Near-infrared Fluorescent Peptides with High Tumor Selectivity: Novel

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Near-infrared Fluorescent Peptides with High Tumor Selectivity: Novel Probes for Image-Guided Surgical Resection of Orthotopic Glioma Yi Liu, Zhengjie Wang, Xiang Li, Xiaowei Ma, Shuailiang Wang, Fei Kang, Weidong Yang, Wenhui Ma, and Jing Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00888 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Molecular Pharmaceutics

Near-infrared Fluorescent Peptides with High Tumor Selectivity: Novel Probes for Image-Guided Surgical Resection of Orthotopic Glioma Yi Liu# 1, Zhengjie Wang# 2, Xiang Li1, Xiaowei Ma1, Shuailiang Wang1, Fei Kang1, Weidong Yang1, Wenhui Ma* 1, Jing Wang* 1 1

Department of Nuclear Medicine, Xijing Hospital, Fourth Military Medical University,

Xi’an 710032, China 2

Department of Nuclear Medicine, The First Affiliated Hospital of Chongqing Medical

University, Chongqing 400016, China

ABSTRACT: The complete excision of glioblastomas with maximal retention of surrounding normal tissues can have positive effect on survival status and quality life of patients. Near-infrared fluorescence (NIRF) optical imaging of the tumor vasculature offers a non-invasive method for detection of early-stage glioblastoma and efficient monitoring of therapeutic responses. The aim of this study was to develop a novel NIRF imaging probe as a visualization tool for image-guided surgical resection of orthotopic glioblastoma. In this study, Cy5.5-RKL, Cy5.5-NKL and Cy5.5-DKL probes were successfully synthesized and their properties investigated in vitro and in vivo. In vivo, Cy5.5-RKL and Cy5.5-NKL were able to detect U87MG xenografts for at least 8 h p.i.. The maximum tumor to muscle ratios of Cy5.5-RKL and Cy5.5-NKL were 7.65 ± 0.72 and 5.43 ± 0.72, respectively. Of the probes, Cy5.5-RKL displayed the best delineation of the boundaries between orthotopic glioblastomas and normal brain tissue at 8 h p.i. In conclusion, NIRF imaging using Cy5.5-RKL is promising not only for diagnostic purposes

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but also for use in image-guided surgery for orthotopic glioblastoma or other superficial tumors.

Key words: Near-infrared fluorescence imaging, glioblastoma, RGD, NGR, anti-cancer peptide

INTRODUCTION The treatment of glioblastoma patients often involves surgical resection and concurrent chemoradiation.1 Because of the diffuse and infiltrative growth of brain tumors, the survival of glioblastoma patients is related to difficulties achieving complete surgical resection.2 It has been demonstrated previously that the survival status the quality life of patients is related to the extent of tumor resection.3 Complete resection of the tumor, while preserving the surrounding brain tissue, can be achieved by surgical navigation and is critical for prolonging survival of the patients.4 The wavelength used for near-infrared fluorescence (NIRF) imaging is primarily in the range of 700–900 nm, at which the absorbance and autofluorescence of biomolecules is minimal. NIRF imaging can be employed for intraoperative tumor visualization using molecular imaging agents.5 Through this approach, delineation of tumor tissues using a targeted fluorescent agent increases the chance of complete resection. Several studies have demonstrated the use of molecules overexpressed specifically in tumor tissue as targets for surgical navigation. Integrin αvβ3 is highly expressed on cytokine-activated vascular endothelial cells and surrounding solid tumor cells.6 CD13 is


