99mTc-HisoDGR as a Potential SPECT Probe for Orthotopic Glioma

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Tc-HisoDGR as a Potential SPECT Probe for Orthotopic Glioma Detection via Targeting of Integrin α5β1 Haitao Zhao,† Hannan Gao,† Luoping Zhai,† Xujie Liu,† Bing Jia,† Jiyun Shi,*,‡ and Fan Wang*,†,‡ †

Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, China ‡ Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China S Supporting Information *

ABSTRACT: Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5β1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported. Developing α5β1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvβ3/αvβ5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5β1-specific probe 99mTc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5β1 in vivo, the mouse models of α5β1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of 99mTc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that 99m Tc-HisoDGR represented a powerful molecular probe for integrin α5β1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5β1-targeted drugs, and assessing and monitoring the response to integrin α5β1-targeted drugs in clinical trials.

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INTRODUCTION

becoming a pertinent therapeutic target and a promising prognostic biomarker for glioma cancer patients.4,9 The growing interest in α5β1 leads to a steadily increasing demand for highly active and selective compounds, especially small-molecule inhibitors. Until now, few have been developed as α5β1-specific radiotracers. Neubauer et al.14 recently reported a 68 Ga-labeled aza-glycine-derived α5β1 antagonist that was initially characterized by Heckmann et al.15,16 based on a nonpeptidic tyrosine scaffold. D’Alessandria et al.17 and Notni et al.18 reported a 68Ga-labeled α5β1 integrin targeted highly active antagonist ligand FR366 and a trimeric pseudopeptide, respectively. All three peptidomimetic radiotracers showed promising PET imaging results in α5β1-positive tumor bearing models and dominant selectivity from αvβ3. However, the PET imaging time point for those tracers is 70−90 min post-injection (p.i.), which may require a relatively high injection dose due to the short half-life of 68Ga (68 min). Koivunen et al.19 isolated a

Glioma is a highly aggressive malignant primary brain tumor associated with a poor prognosis.1,2 Invasion of glioma cells into the healthy brain tissue requires the interaction of extracellular matrix (ECM) molecules with surrounding cells.3 Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy due to their expression on the surface of cancer cells and the tumor neovasculature, as well as their proposed role in mediating angiogenesis, tumor growth, and metastasis.4−7 Among them, αvβ3/αvβ5 have been studied most extensively as diagnosis and therapy targets in recent decades. Although the critical role of α5β1 integrin in physiological angiogenesis and development has been recognized for over two decades, research focusing on α5β1 is far from adequate.8,9 Recently, α5β1 has been highlighted as a prognostic and diagnostic marker in several solid tumors, including glioma.9−13 Integrin α5β1 expression is correlated with a worse prognosis in high-grade glioma, and thus it is © XXXX American Chemical Society

