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Aminopeptidase N (CD13) targeted MR and NIRF dual-modal imaging of ovarian tumor xenograft
Materials Science & Engineering C 93 (2018) 968–974
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Aminopeptidase N (CD13) targeted MR and NIRF dual-modal imaging of ovarian tumor xenograft ⁎
Ying Menga,1, Zixin Zhangb,1, Kang Liuc, Ling Yec, Yuting Lianga, , Wei Guc,
T
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a
Department of Radiology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, PR China Department of Radiology, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, PR China c School of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian tumor xenograft Aminopeptidase N (APN/CD13) Asparagine–glycine–arginine (NGR) MR imaging NIRF imaging
The development of tumor-specific imaging nanoprobes with the potential to improve the accuracy of cancer diagnosis has become an area of intense research. Aminopeptidase N (CD13) predominantly expresses on the surface of ovarian tumor cells and can be specifically recognized by Asn-Gly-Arg (NGR) peptide. The applicability of CD13 as a target for specific ovarian tumor imaging, however, remains unexploited so far. In this study, Cy5.5-labeled, NGR-conjugated iron oxide nanoparticles (Cy5.5-NGR-Fe3O4 NPs) were prepared as an ovarian tumor specific bimodal imaging nanoprobe. It is demonstrated that the conjugation of NGR targeting moiety leads to a higher cellular uptake toward ES-2 cells, the human ovarian carcinoma cells that highly express CD13. Moreover, magnetic resonance imaging of ovarian tumor xenograft reveals that the Fe3O4-Cy5.5-NGR NPs results in a significant T2* signal reduction in the tumor. Meanwhile, near infrared fluorescence imaging indicates a higher accumulation of Fe3O4-Cy5.5-NGR NPs in the tumor xenograft. Therefore, CD13 could be applied as a novel and efficient target for constructing ovarian tumor specific nanoprobes with improved accuracy for ovarian tumor diagnosis.
1. Introduction Ovarian cancer is the leading cause of death among the gynecological malignancies [1,2]. The high mortality rate is largely due to the lack of early clinical symptoms and the rapid progression to peritoneal metastases [3,4]. Therefore, the preoperative qualitative diagnosis and early detection of ovarian tumors are considered as key steps for optimal clinical treatment strategy and improved patient prognosis. Although transvaginal ultrasonography (US) and computed tomography (CT) are conventional imaging techniques currently used to evaluate ovarian tumors in clinic, non-invasive magnetic resonance (MR) imaging is increasingly being used in patients with gynecological disorders due to its high contrast resolution compared to US and CT. Particularly, when US findings are suboptimal or indeterminate, MR imaging is of great help in preoperatively identifying malignant lesions. Moreover, the detection accuracy of ovarian tumors could be further improved by using MR contrast agents. For instance, superparamagnetic iron oxide NPs have been extensively investigated as an MR contrast agent for cancer diagnosis due to their excellent biocompatibility and magnetic properties.
Recently, development of tumor specific nanoprobes, which generally involves the use of active targeting strategies that rely on specific receptor–ligand interactions at the cell surface, has emerged as one of the most promising way to achieve specific accumulation of nanoprobes within tumors for broader and more effective diagnosis in various cancer types. It is well known that tumor cells express a variety of biomarkers on their surface [5–7]. These biomarkers are critical in the growth, progression, and metastasis of solid tumors and have been used to confirm diagnosis and disease stage. On the other hand, these unique biomarkers could be utilized for constructing tumor-specific NPs through receptor–ligand interactions. In the case of ovarian tumor cells, one of the most promising biomarkers is CD13, also known as aminopeptidase N [8]. As a Zn2+-dependent transmembrane ectopeptidase expressed on cell surface of the myeloid lineage and on nonhematopoietic tissue [9–11], CD13 possesses a number of functions, such as signal transduction, neuropeptide and cytokine degradation, antigen processing and presentation and extracellular matrix degradation associated with the progression of cancers and inflammatory reaction [12–14]. Moreover, accumulated experimental evidence suggests that CD13 is implicated in the pathogenesis of tumor. On this
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Corresponding authors. E-mail addresses: [email protected] (Y. Liang), [email protected] (W. Gu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.msec.2018.09.002 Received 28 January 2018; Received in revised form 9 August 2018; Accepted 1 September 2018 Available online 02 September 2018 0928-4931/ © 2018 Published by Elsevier B.V.
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obtained Fe3O4-Cy5.5 NPs were purified by centrifugal filter (MWCO = 5000 Da) and subjected to freeze-drying.
occasion, the interest in this enzyme as a therapeutic target has been progressively increasing. However, the applicability of CD13 as a target for ovarian tumor diagnosis has not been exploited so far. It is reported that CD13 can be recognized by peptides containing Asn-Gly-Arg (NGR) [15,16], a motif originally discovered by screening peptide-phage libraries in tumor-bearing mice. In this study, we synthesized NGR peptide conjugated, Cy5.5 labeled iron oxide (Fe3O4Cy5.5-NGR) NPs as a targeted MR/NIRF dual-modal imaging nanoprobe to test the applicable of CD13 as a target for ovarian tumor imaging with enhanced contrast effects. Poly(ethyleneglycol) (PEG) was also integrated to serve both as a biocompatible coating and linking molecule for the covalent attachment of the Cy5.5 and NGR to the iron oxide NPs. Cytotoxicity of Fe3O4-Cy5.5-NGR NPs was evaluated by CCK-8 cell viability assays, while their tumor targeting ability was examined by in vitro cellular uptake and in vivo MR and NIRF imaging of ovarian tumor xenograft. It is disclosed that CD13 could be utilized as a suitable target for ovarian tumor specific imaging with an enhanced contrast effect.
2.5. Synthesis of Fe3O4–Cy5.5-NGR NPs To 3 ml of PBS (pH = 6), 60 mg of Fe3O4–Cy5.5 NPs, 9.0 mg of NHS, and 6.0 mg of EDC were added. After stirring for 30 min, 3.0 mg of NGR and 66 mg of H2N–PEG3500–NH2 were added (pH = 8). Note that the weight ratio between Cy5.5-NGR and Fe3O4 NPs is 6%. The reaction was allowed for 12 h under stirring. Then, the product was purified using a centrifugal filter (MWCO = 5000 Da) and lyophilized to yield Fe3O4–Cy5.5–NGR NPs. 2.6. Characterization TEM images of NPs were obtained on a JEM-2010 (JEOL, Japan) at an operating voltage of 160 kV. Fourier transform infrared spectroscopy (FTIR) spectra were acquired on an ATR diamond-crystal equipped miniature FTIR spectrometer (iS5 FT-IR spectrometer, Thermo Nicolet Instrument Corp). The content of Fe was measured on an inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710ES, USA).