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a biomarker selectively expressed on the surfaces of tumor cells and endothelial cells in proliferating vessels producing angiogenic signals.7 Thus, both integrin αvβ3 and CD13 are promising targets for the early detection of tumors. Several studies have shown that peptides containing arginine–glycine–aspartic (RGD) or asparagine–glycine–arginine (NGR) motifs specifically bind to neovascular tissue mainly via αvβ3 or CD13, respectively.8 Previous studies have demonstrated that peptides containing RGD or NGR labeled with Cy5.5 can be used to detect tumors by NIRF imaging.9 However, the rapid clearance of Cy5.5-RGD in intracranial tumors and the limited tumor-targeting ability of NGR-containing peptides resulted in unfavorable imaging results when implemented in vivo. Therefore, it is desirable to improve the tumor-targeting ability of NGR- or RGD-containing peptides via chemical modifications to increase their efficacy as tumor probes. Cationic antimicrobial peptides (AMPs) play critical roles in the innate immunity of various organisms, including mammals, insects, bacteria, fungi, and plants. These peptides strongly bind to negatively charged membranes10 and subsequently lyse them.11 The outer membrane of cancer cells contain negatively charged phosphatidylserine,12 which may contribute to the anticancer properties of AMPs.13 However, the utilization of native AMPs is limited, as their function is compromised in serum due to enzymatic degradation and binding to serum components.14 DKL (LKlLKlLlkKLLkLL-NH2), a synthesized anticancer peptide,15 has been shown to bind selectively to tumor cells in serum and cause complete arrest of 22Rv1 prostate cancer cell growth at the threshold concentration, along with decreased tumor marker expression.16



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We hypothesized that the combination of the tumor-specific RGD or NGR motif with the tumor-selective DKL peptide will effectively increase tumor-binding ability compared with single RGD, NGR, or DKL alone. Therefore, we investigated the combination of RGD or NGR with DKL (RKL and NKL, respectively). Cy5.5-labeled probes were synthesized for NIRF imaging and evaluated for their utility in image-guided surgical navigation in an orthotopic glioblastoma model.

MATERIALS AND METHODS General. Chemicals (analytical grade) were obtained commercially and used without further purification. DKL (LKlLKlLlkKLLkLL-NH2; K = Lys, L = Leu, k = D-Lys, l = D-Leu) was purchased from Science Peptide Biological Technology Co., Ltd. (Shanghai, China). NKL [COOH-CNGRCK (DOTA)-(PEG2)-LKlLKkLlkKLLkLL-NH2; disulfide Cys:Cys = 1–5, K

=

Lys,

L

=

Leu,

k

=

D-Lys,

l

=

D-Leu]

and

RKL

[COOH-RGDK(DOTA)-(PEG2)-LKlLKkLlkKLLkLL-NH2; K = Lys, L = Leu, k = D-Lys, l = D-Leu]

were

purchased

from

ChinaPeptides

Co.,

Ltd.

(Shanghai,

China).

Sulfo-Cy5.5–NHS ester was purchased from Lumiprobe Corporation (Hallandale Beach, FL, USA).

Synthesis of Cy5.5 Labeled Peptides. DKL (78.9 μg), NKL (131.8 μg), and RKL (122.8 μg) were added to sodium bicarbonate-buffered saline (200 µL, pH 8) containing sulfo-Cy5.5–NHS ester (10 µL), which had been dissolved in DMF to a concentration of 10 mg/ml. The mixtures were stirred in the dark at 4°C overnight. The crude peptides


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were purified by HPLC. The peaks containing the desired products were collected, lyophilized, and stored in the dark at −20°C until use.

HPLC Methods. The reaction mixtures were purified by semi-preparative HPLC with gradient elution starting with 95% A (0.1% TFA in water) and 5% B (0.1% TFA in acetonitrile) for 5 min and then increasing to 65% B for 35 min, at a flow rate of 12 mL/min. The fractions containing the desired products were collected and lyophilized.

Absorption and Emission Spectra. The absorption spectra of Cy5.5 labeled peptides were recorded on a UV-VIS-NIR Spectrometer (PE Lambda950). The spectra were scanned from 500 to 700 nm in increments of 1 nm. The fluorescence emission of Cy5.5-labeled peptides was measured using a transient/steady-state fluorescence spectrometer (Edinburgh FLS9), and the spectra were scanned from 600 to 850 nm in increments of 1 nm. The wavelength of excitation light was set at 650 nm.