Received: February 22, 2016 Revised: April 18, 2016

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DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry lead peptide c(C*RRETAWAC*) (asterisks indicate a disulfide bridge) from a phage display library with a high specificity and affinity for α5β1. However, the biodistribution studies of 18Flabeled tracer based on this peptide demonstrated that it was not suitable for in vivo tumor imaging due to its considerably high and constant radioactivity accumulation in the blood and other major organs.20 Zhaohui Jin et al.21 functionalized a α5β1-specific fibronectin mimetic peptide sequence KSSPHSRN(SG)5RGDSP (called PR_b) as a PET imaging probe. The α5β1-positive tumors could be clearly visualized by 18F-PR-b PET imaging. However, the linear peptide degraded and metabolized rapidly in vivo, resulted in a relatively high kidney uptake. Therefore, a need remains to develop efficient molecular probes for noninvasive in vivo imaging of α5β1 expression. More recently, isoAsp-Gly-Arg (isoDGR)-derived peptides, working as novel integrin binders, have attracted increasing attention.22−26 As a mimetic of Arg-Gly-Asp (RGD), isoDGR can recognize RGD-dependent integrins (such as αvβ3, αvβ5, αvβ6, αvβ8, and α5β1) with different affinity and selectivity, depending on the isoDGR conformation and molecular scaffold.24−27 Among the isoDGR derivatives, the head-to-tail cyclic hepa-peptide c(phg-isoDGRk) with the flanking residues D-phenylglycine (phg) and D-lysine (k) showed outstanding selective activity for α5β1 (IC50 = 8.7 ± 0.7 nmol) while retaining inactivity toward αvβ3, thus demonstrating great potential for development as a novel integrin α5β1-targeted molecular imaging probe for tumor detection.24 In this study, we intended to develop the c(phg-isoDGRk) (shorted as isoDGR) as a SPECT (single-photon emission computed tomography) imaging probe for in vivo glioma detection. First, the bifunctional chelator 6-hydrazinonicotinyl (HYNIC) was conjugated to c(phg-isoDGRk) via the amino group of the lysine side chain to obtain HYNIC-c(phg-isoDGRk) (named HisoDGR). For 99mTc labeling, TPPTS and tricine were used as coligands to prepare the tracer 99mTc-HisoDGR. To evaluate the targeting capability of this novel SPECT imaging probe, subcutaneous and orthotopic U87MG human glioma xenograft mouse models were employed. In vivo biodistribution and SPECT imaging of 99mTc-HisoDGR were performed in U87MG tumor models. In vitro cell binding and ex vivo tissue staining were also determined to verify the specificity and selectivity of 99mTc-HisoDGR for integrin α5β1. As a result, 99m Tc-HisoDGR represented a promising molecular probe for the detection of integrin α5β1 expression in cancer.

Figure 1. Chemical structures of (A) c(phg-isoDGRk) peptide and (B) 99m Tc-radiolabeled compound 99mTc-HisoDGR. (C) Typical radioHPLC chromatogram of 99mTc-HisoDGR.

The stability of the radiotracer in physiological medium is essential for further investigation in vitro and in vivo. Therefore, the in vitro stability of 99mTc-HisoDGR was assessed for 4 h at room temperature in saline. The results revealed that 99mTcHisoDGR remained intact after 4 h incubation in saline. Moreover, the in vivo stability of 99mTc-HisoDGR was also tested, and it was found to remain intact in the urine sample at 4 h p.i., as shown in Radio-HPLC chromatography (Figure S2). Therefore, 99mTc-HisoDGR showed a potent resistance to degradation or transchelation, which warrants its further exploration for targeting integrin α5β1 in vitro and in vivo. Moreover, the in vitro chemical properties of 99mTc-HisoDGR was also evaluated in an equal volume mixture of n-octanol and 25 mM phosphate buffer (pH 7.4). The partition coefficient (log P) of 99mTc-HisoDGR was calculated to be −3.84 ± 0.06. In Vitro Integrin α5β1 Specificity. Previous research has shown that the isoDGR sequence can potently bind to integrin α5β1 while also holding binding potentiality to integrin αvβ6.24 Thus, the expression level of these two integrins in the U87MG human glioma cell line was determined. As shown in Figure 2A, fluorescence-activated cell sorting analysis clearly showed that U87MG tumor cells were integrin α5β1-positive (98%) and integrin αvβ6-negative (0%). The binding specificity of isoDGR toward U87MG tumor cells was investigated in vitro, and the results are shown in Figure 2B. There was a statistically significant (P < 0.01) difference between the 99mTc-HisoDGR binding group and blocking group (with excess cold peptide), indicating that this isoDGR peptide could specifically bind to U87MG tumor cells in vitro. To analyze the specific interactions between isoDGR peptides and integrin α5, a surface plasmon resonance (SPR) assay was performed to characterize the affinity. As shown in Figure S3, both HisoDGR and isoDGR can specifically bind to the integrin α5 protein immobilized on the CM5 chip. The KD of HisoDGR to the integrin α5 was almost