2. Experimental 2.1. Materials
2.7. Cell culture
Iron (III) acetylacetonate (Fe(acac)3), trioctylamine, benzyl ether, 1,2-hexadecanediol, oleic acid (OA) and oleylamine were purchased from Sigma-Aldrich. NGR (c-RYKNG) was purchased from GL Biochem Ltd. (Shanghai, China). Polyethylene glycol (H2N–PEG2000–NH2 and H2N–PEG3500–NH2) were received from JenKem Technology Co. Ltd. (Beijing, China). N-(Trimethoxysilylpropyl) ethylene diamine triacetic acid, trisodium salt (TETT, 45% in water) was obtained from Gelest Inc. All chemicals were in analytical grade and used without further purification. The water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA).
ES-2 cells, a type of human ovarian carcinoma cells highly expressed CD13, were obtained from China infrastructure of cell line resources and cultured in McCoy's 5A media with 10% fetal bovine serum (FBS) 4, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in the presence of 5% CO2 in humidified incubator. 2.8. Cytotoxicity assay
The Fe3O4–OA NPs were prepared through the thermal decomposition method [17]. Firstly, 1.413 g of Fe(acac)3, 5.169 g of 1,2hexadecanediol, 4 ml of oleic acid, 4 ml of oleylamine, and 40 ml of benzyl ether were mixed and preheated at 100 °C for 1 h under vacuum, then heating at 200 °C for 2 h, and 300 °C for 1 h under a nitrogen flow. After cooling to room temperature, ethanol was added to precipitate the product. The precipitation was washed twice with hexane containing 0.5 ml of oleic acid and 0.5 ml of oleylamine. The purified Fe3O4–OA NPs were collected by centrifugation.
The cytotoxicity of NPs was evaluated via a Cell Counting Kit-8 (CCK-8, water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzene disulfonate) F-2H-tetrazolium monosodium salt]) cell cytotoxicity assay. ES-2 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well. The cells were cultured in McCoy's 5A media for 24 h and incubated with NPs at concentrations of 0, 12.5, 25, 50, and 100 μg/ml (10 μl per well) for another 12 h. After removing the media and washing by PBS, 10 μl of CCK was added to each well and the cells were incubated for 4 h. The optical density (OD) value was measured at wavelengths of 450 nm on a Thermo Scientific Microplate Reader and the cell viability was calculated between the experimental and the control groups.
2.3. Synthesis of TETT-Fe3O4 NPs
2.9. Flow cytometry analysis
A ligand-exchange method was used to synthesize TETT-Fe3O4 NPs [18]. 100 mg of Fe3O4–OA NPs were dissolved into the mixture of 60 ml of anhydrous toluene and 60 μl of acetic acid, followed by sonicating for 10 min, adding 3 ml of TETT dropwisely, and stirring and heating at 70 °C for 48 h. Then, the precipitate was collected, washed with toluene and ethanol for three times. After centrifuging, the product was purified by dialysis (MWCO = 5000 Da) and subjected to lyophilization.
ES-2 cells were seeded in a 6-well plate with a density of 1 × 105 per well and cultured in McCoy's 5A media for 24 h. Afterwards, Fe3O4–Cy5.5–NGR or Fe3O4–Cy5.5 NPs at different Fe concentrations (40, 80, 160 μΜ) were added and incubated for 12 h, respectively. After the removal of media, cells were washed by PBS, harvested by trypsinization, collected by centrifugation, and re-suspended for flow cytometry analysis, which was conducted on a cytometry system (Cyan-LX, DakoCytomation).
2.2. Synthesis of oleic acid-capped Fe3O4 (Fe3O4–OA) NPs
2.4. Synthesis of Cy5.5 labeled Fe3O4 NPs 2.10. Relaxivity measurement The H2N–PEG2000–Cy5.5 NPs was firstly prepared by dissolving 1 mg of Cy5.5-NHS and 60 mg of H2N–PEG2000–NH2 in PBS (pH = 8), followed by stirring at room temperature for 24 h. Next, the activated Fe3O4-TETT NPs was prepared as follows, 100 mg of Fe3O4-TETT NPs, 16.2 mg of EDC, 16 mg of NHS, and 110 mg of H2N–PEG3500–NH2 were mixed in 10 ml of PBS (pH = 5.5). After stirring for 30 min, the pH of the mixture was adjusted to 8 and H2N–PEG2000–Cy5.5 was added. The reaction was preceded under stirring for another 24 h. Finally, the
NPs dispersions with different Fe concentrations of 500, 250, 125, 62.5, and 31.25 μΜ were prepared and scanned on a 7.0 T MRI (Bruker) to obtain the relaxation times and corresponding R2 maps. The parameters for MR T2 measurements were set as follows: TR = 3000 ms, TE = 45 ms, matrix size = 256 × 256, FOV = 4.0 × 4.0 cm and slice thickness = 1 mm. The r2 relaxivity was determined from the slope of linear 1/T2 against the Fe concentration. 969
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B frequency (%)
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size (nm) Fig. 1. TEM image (a) and size distribution (b) of Fe3O4-Cy5.5-NGR NPs.
2.11. Xenograft model
3. Results and discussion
All animal experiments were performed according to protocols evaluated and approved by the ethical committee of Capital Medical University (Beijing, China). The female BALB/c mice (6 weeks of age) were inoculated subcutaneously (right lateral thigh) with 5 × 106 ES-2 cells. All mice were maintained under specific pathogen-free (SPF) conditions, allowing free access to drinking water in a light/dark cycle at 22 ± 2 °C. When tumors reached a diameter of 0.8 mm (~7 days), the mice were subjected for imaging procedures.