Cell Line and Culture Conditions. The U87MG human glioblastoma cell line was kindly donated by the School of Pharmacy and Gene Technology, the Fourth Military Medical University. U87MG cells were grown in Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone) at 37°C in a humidified atmosphere containing 5% CO2. The cell lines were passaged for less than 3 months, and cell stocks were stored in liquid nitrogen.



In Vitro Fluorescence Imaging of Cy5.5-Labeled Peptides. U87MG cells were seeded into chamber slides (Millicell EZ) at 5 × 104/well and cultured overnight. After washing with phosphate-buffered saline (PBS) (three times, 3 min/wash), the cells in each well were fixed with 4% paraformaldehyde for 20 min and then washed with PBS (three times, 3 min/wash). The cells were incubated with 50 nM Cy5.5-labeled peptides in 500 µL PBS at 37°C in the dark for 1 h, followed by PBS washing (three times, 5 min/wash). For the blocking group, the U87MG cells were incubated with 50 nM Cy5.5-labeled peptides and 50 μM unlabeled peptides, respectively. The chamber slides were then mounted using mounting medium containing 4’ 6-diamidino-2-phenylindole. The slides were scanned under a confocal laser-scanning microscope (FluoView FV1000; Olympus, Japan). The images were analyzed using Image-pro 3D Suite software.

Animal Model. All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Male Balb/c nude mice (4–6 weeks old, body weight 20–25 g) were used. The subcutaneous tumor model was established by injecting 5 × 106 U87MG cells suspended in 50 µL cell culture medium and 50 µL BD Matrigel (BD Biosciences, San Jose, CA, USA) subcutaneously into the right shoulder. The orthotopic tumor model was established after anesthetizing the mice with 3% sodium pentobarbital and connecting the mice to a stereotaxic system (Kopf Instruments, Sunland, CA, USA). After disinfection and incision of the skin, a midline incision was made through the skin overlying the cranium. A small hole was created in the skull using a bone drill, and then 2. 5 × 105 U87MG cells in 5 µL


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cell culture medium were implanted into the brain (2 mm posterior, 2 mm lateral, and 2.5 mm behind the bregma) using a microliter syringe (Gaoge Industry & Trade Co., Ltd. Shanghai, China). The needle was slowly withdrawn after the cell infusion, and the scalp was closed with sutures. The cells were allowed to grow for 2 weeks. Subcutaneous tumor growth was measured using calipers in orthogonal dimensions, which reached to 0.5–1 cm3. The growth of orthotopic tumor was verified by hematoxylin and eosin (H&E) staining after imaging.

In Vivo Near-infrared Fluorescence Imaging and Biodistribution Study. In vivo fluorescence imaging was performed using the Caliper Lumina XR Imaging System (PerkinElmer, Waltham, MA, USA) equipped with near-infrared fluorescent filter sets the IVIS Lumina II imaging station (Caliper Life Sciences) and analyzed using the IVIS Living Imaging 4.3.1 software (PerkinElmer Inc., Alameda, CA, USA). A Cy5.5 filter set was used to measure the fluorescence of the Cy5.5-labeled peptides. Identical illumination settings (lamp voltage, filters, f/stop, field of views, binning) were used to acquire all images. Fluorescence emission values were normalized and expressed as photons per second per centimeter squared per steradian (p/s/cm2/sr). The mice in the non-blocking group (n = 3) received 1.5 nmol Cy5.5-labeled peptides intravenously and were subjected to optical imaging at various time points post-injection (p.i.). The mice in the blocking group (n = 3) were injected with a mixture of Cy5.5-labeled peptides (1.5 nmol) and corresponding unlabeled peptides (20 mg/kg). All near-infrared fluorescence images were acquired using a 1 s exposure time (f/stop = 4). Mice from the non-blocking and



blocking groups were euthanized at 8 h p.i. The tumors, tissues, and organs were dissected and subjected to ex vivo fluorescence imaging. The mean fluorescence in each sample was recorded.