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RESULTS Chemistry and Radiochemistry. HisoDGR was prepared by direct conjugation of isoDGR peptide with HYNIC-NHS (Figure 1A,B). The product was obtained in approximately 60% yield and confirmed by HPLC (retention time = 22.4 min, purity >98%). The MALDI-TOF-MS measured molecule weight (MW) ([M + H]+ = 893.4) and was consistent with the expected MW (892.95). HisoDGR (Figure S1) was radiolabeled with Na99mTcO4 via a robust method28,29 using TPPTS and tricine as coligands with a yield of >95% and specific activity of >30 GBq/μmol. Although the purification appears to not be necessary, the tracer underwent purification with Sep-Pak C18 cartridges, which improved the radiochemical purity of the tracer to >99%. The Radio-HPLC chromatography of the purified tracer is shown in Figure 1C. The well-prepared probe was used in the following experiments directly after the quality control, without further purification. B

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. (A) Representative flow cytometry histograms of U87MG cells without (black-outlined spectrum) and with (green-outlined spectrum) the addition of anti-integrin α5β1 and αvβ6 antibody; “+” represents positive, and “−” represents negative. (B) Binding of 99mTc-HisoDGR to U87MG cells without or with isoDGR peptide blocking; **P < 0.01.

Figure 3. (A) Biodistribution of 99mTc-HisoDGR in U87MG glioma tumor-bearing mice at 0.5, 1, and 2 h p.i. (B) Biodistribution of 99mTc-HisoDGR in blocking group at 0.5 h p.i. Blocking group was coinjected with cold isoDGR peptide as a blocking agent; **P < 0.01.

identical to that of isoDGR with the KD values of 7.74 × 10−7 and 7.79 × 10−7 mol/L, respectively. Biodistribution. Biodistribution studies were performed in the U87MG glioma tumor-bearing model. The blocking experiment was performed by coinjecting 400 μg of cold isoDGR peptide. As shown in Figure 3A, the uptake values of 99m Tc-HisoDGR in U87MG tumors were 1.71 ± 0.04, 0.43 ± 0.03, and 0.22 ± 0.02%ID/g at 0.5, 1, and 2 h p.i., respectively. The uptake in other normal organs, such as the heart, bone, muscle, and liver, demonstrated low background uptake in vivo

(Figure 3A). For example, tumor-to-liver and tumor-to-muscle ratios were 2.2 and 3.0 at 0.5 h p.i. and 1.7 and 4.2 at 1 h p.i., respectively, indicating that in vivo visualization of the tumors with good contrast should be feasible. The uptake was highest in the kidneys, which revealed that renal clearance was the main route of 99mTc-HisoDGR elimination from the body. In the blocking group, the tumor uptake was remarkably inhibited to 0.77 ± 0.16%ID/g (Figure 3B), with a statistically significant reduction compared to the uptake in the nonblocking group at 0.5 h p.i. (p < 0.01). The results demonstrated that the 99mTcC

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. Representative small-animal NanoSPECT/CT images of 99mTc-HisoDGR in subcutaneous U87MG glioma xenograft model obtained at 0.5, 1, and 2 h post-injection. The blocking group was coinjected with excess cold isoDGR peptide as a blocking agent. Arrows indicate tumor location. For all the SPECT images, bladder was excluded during imaging reconstruction. (P represents posterior view.)

HisoDGR specifically targeted to U87MG tumors and possessed high tumor-to-background ratios in vivo. Small-Animal SPECT/CT Imaging. The in vivo targeting ability of 99mTc-HisoDGR was also evaluated by small-animal SPECT/CT imaging in mice bearing subcutaneous U87MG human glioma xenografts, which were classified into two groups of the imaging group and the blocking group. Representative images at 0.5, 1, and 2 h are shown in Figure 4. The tumors were clearly visualized with low background in the imaging group. The tracer was mostly undetectable in normal organs except for the kidneys. In the blocking group, injection of 99mTc-HisoDGR together with excess cold peptide only permitted indistinct tumor imaging compared to the imaging group at all tested time points. Whole body imaging was consistent with the biodistribution results. High accumulation of radioactivity was observed in the kidneys, which confirmed that the probe was excreted by the kidneys. The results convincingly verified the specificity of this probe targeting U87MG tumors, suggesting that 99mTc-HisoDGR could be an efficient molecular probe for SPECT imaging of integrin α5β1 expression. In the orthotopic tumor model, the maximum intensity projection (MIP) images of the nanoscanSPECT scans showed that 99mTc-HisoDGR was notably localized in the brain area with a high ratio of tumor-to-background (Figure 5). Furthermore, transverse, coronal and sagittal brain section images were taken to further characterize the localization of 99mTc-HisoDGR in the brain area (Figure 5). Immunofluorescent Staining. Immunofluorescent staining was performed to characterize the microdistribution of isoDGR in U87MG tumors. As shown in Figure 6, U87MG tumor tissues were stained positively for human integrin α5β1 and αvβ3. Overlying imaging revealed that most of the isoDGRFITC-positive areas coexpressed integrin α5β1 but little αvβ3, indicating that isoDGR specifically and selectively targeted