3.1. Synthesis and characterization of Fe3O4-Cy5.5-NGR NPs CD13 is highly and stably expressed on endothelial and subendothelial cells in angiogenesis, which is an important process in tumor growth, progression and metastasis. In this study, NGR peptide that could specifically bind the CD13 was used as the targeting moiety to synthesize Fe3O4-Cy5.5-NGR as an MR/NIRF dual-modal imaging nanoprobe. Specifically, Fe3O4–OA NPs were first prepared through the thermal decomposition method [17]. Next, the Fe3O4-Cy5.5 NPs were prepared by replacing oleate with carboxylate silane (TETT) and subsequent conjugation of PEGylated Cy5.5. Finally, the conjugation of NGR yields the Fe3O4-Cy5.5-NGR NPs. The morphology Fe3O4-Cy5.5-NGR NPs were observed on TEM image (Fig. 1a). The TEM image reveals that the NPs are well separated and in nearly cubic shape. The average diameters of Fe3O4-Cy5.5-NGR NPs as calculated from 100 individual NPs are 8.93 ± 0.77 nm (Fig. 1b). Furthermore, to verify the crystallinity of NPs, the XRD pattern was obtained and is shown in Fig. S1. The diffraction peaks can be indexed to be magnetite (PDF card # 85-1436), confirming the crystallinity of NPs. The successful conjugation of NGR peptide was confirmed by FTIR. As illustrated in Fig. 2, the IR spectrum of Fe3O4-Cy5.5-NGR NPs shows a band with moderate intensity around 1632 cm−1 originates from the characteristic C]O band of amide groups in the NGR peptide. In addition, intensive bands at 1428 and 2882 cm−1 are assigned to the vibrations of CeH from the PEG.
2.12. In vivo MR and NIRF imaging The Fe3O4–Cy5.5 and Fe3O4–Cy5.5–NGR NPs at a dosage of 5.5 mg Fe per kg body weight (0.2 ml) were intravenously injected into the tumor-bearing mice through tail vein. The mice were anesthetized by CO2 and imaged on a 7.0 T MR scanner before and after injection of NPs at 60 min, 120 min, and 24 h, respectively. MR parameters were set as follows: TR/TE = 3500/33 ms(T2WI), TR/TE = 600/8 ms(T2⁎WI), matrix size = 256 × 256, FOV = 3.5 × 3.0 cm and slice thickness = 0.5 mm. NIRF images of the tumor-bearing mice were obtained before and after injection of NPs on a NightOWL LB 983 imaging system set with a 630 nm excitation filter and a 680 nm emission filter.
2.13. Ex vivo confocal laser scanning microscopy (CLSM) imaging Tumor-bearing BALB/c mice were sacrificed immediately after MR and NIR scanning. Tumors were excised and fixed in 4% formalin and dehydrated in 30% sucrose solution for 24 h. Next, the tumors were embedded with paraffin, snap-frozen, cryosectioned into 20 μm, and stained with DAPI. All sections were sealed by neutral gum and observed on a LEICA TCS SP5 confocal microscope (Leica Microsystems, Germany) with excitation at 405 and 630 nm for DAPI and Cy5.5, respectively.
3.2. Cytotoxicity To evaluate the cytotoxicity Fe3O4-Cy5.5-NGR NPs, cell counting kit based on water-soluble tetrazolium salt WST-8 was used. For comparison purpose, the cytotoxicity of Fe3O4-Cy5.5 NPs was evaluated as well. ES-2 cells were incubated with Fe3O4-Cy5.5 or Fe3O4-Cy5.5-NGR NPs at different concentrations (12.5, 25, 50 and 100 μg/ml) for 24 h and the cell viability was determined. As shown in Fig. 3, the viability of ES-2 cells is over 90% upon the treatment of either Fe3O4-Cy5.5 or Fe3O4-Cy5.5-NGR NPs at all tested concentrations, suggesting the negligible cytotoxicity of these NPs toward ES-2 cells.
2.14. Histological and immunohistochemical analysis Tumors were excised, fixed, dehydrated, embedded with paraffin, and cryosectioned into 20 μm slices. The slides were stained with hematoxylin and eosin (H&E) and CD13 immunohistochemistry according to standard clinical pathology protocols and were examined with 40× and 100× lens on a microscope (Olympus-BX53, Olympus Inc., Japan).
3.3. Tumor targeting specificity The targeting specificity of Fe3O4-Cy5.5-NGR NPs toward E2 cells was evaluated by a quantitative monitor of cellular uptake by flow 970
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Fig. 5. The linear fitting of the R2 against the Fe concentrations for r2 relaxivity measurement and T2-weighted maps of Fe3O4-Cy5.5-NGR NPs.
Wavenumber (cm ) Fig. 2. FTIR spectrum of Fe3O4-Cy5.5-NGR NPs.
Fe3O4-Cy5.5 Fe3O4-Cy5.5-NGR
125 Cell viability (%)
cytometry. Mean fluorescence intensities of ES-2 cells were measured at 24 h after incubation with Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs, respectively. As shown in Fig. 4, the cells treated with NPs have a significantly increased fluorescence signal, suggesting that both NPs can be internalized by ES2 cells. However, NGR conjugated Fe3O4Cy5.5 NPs clearly show greater fluorescence intensity than that of NPs without NGR conjugation at all tested concentrations, indicating a higher cellular uptake resulting from the specific interaction between CD13 and NGR.
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3.4. r2 Relaxivity To investigate the feasibility of Fe3O4-Cy5.5-NGR NPs as MR contrast agents, transverse relaxivity (r2) of Fe3O4-Cy5.5-NGR NPs was measured by linear fitting the inverse relaxation time against the Fe concentration. As plotted in Fig. 5, the r2 value of Fe3O4-Cy5.5-NGR NPs is 155.49 mM−1 s−1. Such r2 relaxivity value is comparable with that reported previously [19–21]. This together with the T2-weighted maps of NPs dispersion, which exhibit a concentration dependant darken effect, confirms the applicable of Fe3O4-Cy5.5-NGR NPs as effective T2-weighted contrast agents.
25 0
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concentraion ( g/mL) Fig. 3. CCK-8 cytotoxicity assay of Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs toward ES-2 cells at different concentrations for 24 h.