In Vivo NIRF Image-Guided Intraoperative localization of Orthotopic Glioblastoma. NIRF imaging was performed in orthotopic glioblastoma mice after intravenous injection of the Cy5.5-RKL probe on day 14 after tumor cell implantation. The mice were anesthetized with 2% isoflurane and placed on a flat plate perpendicular to the turntable. At 8 h after injection of Cy5.5-RKL, the mice bearing orthotopic glioblastoma were sacrificed and subjected to craniotomy. NIRF imaging was used to evaluate the ability of Cy5.5-RKL to detect the localization of glioma in the brain intraoperatively.

Histological Examination of Tumor Tissue. After surgery, the mice were euthanized, and the brain and tumor tissues were excised, fixed, and embedded in paraffin. Continuous sections (4 μm thick) were obtained and subjected to hematoxylin and eosin (H&E) staining. For immunohistochemistry, tumor tissue slides were deparaffinized in xylene baths and rehydrated in an alcohol gradient. The slides were then boiled in sodium citrate buffer (10 mmol/L, pH 9.0) for 20 min and washed in PBS for antigen retrieval. Quenching of endogenous peroxidase was performed using 1.5% hydrogen peroxide, and samples were blocked in 5% goat serum for 20 min. Slides were incubated with an integrin αv or CD13 mouse monoclonal antibody (1:100; Abcam, MA, USA) overnight, washed in PBS, and incubated with a HRP-conjugated secondary antibody for 1 h at


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room temperature. DAB reagent was used for chromogenic staining. Slides were dehydrated in an alcohol gradient followed by xylene. Finally, the slides were mounted, covered with cover slips, and scanned using the Olympus Imaging System Microscope.

Data Processing and Statistical Analysis. All data are presented as means ± standard deviation of n independent measurements. Statistical analyses were performed by Student’s t test using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Statistical significance was set at P < 0.05. To determine tumor contrast, the mean fluorescence intensities of the tumor tissue and normal surrounding tissue were calculated using the region-of-interest (ROI) function in the IVIS Living Image 4.3.1 software.

RESULTS Synthesis of Cy5.5-Labeled Peptides. The molecular structures of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL are shown in Figure 1. Preparation of Cy5.5-labeled peptides involved two steps. First, sulfo-Cy5.5-NHS ester was coupled to the amino group of DKL, NKL, or RKL. Second, azide-containing Cy5.5 fluorophore (Cy5.5-N3) was reacted with peptides in sodium bicarbonate buffer. The retention times of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL in analytical HPLC were 19.39, 19.44, and 19.40 min, respectively. The yields of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were 84.95%, 96.12%, and 76.78%, respectively. HPLC purification resulted in the purity were greater than 95% for all three peptides.



Absorption and Emission Spectra. The absorption and fluorescence emission spectra of Cy5.5-RKL, Cy5.5-NKL and Cy5.5-DKL are shown in Figure 2. The maximum absorption wavelengths were 675, 673, and 673 nm and the maximum fluorescence emission wavelengths 693, 695, and 693 nm for Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL, respectively.

In Vitro Fluorescence Imaging of Cy5.5-Labeled Peptides. To determine the binding specificity and subcellular localization of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL, the probes were incubated with U87MG tumor cells at 37°C for 1 h, followed by laser confocal microscopy. Representative images are shown in Figure 3a, b, and c. An intense fluorescent signal was observed in the cell membrane after Cy5.5-RKL incubation. Fluorescence emitted by Cy5.5-NKL in the membrane of U87MG cells was moderate, and that by Cy5.5-DKL was the lowest. The signal intensities were significantly reduced in U87MG cells incubated with excess unlabeled peptides (50 μM). The fluorescent intensities in U87MG cells incubated with all three probes are shown in Figure 3d. Taken together, the laser confocal microscopy results demonstrated that Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL bind specifically to U87MG cells, with the greatest membrane association seen with Cy5.5-RKL.