Figure 5. Representative small-animal NanoSPECT/CT MIP images and brain slice images (including transverse, coronal, and sagittal sections) of 99mTc-HisoDGR in an orthotopic U87MG glioma tumor model obtained at 0.5 h p.i. Arrows and crosshairs indicate tumor location. For all the SPECT images, bladder was excluded during imaging reconstruction. (P represents posterior view; R represents right lateral view.)

U87MG human glioma tumors, mainly via the mediation of integrin α5β1 rather than αvβ3.

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DISCUSSION Integrins play an important role in glioma proliferation and invasion, and they represent attractive targets for glioma D

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 6. Immunofluorescence staining of FITC-isoDGR colocalization with human integrin α5β1 and αvβ3 in ex vivo U87MG tumor tissues harvested from tumor-bearing mice. Red represents secondary antibodies conjugated dylight-649, green represents FITC-isoDGR, and blue represents DAPI for visualization of nuclei. Arrows indicate the overlaid area.

diagnosis and antitumor therapy. Clinically, integrin α5β1 expression was associated with a more aggressive phenotype in brain tumors, and high integrin α5 gene expression was associated with decreased survival of patients with high-grade glioma.3,30,31 Therefore, imaging of integrin α5β1 may be particularly useful for detecting highly aggressive glioma tumors. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported.14,19−21 More recently, a novel cyclic pentapeptide c(phg-isoDGRk) has been developed and proven to exhibit high binding affinity for integrin α5β1, providing an excellent platform for developing integrin α5β1-specific imaging probes.24 In this study, the c(phg-isoDGRk) peptide (shorted as isoDGR) was modified with bifunctional chelator and designed as an imaging probe for glioma detection. The amino group of the lysine side chain in c(phg-isoDGRk) provides great convenience for chelator conjugation. First, 99mTc was chosen as the imaging nuclide because of its high availability and low cost compared to other nuclides. Then, HYNIC-NHS was chosen as a chelator for 99mTc-labeling and conjugated to the isoDGR peptide to obtain HisoDGR, while TPPTS was used as a reducing agent and together with tricine as coligands to prepare 99m Tc-HisoDGR (Figure 1).28,29,32 The radiotracer was prepared with high radiolabeling yield and high specific activity, which allows the formulation of a kit for the rapid preparation of radiotracer for nuclear medicine applications, according to our previous experience on a kit formularized with HYNICconjugated RGD peptides.29,33 Furthermore, 99mTc-HisoDGR showed excellent stability both in vitro and in vivo for at least 4 h, possibly because it is a cyclic peptide with D-phenylglycine (phg) and D-lysine (k) in the loop, and one is modified as a non-nature amino acid (phg). The tracer 99mTc-HisoDGR also showed high hydrophilic properties according its log P value (−3.84 ± 0.06), which may indicate the tracer would be washed preferentially via kidney and urinary system. For the in vitro and in vivo bioevaluation of 99mTc-HisoDGR, an integrin α5β1-positive U87MG human glioma cell line was employed for cell study and the establishment of animal models. In previous research, this isoDGR peptide also showed binding affinity to integrin αvβ6 in addition to integrin α5β1.24 However, the affinity of integrin αvβ6 has been mostly excluded in this study, as the U87MG cell was α5β1-positive and αvβ6-negative according to the results of flow cytometry analysis (Figure 2A).