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3.5. In vivo MR imaging of ovarian tumor xenograft
Fe3O4-Cy5.5
Next, in vivo T2-weighted MR imaging of mice bearing ovarian tumor xenograft was performed before and after intravenously injection of the NPs via tail vein at a dosage of 5.5 mg Fe per kg body weight. The T2*-weighted images obtained at different time points are presented in Fig. 6. In the case of Fe3O4-Cy5.5-NGR NPs group, the intensity of MR signal in ovarian tumor xenograft gradually increased (darken effect) after injection and the highest contrast enhancement was observed at 24 h after injection. In contrast, the change of MR contrast enhancement in the Fe3O4-Cy5.5 NPs group was less significant. The MR contrast enhancement was further quantitatively analyzed by calculating the contrast-to-noise ratio (CNR) [CNR = (Stumor – Stissue) / σ]. As shown in Fig. 7, the CNR values of NGR-conjugated NPs are higher at each time point than that of nontargeting NPs and it reaches the maximum at 24 h post-injection. However, it is noted that, the improvement of CNR of the NGR-conjugated NPs at 60 min and 120 min post-injection is minor. This might be due to the fact that the non-targeting NPs could also accumulate in the turmor region by the EPR effect. Nevertheless, due to the specific interaction between CD13 and NGR, the NGR-functionalized NPs could not only accumulate in the tumor region, but also could be uptake by tumor cells. As a result, a significant enhancement in CNR is evidenced
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Fig. 6. In vivo MR T2* weighted imaging of ovarian tumor xenograft pre- and post-injection of Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs at a dose of 5.5 mg Fe per kg body weight.
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at 24 h post-injection, signifying the applicable of CD13 as an effective target for NGR-based nanoprobe. Moreover, considering the fact that multiple types of receptors are typically overexpressed on the surface of tumor cells, dual-targeting or multi-targeting systems have been demonstrated to further enhance the targeting specificity [22,23]. On this occasion, the verification of CD13 as a novel and effective target for ovarian tumor would facilitate the rational design multi-targeted nanoprobes for more accurate and earlier diagnosis of ovarian tumors.
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3.6. In vivo NIRF imaging of ovarian tumor xenograft
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The conjugation of fluorescent dye Cy5.5 onto Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs imparts the NPs NIRF imaging modality. The NIRF images of tumor-bearing mice were acquired pre- and post-injection of NPs. As presented in Fig. 8, Fluorescence signals are primarily observed at spine, heart, lung and kidney at 30 min post-injection of NPs. Nevertheless, the fluorescence signal intensity at the tumor site gradually increased as time passing and show a maximum intensity at
24 h
Fig. 7. CNR analysis (n = 3).
Fig. 8. In vivo NIRF images of ovarian tumor xenograft before and post intravenous injection of Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs with a dose of 5.5 mg Fe per kg body weight. 972
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NPs
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Fig. 9. CLSM images of tumor sections that obtained from BABL/c xenograft model injected with Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs at a dosage of 5.5 mg Fe per kg body weight. Cell nuclei were stained with DAPI and red fluorescence indicates the localization of NPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 10. Histopathological staining of tumor xenograft under 40× (A) and 100× (B) microscope shows the obvious atypia, and CD13 immunohistochemical staining under different magnifications (C and D), indicating the presence of CD13 on tumor cells.
conjugation. The discrepancy of fluorescence intensity found in both flow cytometry and CLSM verifies the tumor specificity of Fe3O4-cy5.5NGR NPs.
24 h post-injection, indicating a higher accumulation of NPs at the targeting tissue while NPs are metabolized at other organs. Obviously, the NIRF imaging result is consistent with in vivo MR scanning. The high accumulation of Fe3O4-cy5.5-NGR NPs in tumor was further supported by CLSM. As illustrated in Fig. 9, NGR-conjugated NPs display much stronger red fluorescence signal than that of NPs without NGR. More importantly, the Fe3O4-cy5.5-NGR NPs indeed reside in cytoplasm, indicating the efficient cellular uptake of NPs upon NGR
3.7. Histology analysis The ovarian tumor xenograft identified by Fe3O4-Cy5.5-NGR NPs was excised and subjected to H&E histopathological and CD13 973
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immunohistochemical staining. Fig. 10A and B present the H&E staining results observed under 40× and 100× microscope. The observed atypia and the loss of their original shape clearly validate the successful establishment of ovarian tumor xenograft. Additionally, the presence of CD13 on the surface of tumor cells was confirmed by the immunohistochemical staining. Fig. 9 C and D provide CD13 immunohistochemical staining images of tumor sections. As can be seen, scattered brown punctate shadow distributes around the tumor cells, while only slightly flocculent sandy silhouettes in stromal cells, confirming the presence of CD13 on tumor cells as the target for NGR conjugated MR nanoprobes.
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4. Conclusion In conclusion, the NGR-conjugated, Cy5.5-labeled Fe3O4 NPs were synthesized and applied as a targeted MR and NIRF dual-modal nanoprobe for ovarian tumor imaging. The specific targeting ability of Fe3O4-Cy5.5-NGR toward ovarian tumor cells expressing CD13 is confirmed by in vitro flow cytometry. In vivo MR and NIRF imaging of ovarian tumor xenograft demonstrate the specificity of Fe3O4-Cy5.5NGR NPs toward ovarian tumor. These results suggest CD13 is a suitable target for constructing specific imaging nanoprobes for ovarian tumor diagnosis with enhanced contrast effects and Fe3O4-Cy5.5-NGR as CD13 target-specific dual-modal imaging nanoprobes has great potential in more accurate and earlier diagnosis of ovarian tumor. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2018.09.002. Acknowledgement This work is financially supported by the Basic-clinical Key Research Grant (15JL07) from Capital Medical University and the Beijing Natural Science Foundation (7162064). References [1] G.C. Jayson, E.C. Kohn, H.C. Kitchener, et al., Ovarian cancer, Lancet 384 (9951) (2014) 1376–1388. [2] N. Auersperg, A.S. Wong, K.C. Choi, et al., Ovarian surface epithelium: biology, endocrinology, and pathology, Endocr. Rev. 22 (2) (2001) 255–288. [3] R. Raave, R.B. de Vries, L.F. Massuger, et al., Drug delivery systems for ovarian cancer treatment: a systematic review and meta-analysis of animal studies, PeerJ 3 (2015) e1489. [4] W. Lim, W. Jeong, G. Song, Coumestrol suppresses proliferation of ES2 human epithelial ovarian cancer cells, J. Endocrinol. 228 (3) (2015) 149–160.