In Vivo and Ex Vivo Near-infrared Fluorescence Imaging. NIRF imaging in nude mice bearing subcutaneous U87MG tumors was performed after intravenous injection of 1.5


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nmol Cy5.5-RKL, Cy5.5-NKL, or Cy5.5-DKL (Figure 4a). All probes exhibited rapid U87MG tumor binding, as early as 0.5 h p.i. High tumor-to-background signal ratios for Cy5.5-RKL and Cy5.5-NKL in U87MG tumors were observed from 0.5 to 8 h p.i. However, the retention time of Cy5.5-DKL in U87MG tumors was much shorter, no more than 6 h. The highest tumor-to-background signal ratios for Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were observed at 3, 7.5, and 3 h p.i., respectively. Fluorescence intensities in U87MG tumor and muscle tissues were plotted as a function of time (Figure 4b, c, and d). Tumor uptake of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL reached a maximum at 2 h p.i., and the probes were slowly washed out over time. In contrast, the normal tissue showed more rapid probe binding and washout. The overall uptake of the three probes was significantly lower in muscle than in tumor tissues during the 8 h evaluation period. The tumor specificity of Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were verified in a blocking experiment. For the blocking treatments, each U87MG tumor-bearing mouse was intravenously co-injected with 1.5 nmol Cy5.5-RKL, Cy5.5-NKL, or Cy5.5-DKL and their corresponding unlabeled peptides (20 mg/kg). Mice in the non-blocking group were injected without excess unlabeled RKL, NKL, or DKL. The results showed that the unlabeled peptides significantly reduced U87MG tumor uptake and contrast at all imaging time points. The optical images obtained from U87MG tumor-bearing mice in the non-blocking and blocking groups at 3 h p.i are presented in Figure 5. Tumor contrast was quantified by ROI analysis of each image. The tumor-to-muscle tissue ratios of the fluorescence emitted by Cy5.5-RKL at 3 h p.i was decreased with blocking from 7.65 ± 0.72 to 0.73 ± 0.06 (P < 0.05) (Figure 5). Likewise, the tumor-to-muscle tissue ratios of



the fluorescence emitted by Cy5.5-NKL and Cy5.5-DKL were decreased with blocking from 5.43 ± 0.72 and 4.89 ± 0.26 to 1.07 ± 0.04 and 1.40 ± 1.23, respectively. Furthermore, the ex vivo imaging findings were consistent with the in vivo findings. In the absence of blocking, predominant Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL probe uptake in the U87MG tumors was observed in the excised organs at 8 h p.i ex vivo. Conversely, blocking caused a significant reduction in U87MG tumor uptake (Figure 6). Probe uptake was higher in digestive organs, including the liver, stomach, and intestine, than in other major organs, suggesting that these probes were excluded mainly from the biliary system in liver. Quantification values of the ROIs in these organs are plotted in Figure 6b, c, and d. In the non-blocking group, these three probes showed significantly higher U87MG tumor uptake compared with the blocking group (P < 0.05) which supports tumor specificity for Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL. Based on the quantitative analysis of ex vivo imaging, the tumor-to-muscle contrast ratios in the non-blocking and blocking groups were calculated. The tumor-to-muscle ratios for Cy5.5-RKL at 8 h p.i. in the non-blocking and blocking groups were 46.17 ± 10.39 and 9.41 ± 0.31, respectively.

In Vivo NIRF Image-Guided Intraoperative localization of Orthotopic Glioblastoma. In the orthotopic tumor model, optical imaging revealed localization of Cy5.5-RKL in the brain from 1 to 8 h p.i. using 1.5 nmol of the probe (Figure 7). The U87MG tumor showed a significantly higher fluorescent signal that was clearly observed in the surrounding normal tissue, lasting for at least 8 h p.i. NIRF image-guided intraoperative detection of glioblastoma at 8 h is shown in Figure 8. The border of the tumor was clearly visualized


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in situ by NIRF imaging (Figure 8a). The highest value of tumor/normal brain tissue, which was occurred in 3 hours after the injection of Cy5.5-RKL, was 4.82 ± 0.80. The data suggest that NIRF imaging using Cy5.5-RKL is useful for intraoperative detection of glioma. The brain tumors were further analyzed by H&E staining (Figure 8b). Immunohistochemical staining showed high expression of integrin αvβ3 and CD13 in tumor tissues (Figure 8c and 8d), further supporting the specific targeting abilities of Cy5.5-RKL and Cy5.5-NKL.