Cell binding results demonstrated that the tracer 99mTcHisoDGR could specifically bind to U87MG cells in vitro (Figure 2B). Therefore, the U87MG tumor-bearing mice were further used for evaluating the in vivo biobehavior of 99mTcHisoDGR. Like the biodistribution data shown in Figure 3, 99m Tc-HisoDGR highly accumulated in U87MG tumors, and its uptake could be significantly inhibited by coinjection of excess cold isoDGR peptide, meaning that the uptake of radiotracer in the tumors was specifically mediated by the receptor. However, the tumor uptake decreased dramatically at 1 and 2 h p.i., which may be partially due to the relative low KD of HisoDGR. Uptake in normal organs was relatively low compared to previously reported α5β1-specific radiotracers. The highest uptake was found in the kidneys, suggesting that the tracer was mainly excreted via renal clearance. SPECT imaging of 99mTc-HisoDGR in U87MG tumor-bearing mice was consistent with the biodistribution results. Tumors were clearly visualized in MIP images of 99mTc-HisoDGR SPECT/CT with low background in all normal organs except the kidneys, as shown in Figure 5. The blocking group, which was coinjected with excess cold isoDGR, showed notable reduction of 99mTc-HisoDGR radioactivity in the tumor area, resulting in indistinct tumor imaging. These findings confirmed that the 99mTc-HisoDGR could specifically target U87MG glioma tumor. To investigate the potential of 99m Tc-HisoDGR for the detection of orthotopic glioma, an orthotopic U87MG human glioma mouse model was established. With the clear background of 99mTc-HisoDGR in the brain, the orthotopic brain tumors were clearly visualized in MIP images of 99mTc-HisoDGR SPECT/CT and further distinctly localized by its SPECT imaging in coronal, transverse, and sagittal brain section images, suggesting that 99mTc-HisoDGR has the potential for glioma detection in clinical applications. The relatively low background and favorable pharmacokinetic properties of 99mTc-HisoDGR in most normal organs also warrant the detection of metastasis sites in the whole body. Furthermore, the in vivo targeting mechanism of 99mTcHisoDGR was confirmed by immunofluorescence staining of FITC-conjugated isoDGR (isoDGR-FITC), which colocalized with integrin α5β1 staining in ex vivo U87MG tumor tissue. Because the U87MG tumors show well-known overexpression of integrin αvβ3, isoDGR-FITC colocalization with integrin αvβ3 staining was also performed as a control for integrin α5β1 staining. As shown in Figure 6, overlaid areas (shown in yellow) E

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry of isoDGR-FITC (green) and integrin α5β1 staining (red) were magnified and indicated with arrows, which revealed that most of the isoDGR-FITC-positive areas coexpressed integrin α5β1, while almost no overlaid areas of isoDGR-FITC (green) and integrin αvβ3 staining (red) were observed. It showed that isoDGR targeted to U87MG tumor mainly through integrin α5β1.

