4. 4.

    Inject 1.5 nmol of Cy5.5-NGR2 into a tumor-bearing mouse via the tail vein while the mouse is under isoflurane anesthesia.

  5. 5.

    For the blocking experiment, inject 1.5 nmol of Cy5.5-NGR2 with a non-labeled monomeric NGR peptide (20 mg/kg) into a tumor-bearing mouse via the tail vein.

  6. 6.

    Scan the animal at 0.5, 1, 2, 3, and 4 h (see Note 10 ). The identical illumination settings (lamp voltage, filters, f/stop, field of views, binning) are used to acquire all images. All NIRF images are acquired using 1-s exposure time (f/stop = 4). Fluorescence emission images are normalized and reported as photons per second per centimeter squared per steradian (p/s/cm2/sr) (see Note 11 ).

Representative NIRF images are shown in Fig. 5 after HT-1080 tumor-bearing mice are injected with 1.5 nmol of Cy5.5-NGR2. To determine tumor contrast, mean fluorescence intensities of the tumor area at the right shoulder of the animal and of the normal tissue at the surrounding tissue are calculated using the region-of-interest (ROI) function of the IVIS Living Image 4.4 software (PerkinElmer Inc., Alameda, CA, USA). The fluorescence signal resulting from Cy5.5-NGR2 is clearly visible and the tumor fluorescence intensity reaches maximum at 2 h post-injection (Fig. 6). The NIRF images of HT-1080 tumor-bearing mice at 2 h pi from the non-blocking and blocking group are presented in Fig. 7. Tumor contrast as quantified by the ROI analysis of images shows that the tumor-to-muscle value at 2 h pi is reduced from 2.65 ± 0.13 to 1.05 ± 0.06 (P < 0.05) by blocking CD13 receptor with a non-labeled NGR peptide (Fig. 8) (see Note 12 ).

Fig. 5
figure 5

Time-course fluorescence imaging of subcutaneous HT-1080 tumor-bearing nude mice after intravenous injection of 1.5 nmol of Cy5.5-NGR2. The tumors are clearly visualized as indicated by an arrow from 0.5 to 4 h pi. The fluorescence intensity is recorded as per second per centimeter squared per steradian (p/s/cm2/sr). Reproduced from Ref. 6 with permission from Springer

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Fig. 6
figure 6

Quantification and kinetics of in vivo targeting character of Cy5.5-NGR2 in the HT-1080 tumor vs. muscle. The Cy5.5-NGR2 uptake in HT-1080 tumor at various time points is significantly higher than that in muscle. Error bar is calculated as the standard deviation (n = 3). Reproduced from Ref. 6 with permission from Springer

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Fig. 7
figure 7

Representative optical imaging (at 2 h pi) of mice bearing HT-1080 tumor on the right shoulder demonstrating blocking of Cy5.5-NGR2 (1.5 nmol) uptake by co-injection with a non-labeled monomeric NGR peptide (20 mg/kg). Reproduced from Ref. 6 with permission from Springer

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Fig. 8
figure 8

Fluorescence intensity ratio of tumor to muscle based on the ROI analysis of Cy5.5-NGR2 uptake at 2 h pi in HT-1080 tumors without (non-blocking) or with (blocking ) co-injection of a non-labeled monomeric NGR peptide (20 mg/kg). Error bar is calculated as the standard deviation (n = 3). Reproduced from Ref. 6 with permission from Springer

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4 Notes

  1. 1.

    It is critical to adjust the pH value of mixture to 8.5 for amide bond formation between a succinimide ester and an amino group.

  2. 2.

    The flow rate is 1 mL/min with the mobile phase starting from 100 % solvent A (0.1 % TFA in water) to 40 % solvent A and 60 % solvent B (0.1 % TFA in acetonitrile) at 22.5 min.

  3. 3.

    A typical yield is between 70 and 80 %.

  4. 4.

    The selection of NIR optical dyes for in vivo cancer imaging is referred to the literature [36].

  5. 5.

    The flow rate is 1 mL/min with the mobile phase starting from 100 % solvent A (0.1 % TFA in water) to 5 % solvent A and 95 % solvent B (0.1 % TFA in acetonitrile) at 30 min.

  6. 6.

    A typical yield is between 90 and 95 %. The fluorescent peptide is stored as a dry powder in the dark at −20 °C until use.

  7. 7.

    To achieve the blocking experiment, various concentrations of non-labeled NGR peptide are tested. However, if the concentration of blocking NGR peptide is too high, the cells may detach from the chamber slide.

  8. 8.

    The receptor binding affinity of fluorescent probe can also be determined by cell-based receptor-binding assay.

  9. 9.

    Animals are fed with alfalfa-free diet, such as 2018 Teklad global 18 % protein rodent diet (Harlan Laboratories, Livermore, CA, USA) for 2 weeks prior to tumor implantation and maintained on the same diet throughout the study to minimize autofluorescence and improve imaging clarity.

  10. 10.

    Based on the pharmacokinetics of fluorescent probe , the imaging time points can be varied.

  11. 11.

    To investigate the biodistribution of fluorescent probe , ex vivo NIRF imaging can be performed. After injection of fluorescent probe at a certain time point, tumors, tissues, and organs are dissected and subjected to ex vivo NIRF imaging. The uptake of fluorescent probe can be quantified by the ROI analysis of images.

  12. 12.

    Statistical analysis is performed with a Student’s t test . Statistical significance is assigned for P values < 0.05.