DISCUSSION Molecular imaging, including positron emission tomography–computed tomography, Cerenkov luminescence imaging, and fluorescence imaging, are used to diagnose early-stage tumors.17 These molecular imaging modalities can be used to delineate tumor boundaries during radical surgery used specific molecular probes.18 However, traditional molecular imaging techniques still have some drawbacks such as radioactivity, lack of specificity, or weak signal intensity.19 The probes used for optical imaging are non-radioactive, and the imaging instrument is portable. Both of these features are advantageous for use in surgical navigation.20 Therefore, optical imaging techniques have been actively explored for optical image-guided tumor surgeries in pre-clinical and clinical settings. The recurrence of glioma is related to its resection rate. Radical resection of tumors with minimal effect on the surrounding normal tissues is most effective at improving the survival rate of cancer patients.21 Patients with complete resection derived the most



benefit from a temozolomide regimen compared with those with incomplete resection (4.1 months vs. 1.8 months overall survival).22 However, the boundary between gliomas and the normal surrounding brain tissue is not always clear due to the fingerlike tentacles formed by infiltrating gliomas. This convoluted boundaries constitute a major obstacle to the complete removal of gliomas.23 The use of optical molecular imaging probes that can specifically target gliomas and the neovascular enables effective imaging of gliomas.24 Therefore, intraoperative localization of gliomas can greatly assist in complete glioma removal. Since this optical imaging method is not radioactive, this imaging modality can also be used for intraoperative positioning of gliomas and to improve the prognosis of patients.25 Imaging probes are the basis of molecular imaging. Advances in molecular and cell biology have facilitated the discovery of potential molecular targets for cancer such as NGR or RGD, which can be used in conjunction with molecular imaging-based probes.26 During the past decade, RGD and NGR peptides targeting integrin αvβ3 and CD13 proteins, expressed during neovascularization and in several tumor cells, respectively, have been widely used in imaging studies.27 However, the distribution of tumor neovascular biomarkers is diverse, and these biomarkers may not be present in some tumors.25,28 Therefore, this study aimed to modify RGD and NGR and further optimize their pharmacokinetics. The ability to kill cancer cells demonstrates the tumor selectivity of anti-cancer peptides.29-32 Using D amino acids, the stability and tumor selectivity of DKL are obviously increased.26 DKL combined with NGR or RGD is expected to increase the tumor-targeting ability.


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Our novel probes exhibited good NIRF characteristics, with roughly the same maximum absorption and emission wavelength (Figure 2) values among all three probes. To determine the tumor-targeting efficacy of these three probes, Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were evaluated in subcutaneous U87MG glioblastoma mouse xenografts (Figure 4). In vivo optical imaging studies showed that all three peptides exhibited rapid tumor targeting, as early as 0.5 h p.i. The tumor-to-background contrast ratios for all three probes were excellent, even though the highest value of each probe was observed at different time points p.i. (Figure 4). In the blocking experiment, Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were co-injected with their corresponding unlabeled peptides (20 mg/kg). The tumor uptake (P < 0.05) was significantly reduced in the blocking groups at 3 h p.i compared with the non-blocking groups (Figure 5), indicating that these novel peptides are target specific. The uptake of probes in digestive organs, including the liver, stomach, and intestine, was higher than that in other major organs, suggesting that the hepatic pathway is the likely route of probe excretion (Figure 6). The tumor-to-muscle ratio of Cy5.5-RKL was greater than 4 at all time points p.i. and was much higher than that in Cy5.5-RGD group alone.26 As demonstrated in Chen’s paper, the tumor contrast of the 0.5-nmol Cy5.5-RGD, higher than 1.5-nmol Cy5.5-RGD, was just 3.02 ± 0.45. However, the tumor-to-muscle tissue ratio of the fluorescence emitted by Cy5.5-RKL was up to 7.65 ± 0.72.