Determination of Log P. The radiotracer 99mTc-HisoDGR was prepared and purified by Sep-Pak C18 cartridges (Waters). Volatiles in the purified product were removed under vacuum. The residue was dissolved in an equal volume (500 μL:500 μL) mixture of n-octanol and 25 mM phosphate buffer (pH 7.4). After stirring vigorously for 30 min, the mixture was centrifuged at a speed of 10 000 rpm for 5 min. Samples (in triplets) from both n-octanol and aqueous layers were obtained and measured in a γ-counter. Partition coefficient was tested three times. The log P value was reported as the average of three independent measurements plus the standard deviation. Cell Culture and Establishment of Animal Models. The human glioma cell line U87MG (ATCC HTB-14) was purchased from American Type Culture Collection (Manassas, VA). The U87MG cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/ v) fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. Female BALB/c nude mice (4−6 weeks of age) were purchased from the Department of Experimental Animals, Peking University Health Science Center. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University. The xenografted tumor model was established by subcutaneous injection of 2 × 106 tumor cells into the right flank. When the tumor volume reached 200−300 mm3 (3−4 weeks after inoculation), the tumor-bearing nude mice were used for biodistribution and imaging studies. The orthotopic tumor model was established by intracranial injection of ∼105 U87MG tumor cells into the frontal white matter of nude mice and was used for SPECT imaging at 10−12 days post-inoculation. Flow Cytometry Analysis. The expression level of integrin α5β1 in the U87MG cell line was validated by fluorescenceactivated cell sorting (FACS) analysis. Briefly, the cells were harvested and suspended in PBS supplemented with 5% bovine serum albumin (BSA). The cells were incubated with the mouse anti-human integrin α5β1 antibody (Chemicon, Millipore, USA) for 1 h at 4 °C. After washing with cold PBS, the cells were incubated with the FITC-conjugated goat anti-mouse secondary antibody (Invitrogen, Thermo, USA) for 30 min at 4 °C. The cells were washed, resuspended in PBS, and then analyzed using a flow cytometer (Becton Dickinson, Germany). Because the c(phg-isoDGRk) peptide was previously proven to specifically bind to both integrin α5β1 and αvβ6, the expression of integrin αvβ6 in the U87MG cell line was also determined as above. Cell Binding Assay. The in vitro binding specificity of isoDGR toward integrin α5β1 on U87MG cells was evaluated. The tracer 99mTc-isoDGR (74 kBq) was added to the suspension of U87MG cells in 1.5 mL centrifuge tubes with or without an excess amount (10 μg/tube) of cold isoDGR peptide. After incubation at 4 °C for 2 h, the cells were washed and collected. The cell-associated radioactivity of each tube was measured using a γ-counter. Results were expressed as radioactivity counts of per 107 cells. Biodistribution. Twelve U87MG human glioma xenograftbearing mice were randomly divided into three groups of four animals each. A dose of 0.37 MBq 99mTc-HisoDGR (in 0.1 mL saline) was administered intravenously to each mouse. The biodistribution studies were performed by euthanizing the mice at 0.5, 1, and 2 h post-injection (p.i.). The blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain, and tumor were harvested, weighed, and measured for radioactivity in a γ-counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue mass (%ID/g). The

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CONCLUSION In this study, a novel potential SPECT imaging probe, 99mTcHisoDGR with integrin α5β1 specificity, was fabricated and fully evaluated in subcutaneous and orthotopic U87MG human glioma xenograft mouse models. This radiotracer can be easily prepared with high RCP and possesses promising tumor targeting capability and favorable pharmacokinetics properties in vivo. Because integrin α5β1 is overexpressed on both glioma tumor cells and tumor vascular endothelium, it plays important roles in tumor progression, angiogenesis, and metastasis. 99mTcHisoDGR may be a promising imaging probe for glioma tumor in the use of evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to α5β1targeted drugs, and assessing and monitoring the response to α5β1-targeted drugs in clinical trials.