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Aminopeptidase N (CD13) targeted MR and NIRF dual-modal imaging of ovarian tumor xenograft ⁎
Ying Menga,1, Zixin Zhangb,1, Kang Liuc, Ling Yec, Yuting Lianga, , Wei Guc,
T
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a
Department of Radiology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, PR China Department of Radiology, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, PR China c School of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian tumor xenograft Aminopeptidase N (APN/CD13) Asparagine–glycine–arginine (NGR) MR imaging NIRF imaging
The development of tumor-specific imaging nanoprobes with the potential to improve the accuracy of cancer diagnosis has become an area of intense research. Aminopeptidase N (CD13) predominantly expresses on the surface of ovarian tumor cells and can be specifically recognized by Asn-Gly-Arg (NGR) peptide. The applicability of CD13 as a target for specific ovarian tumor imaging, however, remains unexploited so far. In this study, Cy5.5-labeled, NGR-conjugated iron oxide nanoparticles (Cy5.5-NGR-Fe3O4 NPs) were prepared as an ovarian tumor specific bimodal imaging nanoprobe. It is demonstrated that the conjugation of NGR targeting moiety leads to a higher cellular uptake toward ES-2 cells, the human ovarian carcinoma cells that highly express CD13. Moreover, magnetic resonance imaging of ovarian tumor xenograft reveals that the Fe3O4-Cy5.5-NGR NPs results in a significant T2* signal reduction in the tumor. Meanwhile, near infrared fluorescence imaging indicates a higher accumulation of Fe3O4-Cy5.5-NGR NPs in the tumor xenograft. Therefore, CD13 could be applied as a novel and efficient target for constructing ovarian tumor specific nanoprobes with improved accuracy for ovarian tumor diagnosis.
1. Introduction Ovarian cancer is the leading cause of death among the gynecological malignancies [1,2]. The high mortality rate is largely due to the lack of early clinical symptoms and the rapid progression to peritoneal metastases [3,4]. Therefore, the preoperative qualitative diagnosis and early detection of ovarian tumors are considered as key steps for optimal clinical treatment strategy and improved patient prognosis. Although transvaginal ultrasonography (US) and computed tomography (CT) are conventional imaging techniques currently used to evaluate ovarian tumors in clinic, non-invasive magnetic resonance (MR) imaging is increasingly being used in patients with gynecological disorders due to its high contrast resolution compared to US and CT. Particularly, when US findings are suboptimal or indeterminate, MR imaging is of great help in preoperatively identifying malignant lesions. Moreover, the detection accuracy of ovarian tumors could be further improved by using MR contrast agents. For instance, superparamagnetic iron oxide NPs have been extensively investigated as an MR contrast agent for cancer diagnosis due to their excellent biocompatibility and magnetic properties.
Recently, development of tumor specific nanoprobes, which generally involves the use of active targeting strategies that rely on specific receptor–ligand interactions at the cell surface, has emerged as one of the most promising way to achieve specific accumulation of nanoprobes within tumors for broader and more effective diagnosis in various cancer types. It is well known that tumor cells express a variety of biomarkers on their surface [5–7]. These biomarkers are critical in the growth, progression, and metastasis of solid tumors and have been used to confirm diagnosis and disease stage. On the other hand, these unique biomarkers could be utilized for constructing tumor-specific NPs through receptor–ligand interactions. In the case of ovarian tumor cells, one of the most promising biomarkers is CD13, also known as aminopeptidase N [8]. As a Zn2+-dependent transmembrane ectopeptidase expressed on cell surface of the myeloid lineage and on nonhematopoietic tissue [9–11], CD13 possesses a number of functions, such as signal transduction, neuropeptide and cytokine degradation, antigen processing and presentation and extracellular matrix degradation associated with the progression of cancers and inflammatory reaction [12–14]. Moreover, accumulated experimental evidence suggests that CD13 is implicated in the pathogenesis of tumor. On this
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Corresponding authors. E-mail addresses: [email protected] (Y. Liang), [email protected] (W. Gu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.msec.2018.09.002 Received 28 January 2018; Received in revised form 9 August 2018; Accepted 1 September 2018 Available online 02 September 2018 0928-4931/ © 2018 Published by Elsevier B.V.
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obtained Fe3O4-Cy5.5 NPs were purified by centrifugal filter (MWCO = 5000 Da) and subjected to freeze-drying.
occasion, the interest in this enzyme as a therapeutic target has been progressively increasing. However, the applicability of CD13 as a target for ovarian tumor diagnosis has not been exploited so far. It is reported that CD13 can be recognized by peptides containing Asn-Gly-Arg (NGR) [15,16], a motif originally discovered by screening peptide-phage libraries in tumor-bearing mice. In this study, we synthesized NGR peptide conjugated, Cy5.5 labeled iron oxide (Fe3O4Cy5.5-NGR) NPs as a targeted MR/NIRF dual-modal imaging nanoprobe to test the applicable of CD13 as a target for ovarian tumor imaging with enhanced contrast effects. Poly(ethyleneglycol) (PEG) was also integrated to serve both as a biocompatible coating and linking molecule for the covalent attachment of the Cy5.5 and NGR to the iron oxide NPs. Cytotoxicity of Fe3O4-Cy5.5-NGR NPs was evaluated by CCK-8 cell viability assays, while their tumor targeting ability was examined by in vitro cellular uptake and in vivo MR and NIRF imaging of ovarian tumor xenograft. It is disclosed that CD13 could be utilized as a suitable target for ovarian tumor specific imaging with an enhanced contrast effect.
2.5. Synthesis of Fe3O4–Cy5.5-NGR NPs To 3 ml of PBS (pH = 6), 60 mg of Fe3O4–Cy5.5 NPs, 9.0 mg of NHS, and 6.0 mg of EDC were added. After stirring for 30 min, 3.0 mg of NGR and 66 mg of H2N–PEG3500–NH2 were added (pH = 8). Note that the weight ratio between Cy5.5-NGR and Fe3O4 NPs is 6%. The reaction was allowed for 12 h under stirring. Then, the product was purified using a centrifugal filter (MWCO = 5000 Da) and lyophilized to yield Fe3O4–Cy5.5–NGR NPs. 2.6. Characterization TEM images of NPs were obtained on a JEM-2010 (JEOL, Japan) at an operating voltage of 160 kV. Fourier transform infrared spectroscopy (FTIR) spectra were acquired on an ATR diamond-crystal equipped miniature FTIR spectrometer (iS5 FT-IR spectrometer, Thermo Nicolet Instrument Corp). The content of Fe was measured on an inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710ES, USA).