NKL decreased liver uptake compared with the Cy5.5-labeled dimeric NGR

peptide or Cy5.5-DKL. These results demonstrated that integration of DKL effectively improved the imaging ability of NGR or RGD. Cy5.5-RKL displayed the best imaging characteristics in vivo and was selected as the probe for evaluation of intraoperative



localization of orthotopic glioma. This probe targeted intracranial tumors and the neovascular, while its uptake in normal brain tissues was extremely low. In the orthotopic glioblastoma model, the highest value of tumor/normal brain tissue, which was occurred in 3 hours after the injection of Cy5.5-RKL, was 4.82 ± 0.80, indicating that the probe is useful for intraoperative image-guided resection of glioblastoma. Due to the limitations of our experimental conditions, our experiment only simulated the tumor location after craniotomy, but the data are sufficient to support the use of optical imaging in radical glioblastoma resection using Cy5.5-RKL. The results confirmed that the probe accurately displayed the glioma boundary. These peptide probes are easy to synthesize and biocompatible. The tumor targeting abilities of NKL and RKL were improved after modification with DKL. Even though RKL has been successfully used for tumor localization in the orthotopic glioma model, its efficacy in fluorescence image-guided cancer surgery may be compromised by the relatively low signal-to-noise ratio and tissue penetration. Biological safety is another issue for clinical application.33-35 Despite these disadvantages, optical image-guided cancer surgery using these novel highly tumor-selective probes exhibited high performance in detecting microscopic ultra-small tumors and presents future clinical potential

CONCLUSION The novel Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL probes were successfully synthesized and investigated in vitro and in vivo. Optical imaging demonstrated that all


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probes specifically labeled U87MG cells and tumors. The best candidate, Cy5.5-RKL, revealed remarkable accumulation in orthotopic glioblastoma and increased the precise delineation of the tumor boundary. In the future, this may be beneficial for image-guided tumor resection. NIRF imaging using Cy5.5-RKL is very promising for tumor diagnosis with strong potential use in image-guided surgery for orthotopic glioblastoma.

AUTHOR INFORMATION Corresponding Author J.Wang and W.Ma, Tel: +86 029-84771048; Fax: +86 029-83210242; e-mail: [email protected] and [email protected].

ORCID Yi Liu: 0000-0001-8531-9624

Notes No author has any conflict of interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant nos. 81601521 and 81230033). The authors wish to thank The School of Pharmacy and Gene Technology, Fourth Military Medical University for supplying the U87MG cell line and the Department of Biochemistry and Molecular Biology, Fourth Military Medical University for



the imaging work.

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Figure 1. Schematic structures of Cy5.5 labeled probes. RKL, NKL and DKL were added into sodium bicarbonate buffered saline containing sulfo-Cy5.5–NSH ester dissolved in DMF. The mixtures were shaken in the dark at 4°C overnight to obtain the probes. a is NGR. b is RGD. c is sulfo-Cy5.5-NSH ester. d is DKL. e is Cy5.5-RKL. f is Cy5.5-NKL. g is Cy5.5-DKL.


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Figure 2. Absorption and emission fluorescence spectra of the Cy5.5-RKL (a), Cy5.5-NKL (b) and Cy5.5-DKL (c). Green line showed absorption fluorescence spectra and red line displayed emission fluorescence spectra.

Figure 3. Confocal microscopy images of U87MG cells incubated with Cy5.5-RKL (a), Cy5.5-NKL (b) and Cy5.5-DKL (c) (50 nM of Cy5.5 labeled peptides in 500 μL of PBS) (magnification ×60). For the blocking group, the U87MG cells were co-incubated with extra unlabeled peptides (50 μM). The results were scanned under confocal laser-scanning microscopy. The images were analyzed in the software of Image-pro plus 6.0. d represented the fluorescence cumulative optical density of each images and Cy5.5-RKL showed the best optical intensity.