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EXPERIMENTAL SECTION

Materials and Methods. Synthesis of HYNIC-isoDGR Conjugate. To obtain HisoDGR conjugates, a solution of 7 μmol isoDGR peptide in 500 μL of 0.1 M sodium bicarbonate and sodium carbonate buffer (pH 9.0) was mixed with 14 μmol sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-NHS) in 100 μL of N,Ndimethylformamide (DMF). After stirring for 24 h at room temperature, HisoDGR was isolated by semipreparative HPLC. Fractions containing the product were collected and lyophilized. HisoDGR was obtained at approximately 60% yield with >98% purity. The identity of the product was confirmed by mass spectrometry (m/z, 893.4 for [M + H]+). Preparation of 99mTc-HisoDGR. For 99mTc radiolabeling, 20 μg of HisoDGR was added to a combined solution of 100 μL of tricine solution (100 mg/mL in 25 mM succinate buffer, pH 5.0) and 100 μL of TPPTS (trisodium triphenylphosphine-3,3′,3″trisulfonate) solution (50 mg/mL in 25 mM succinate buffer, pH 5.0); 370 MBq Na99mTcO4 was then added to the solution, and the mixture was heated at 100 °C for 20 min. After cooling to room temperature, the sample underwent Radio-HPLC for radiolabeling yield measurement. Purification was performed with Sep-Pak C18 cartridges (Waters) when necessary. After purification, the radiochemical purity of 99mTc-HisoDGR was reanalyzed by radio-HPLC. Stability of 99mTc-HisoDGR. The in vitro stability of 99mTcHisoDGR was tested in saline. 99mTc-HisoDGR (37 MBq) was incubated in 1 mL of saline at room temperature for 4 h. The samples from 0.5, 1, 2, and 4 h were analyzed by radio-HPLC under identical conditions used to analyze the original radiolabeled compound. The percent of intact 99mTc-HisoDGR was determined by quantifying peaks corresponding to the intact and degraded products. The assays were repeated twice. In vivo stability of 99mTc-HisoDGR was also evaluated in nude mice. 99mTc-HisoDGR (37 MBq) was injected into mice via the tail vein. The urine of the mice at 0.5, 1, 2, and 4 h postinjection was collected and filtered. The samples were then analyzed by the above-mentioned methods. The assays were repeated twice. F

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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blocking studies were also performed in U87MG tumor-bearing nude mice (n = 4), and each mouse was coinjected with 400 μg of cold isoDGR peptide and 0.37 MBq of 99mTc-HisoDGR. The mice were sacrificed, and biodistribution of 99mTc-HisoDGR was determined at 0.5 h p.i. Small-Animal SPECT/CT Imaging. Small-animal SPECT/ CT imaging in female nude mice bearing subcutaneous and orthotopic U87MG xenografts was obtained using a NanoScanSPECT/CT imaging system (Mediso Inc.). Each mouse was injected via the tail vein with 37 MBq of 99mTc-HisoDGR. The integrin receptor specificity of 99mTc-HisoDGR was demonstrated by coinjecting excess cold isoDGR peptide (∼400 μg) along with the tracer in mice assigned to the blocking group. The mice were anesthetized by inhalation of 2% isoflurane (Abbott Laboratories, Shanghai) and imaged at 0.5, 1, and 2 h p.i. The SPECT and CT fusion images were obtained using the automatic fusion feature of the Nucline 2.0 program (Mediso Inc.). For all the SPECT images, bladder was excluded during imaging reconstruction. Immunofluorescence Staining. To determine the intratumoral microdistribution of isoDGR peptide, a subset of surgically removed U87MG tumors were immediately frozen in OCT (optimal cutting temperature) medium and cut into 10μm-thick slices. After fixation with ice-cold acetone, tumor sections were incubated with mouse anti-human integrin α5β1 monoclonal antibody (Millipore, Billerica, MA, USA) for 10 h at 4 °C and then incubated with isoDGR-FITC for 2 h at room temperature. The slices were then visualized using dylight-649 conjugated secondary antibodies (Invitrogen, Thermo, USA) under a confocal microscope. The colocalization of isoDGRFITC and integrin αvβ3 was also determined in this assay. Statistical Analysis. Quantitative data are expressed as mean ± SD. Means were compared using the Student’s t test. P values of less than 0.01 were considered statistically significant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00098. Method of Surface plasmon resonance (SPR) and Figures S1−S3 (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported, in part, by the National Natural Science Foundation of China (NSFC) projects (81571727, 81125011, 81321003, 81427802, and 81420108019), grants from the Ministry of Science and Technology of China (2011YQ030114 and 2012ZX09102301018), a grant from Beijing Ministry of Science and Technology (Z141100000214004), and the Beijing Natural Science Foundation (BJNSF) project (7142086),Youth Innovation Promotion Association of Chinese Academy of Sciences (2016090). The authors would like to thank Lijun Zhong for assistance with mass spectrometry. G

DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.bioconjchem.6b00098 Bioconjugate Chem. XXXX, XXX, XXX−XXX