2. Experimental 2.1. Materials
2.7. Cell culture
Iron (III) acetylacetonate (Fe(acac)3), trioctylamine, benzyl ether, 1,2-hexadecanediol, oleic acid (OA) and oleylamine were purchased from Sigma-Aldrich. NGR (c-RYKNG) was purchased from GL Biochem Ltd. (Shanghai, China). Polyethylene glycol (H2N–PEG2000–NH2 and H2N–PEG3500–NH2) were received from JenKem Technology Co. Ltd. (Beijing, China). N-(Trimethoxysilylpropyl) ethylene diamine triacetic acid, trisodium salt (TETT, 45% in water) was obtained from Gelest Inc. All chemicals were in analytical grade and used without further purification. The water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA).
ES-2 cells, a type of human ovarian carcinoma cells highly expressed CD13, were obtained from China infrastructure of cell line resources and cultured in McCoy's 5A media with 10% fetal bovine serum (FBS) 4, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in the presence of 5% CO2 in humidified incubator. 2.8. Cytotoxicity assay
The Fe3O4–OA NPs were prepared through the thermal decomposition method [17]. Firstly, 1.413 g of Fe(acac)3, 5.169 g of 1,2hexadecanediol, 4 ml of oleic acid, 4 ml of oleylamine, and 40 ml of benzyl ether were mixed and preheated at 100 °C for 1 h under vacuum, then heating at 200 °C for 2 h, and 300 °C for 1 h under a nitrogen flow. After cooling to room temperature, ethanol was added to precipitate the product. The precipitation was washed twice with hexane containing 0.5 ml of oleic acid and 0.5 ml of oleylamine. The purified Fe3O4–OA NPs were collected by centrifugation.
The cytotoxicity of NPs was evaluated via a Cell Counting Kit-8 (CCK-8, water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzene disulfonate) F-2H-tetrazolium monosodium salt]) cell cytotoxicity assay. ES-2 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well. The cells were cultured in McCoy's 5A media for 24 h and incubated with NPs at concentrations of 0, 12.5, 25, 50, and 100 μg/ml (10 μl per well) for another 12 h. After removing the media and washing by PBS, 10 μl of CCK was added to each well and the cells were incubated for 4 h. The optical density (OD) value was measured at wavelengths of 450 nm on a Thermo Scientific Microplate Reader and the cell viability was calculated between the experimental and the control groups.
2.3. Synthesis of TETT-Fe3O4 NPs
2.9. Flow cytometry analysis
A ligand-exchange method was used to synthesize TETT-Fe3O4 NPs [18]. 100 mg of Fe3O4–OA NPs were dissolved into the mixture of 60 ml of anhydrous toluene and 60 μl of acetic acid, followed by sonicating for 10 min, adding 3 ml of TETT dropwisely, and stirring and heating at 70 °C for 48 h. Then, the precipitate was collected, washed with toluene and ethanol for three times. After centrifuging, the product was purified by dialysis (MWCO = 5000 Da) and subjected to lyophilization.
ES-2 cells were seeded in a 6-well plate with a density of 1 × 105 per well and cultured in McCoy's 5A media for 24 h. Afterwards, Fe3O4–Cy5.5–NGR or Fe3O4–Cy5.5 NPs at different Fe concentrations (40, 80, 160 μΜ) were added and incubated for 12 h, respectively. After the removal of media, cells were washed by PBS, harvested by trypsinization, collected by centrifugation, and re-suspended for flow cytometry analysis, which was conducted on a cytometry system (Cyan-LX, DakoCytomation).
2.2. Synthesis of oleic acid-capped Fe3O4 (Fe3O4–OA) NPs
2.4. Synthesis of Cy5.5 labeled Fe3O4 NPs 2.10. Relaxivity measurement The H2N–PEG2000–Cy5.5 NPs was firstly prepared by dissolving 1 mg of Cy5.5-NHS and 60 mg of H2N–PEG2000–NH2 in PBS (pH = 8), followed by stirring at room temperature for 24 h. Next, the activated Fe3O4-TETT NPs was prepared as follows, 100 mg of Fe3O4-TETT NPs, 16.2 mg of EDC, 16 mg of NHS, and 110 mg of H2N–PEG3500–NH2 were mixed in 10 ml of PBS (pH = 5.5). After stirring for 30 min, the pH of the mixture was adjusted to 8 and H2N–PEG2000–Cy5.5 was added. The reaction was preceded under stirring for another 24 h. Finally, the
NPs dispersions with different Fe concentrations of 500, 250, 125, 62.5, and 31.25 μΜ were prepared and scanned on a 7.0 T MRI (Bruker) to obtain the relaxation times and corresponding R2 maps. The parameters for MR T2 measurements were set as follows: TR = 3000 ms, TE = 45 ms, matrix size = 256 × 256, FOV = 4.0 × 4.0 cm and slice thickness = 1 mm. The r2 relaxivity was determined from the slope of linear 1/T2 against the Fe concentration. 969
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B frequency (%)
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size (nm) Fig. 1. TEM image (a) and size distribution (b) of Fe3O4-Cy5.5-NGR NPs.
2.11. Xenograft model
3. Results and discussion
All animal experiments were performed according to protocols evaluated and approved by the ethical committee of Capital Medical University (Beijing, China). The female BALB/c mice (6 weeks of age) were inoculated subcutaneously (right lateral thigh) with 5 × 106 ES-2 cells. All mice were maintained under specific pathogen-free (SPF) conditions, allowing free access to drinking water in a light/dark cycle at 22 ± 2 °C. When tumors reached a diameter of 0.8 mm (~7 days), the mice were subjected for imaging procedures.