Figure 4. In vivo fluorescent imaging of the probes. The mice received 1.5 nmol of Cy5.5 labeled peptides intravenously and subjected to optical imaging at various time points post injection (pi). a: the time-course fluorescence imaging of subcutaneous tumor bearing nude mice. Top, middle and bottom are fluorescence imaging of subcutaneous U87MG tumor bearing nude mice after intravenous injection of probes, respectively. To determine tumor contrast, mean fluorescence intensities of the tumor (T) area at the right shoulder of the animal and the normal tissue (N) at the surrounding tissue were calculated using the region-of-interest (ROI) function of the IVIS Living Image 4.3.1 software. Figure b (Cy5.5-RKL), c (Cy5.5-NKL) and d (Cy5.5-DKL) were constructed according to the ROI values of each group. The maximum T/NT values of each group at the time point was displayed at figure e.


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Figure 5. Representative optical imaging of blocking experiment and fluorescence intensity ratio of tumor-to-muscle based on the ROI analysis at 3 h pi. The mice in the non-blocking group only received Cy5.5 labeled peptides (1.5 nmol) intravenously. And the mice in the blocking group were co-injected with a mixture of Cy5.5 labeled peptides



and corresponding unlabeled peptides (20 mg/kg). The optical images of U87MG tumor bearing mice at 3 h pi from the non-blocking and blocking group: Cy5.5-RKL (a), Cy5.5-NKL (b) and Cy5.5-DKL (c). d: Tumor contrasts between the non-blocking and blocking groups

quantified by the ROI analysis.

Figure 6. Ex vivo fluorescent imaging of the probes. a: representative images of dissected organs of mice bearing U87MG tumor 8 h after injection of Cy5.5-RKL (1.5 nmol), Cy5.5-NKL (1.5 nmol) or Cy5.5-DKL (1.5 nmol ) with or without co-injection of the corresponding unlabeled peptides (1, tumor; 2, brain; 3, heart; 4, lung; 5, liver; 6, stomach; 7, intestine; 8, bone; 9, muscle; 10, spleen; 11, kidney; 12, blood). The fluorescence intensity of major tissues of Cy5.5-RKL (b), Cy5.5-NKL (c) or Cy5.5-DKL (d)


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were constructed according to ROI analysis at 8 h pi.

Figure 7. Time-course fluorescence imaging of the orthotopic glioma mouse model. a: fluorescence images of Cy5.5-RKL in the orthotopic U87MG glioblastoma model from 0.5 to 8h post injection. b: in vivo targeting quantification of Cy5.5-RKL in orthotopic U87MG glioblastoma vs. muscle. c: tumor contrast (tumor-to-muscle) calculated from ROI



measurement at different time points after administration of Cy5.5-RKL. d: tumor contrast (tumor-to-normal brain) calculated from ROI measurement at different time points after administration of Cy5.5-RKL.

Figure 8. Near-infrared fluorescence imaging for the delineation of boundary of glioma. a: NIRF imaging delineates boundary of glioblastoma and normal brain tissue at 8 h after intravenous injection of Cy5.5-RKL. The tumor can be clearly visualized in situ through NIRF imaging. b: H&E staining of the brain containing orthotopic glioblastoma (2 ) and the magnification (40 ) of the part surrounded by the blue rectangle, which clearly showing the boundaries between tumor tissue and normal brain tissue. c: immunohistochemical analysis of tumor tissue staining by anti-αv antibody (1:100) (40 ). d: immunohistochemical analysis of tumor tissue with anti-CD13 antibody (1:100) (40 ).


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Table of contents graphic 340x142mm (149 x 149 DPI)


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Near-infrared Fluorescent Peptides with High Tumor Selectivity: Novel

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