3.1. Synthesis and characterization of Fe3O4-Cy5.5-NGR NPs CD13 is highly and stably expressed on endothelial and subendothelial cells in angiogenesis, which is an important process in tumor growth, progression and metastasis. In this study, NGR peptide that could specifically bind the CD13 was used as the targeting moiety to synthesize Fe3O4-Cy5.5-NGR as an MR/NIRF dual-modal imaging nanoprobe. Specifically, Fe3O4–OA NPs were first prepared through the thermal decomposition method [17]. Next, the Fe3O4-Cy5.5 NPs were prepared by replacing oleate with carboxylate silane (TETT) and subsequent conjugation of PEGylated Cy5.5. Finally, the conjugation of NGR yields the Fe3O4-Cy5.5-NGR NPs. The morphology Fe3O4-Cy5.5-NGR NPs were observed on TEM image (Fig. 1a). The TEM image reveals that the NPs are well separated and in nearly cubic shape. The average diameters of Fe3O4-Cy5.5-NGR NPs as calculated from 100 individual NPs are 8.93 ± 0.77 nm (Fig. 1b). Furthermore, to verify the crystallinity of NPs, the XRD pattern was obtained and is shown in Fig. S1. The diffraction peaks can be indexed to be magnetite (PDF card # 85-1436), confirming the crystallinity of NPs. The successful conjugation of NGR peptide was confirmed by FTIR. As illustrated in Fig. 2, the IR spectrum of Fe3O4-Cy5.5-NGR NPs shows a band with moderate intensity around 1632 cm−1 originates from the characteristic C]O band of amide groups in the NGR peptide. In addition, intensive bands at 1428 and 2882 cm−1 are assigned to the vibrations of CeH from the PEG.
2.12. In vivo MR and NIRF imaging The Fe3O4–Cy5.5 and Fe3O4–Cy5.5–NGR NPs at a dosage of 5.5 mg Fe per kg body weight (0.2 ml) were intravenously injected into the tumor-bearing mice through tail vein. The mice were anesthetized by CO2 and imaged on a 7.0 T MR scanner before and after injection of NPs at 60 min, 120 min, and 24 h, respectively. MR parameters were set as follows: TR/TE = 3500/33 ms(T2WI), TR/TE = 600/8 ms(T2⁎WI), matrix size = 256 × 256, FOV = 3.5 × 3.0 cm and slice thickness = 0.5 mm. NIRF images of the tumor-bearing mice were obtained before and after injection of NPs on a NightOWL LB 983 imaging system set with a 630 nm excitation filter and a 680 nm emission filter.
2.13. Ex vivo confocal laser scanning microscopy (CLSM) imaging Tumor-bearing BALB/c mice were sacrificed immediately after MR and NIR scanning. Tumors were excised and fixed in 4% formalin and dehydrated in 30% sucrose solution for 24 h. Next, the tumors were embedded with paraffin, snap-frozen, cryosectioned into 20 μm, and stained with DAPI. All sections were sealed by neutral gum and observed on a LEICA TCS SP5 confocal microscope (Leica Microsystems, Germany) with excitation at 405 and 630 nm for DAPI and Cy5.5, respectively.
3.2. Cytotoxicity To evaluate the cytotoxicity Fe3O4-Cy5.5-NGR NPs, cell counting kit based on water-soluble tetrazolium salt WST-8 was used. For comparison purpose, the cytotoxicity of Fe3O4-Cy5.5 NPs was evaluated as well. ES-2 cells were incubated with Fe3O4-Cy5.5 or Fe3O4-Cy5.5-NGR NPs at different concentrations (12.5, 25, 50 and 100 μg/ml) for 24 h and the cell viability was determined. As shown in Fig. 3, the viability of ES-2 cells is over 90% upon the treatment of either Fe3O4-Cy5.5 or Fe3O4-Cy5.5-NGR NPs at all tested concentrations, suggesting the negligible cytotoxicity of these NPs toward ES-2 cells.
2.14. Histological and immunohistochemical analysis Tumors were excised, fixed, dehydrated, embedded with paraffin, and cryosectioned into 20 μm slices. The slides were stained with hematoxylin and eosin (H&E) and CD13 immunohistochemistry according to standard clinical pathology protocols and were examined with 40× and 100× lens on a microscope (Olympus-BX53, Olympus Inc., Japan).
3.3. Tumor targeting specificity The targeting specificity of Fe3O4-Cy5.5-NGR NPs toward E2 cells was evaluated by a quantitative monitor of cellular uptake by flow 970
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Fig. 5. The linear fitting of the R2 against the Fe concentrations for r2 relaxivity measurement and T2-weighted maps of Fe3O4-Cy5.5-NGR NPs.
Wavenumber (cm ) Fig. 2. FTIR spectrum of Fe3O4-Cy5.5-NGR NPs.
Fe3O4-Cy5.5 Fe3O4-Cy5.5-NGR
125 Cell viability (%)
cytometry. Mean fluorescence intensities of ES-2 cells were measured at 24 h after incubation with Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs, respectively. As shown in Fig. 4, the cells treated with NPs have a significantly increased fluorescence signal, suggesting that both NPs can be internalized by ES2 cells. However, NGR conjugated Fe3O4Cy5.5 NPs clearly show greater fluorescence intensity than that of NPs without NGR conjugation at all tested concentrations, indicating a higher cellular uptake resulting from the specific interaction between CD13 and NGR.
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3.4. r2 Relaxivity To investigate the feasibility of Fe3O4-Cy5.5-NGR NPs as MR contrast agents, transverse relaxivity (r2) of Fe3O4-Cy5.5-NGR NPs was measured by linear fitting the inverse relaxation time against the Fe concentration. As plotted in Fig. 5, the r2 value of Fe3O4-Cy5.5-NGR NPs is 155.49 mM−1 s−1. Such r2 relaxivity value is comparable with that reported previously [19–21]. This together with the T2-weighted maps of NPs dispersion, which exhibit a concentration dependant darken effect, confirms the applicable of Fe3O4-Cy5.5-NGR NPs as effective T2-weighted contrast agents.
25 0
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concentraion ( g/mL) Fig. 3. CCK-8 cytotoxicity assay of Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs toward ES-2 cells at different concentrations for 24 h.
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3.5. In vivo MR imaging of ovarian tumor xenograft
Fe3O4-Cy5.5
Next, in vivo T2-weighted MR imaging of mice bearing ovarian tumor xenograft was performed before and after intravenously injection of the NPs via tail vein at a dosage of 5.5 mg Fe per kg body weight. The T2*-weighted images obtained at different time points are presented in Fig. 6. In the case of Fe3O4-Cy5.5-NGR NPs group, the intensity of MR signal in ovarian tumor xenograft gradually increased (darken effect) after injection and the highest contrast enhancement was observed at 24 h after injection. In contrast, the change of MR contrast enhancement in the Fe3O4-Cy5.5 NPs group was less significant. The MR contrast enhancement was further quantitatively analyzed by calculating the contrast-to-noise ratio (CNR) [CNR = (Stumor – Stissue) / σ]. As shown in Fig. 7, the CNR values of NGR-conjugated NPs are higher at each time point than that of nontargeting NPs and it reaches the maximum at 24 h post-injection. However, it is noted that, the improvement of CNR of the NGR-conjugated NPs at 60 min and 120 min post-injection is minor. This might be due to the fact that the non-targeting NPs could also accumulate in the turmor region by the EPR effect. Nevertheless, due to the specific interaction between CD13 and NGR, the NGR-functionalized NPs could not only accumulate in the tumor region, but also could be uptake by tumor cells. As a result, a significant enhancement in CNR is evidenced
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Fig. 6. In vivo MR T2* weighted imaging of ovarian tumor xenograft pre- and post-injection of Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs at a dose of 5.5 mg Fe per kg body weight.
CNR
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at 24 h post-injection, signifying the applicable of CD13 as an effective target for NGR-based nanoprobe. Moreover, considering the fact that multiple types of receptors are typically overexpressed on the surface of tumor cells, dual-targeting or multi-targeting systems have been demonstrated to further enhance the targeting specificity [22,23]. On this occasion, the verification of CD13 as a novel and effective target for ovarian tumor would facilitate the rational design multi-targeted nanoprobes for more accurate and earlier diagnosis of ovarian tumors.
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3.6. In vivo NIRF imaging of ovarian tumor xenograft
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The conjugation of fluorescent dye Cy5.5 onto Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs imparts the NPs NIRF imaging modality. The NIRF images of tumor-bearing mice were acquired pre- and post-injection of NPs. As presented in Fig. 8, Fluorescence signals are primarily observed at spine, heart, lung and kidney at 30 min post-injection of NPs. Nevertheless, the fluorescence signal intensity at the tumor site gradually increased as time passing and show a maximum intensity at
24 h
Fig. 7. CNR analysis (n = 3).
Fig. 8. In vivo NIRF images of ovarian tumor xenograft before and post intravenous injection of Fe3O4-Cy5.5-NGR and Fe3O4-Cy5.5 NPs with a dose of 5.5 mg Fe per kg body weight. 972
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NPs
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Fig. 9. CLSM images of tumor sections that obtained from BABL/c xenograft model injected with Fe3O4-Cy5.5 and Fe3O4-Cy5.5-NGR NPs at a dosage of 5.5 mg Fe per kg body weight. Cell nuclei were stained with DAPI and red fluorescence indicates the localization of NPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 10. Histopathological staining of tumor xenograft under 40× (A) and 100× (B) microscope shows the obvious atypia, and CD13 immunohistochemical staining under different magnifications (C and D), indicating the presence of CD13 on tumor cells.
conjugation. The discrepancy of fluorescence intensity found in both flow cytometry and CLSM verifies the tumor specificity of Fe3O4-cy5.5NGR NPs.
24 h post-injection, indicating a higher accumulation of NPs at the targeting tissue while NPs are metabolized at other organs. Obviously, the NIRF imaging result is consistent with in vivo MR scanning. The high accumulation of Fe3O4-cy5.5-NGR NPs in tumor was further supported by CLSM. As illustrated in Fig. 9, NGR-conjugated NPs display much stronger red fluorescence signal than that of NPs without NGR. More importantly, the Fe3O4-cy5.5-NGR NPs indeed reside in cytoplasm, indicating the efficient cellular uptake of NPs upon NGR
3.7. Histology analysis The ovarian tumor xenograft identified by Fe3O4-Cy5.5-NGR NPs was excised and subjected to H&E histopathological and CD13 973
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immunohistochemical staining. Fig. 10A and B present the H&E staining results observed under 40× and 100× microscope. The observed atypia and the loss of their original shape clearly validate the successful establishment of ovarian tumor xenograft. Additionally, the presence of CD13 on the surface of tumor cells was confirmed by the immunohistochemical staining. Fig. 9 C and D provide CD13 immunohistochemical staining images of tumor sections. As can be seen, scattered brown punctate shadow distributes around the tumor cells, while only slightly flocculent sandy silhouettes in stromal cells, confirming the presence of CD13 on tumor cells as the target for NGR conjugated MR nanoprobes.
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4. Conclusion In conclusion, the NGR-conjugated, Cy5.5-labeled Fe3O4 NPs were synthesized and applied as a targeted MR and NIRF dual-modal nanoprobe for ovarian tumor imaging. The specific targeting ability of Fe3O4-Cy5.5-NGR toward ovarian tumor cells expressing CD13 is confirmed by in vitro flow cytometry. In vivo MR and NIRF imaging of ovarian tumor xenograft demonstrate the specificity of Fe3O4-Cy5.5NGR NPs toward ovarian tumor. These results suggest CD13 is a suitable target for constructing specific imaging nanoprobes for ovarian tumor diagnosis with enhanced contrast effects and Fe3O4-Cy5.5-NGR as CD13 target-specific dual-modal imaging nanoprobes has great potential in more accurate and earlier diagnosis of ovarian tumor. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2018.09.002. Acknowledgement This work is financially supported by the Basic-clinical Key Research Grant (15JL07) from Capital Medical University and the Beijing Natural Science Foundation (7162064). References [1] G.C. Jayson, E.C. Kohn, H.C. Kitchener, et al., Ovarian cancer, Lancet 384 (9951) (2014) 1376–1388. [2] N. Auersperg, A.S. Wong, K.C. Choi, et al., Ovarian surface epithelium: biology, endocrinology, and pathology, Endocr. Rev. 22 (2) (2001) 255–288. [3] R. Raave, R.B. de Vries, L.F. Massuger, et al., Drug delivery systems for ovarian cancer treatment: a systematic review and meta-analysis of animal studies, PeerJ 3 (2015) e1489. [4] W. Lim, W. Jeong, G. Song, Coumestrol suppresses proliferation of ES2 human epithelial ovarian cancer cells, J. Endocrinol. 228 (3) (2015) 149–160.
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