Background
Ovarian cancer is the sixth most commonly diagnosed cancer in the world, accounting 4 % of all cancers in women [
1], and it is the leading cause of death from gynecologic malignancies in the western world [
2,
3]. Most ovarian cancers are first diagnosed in an advanced stage because patients’ symptoms may be minimal or nonspecific and no reliable biomarkers are available [
4]. Tumor-debulking surgery is the first choice of management for most patients with ovarian cancer [
5], but most ovarian cancers recur after surgery and are intractably drug resistant [
6]. Therefore, although some advances in cytoreductive surgery and case-effective chemotherapy have been made in the last decade, the prognosis for ovarian cancer, especially for epithelial ovarian cancer still is limited.
In most tertiary medical centers, magnetic resonance (MR) imaging is generally performed for imaging assessment of complex ovarian masses [
7,
8] that are indeterminate on either palpation or ultrasonography because of MR’s superb soft-tissue resolution and lack of radiation. The MR diagnostic criteria for ovarian malignancies are based on morphology: thick septum, vegetations, ascites, lymphadenopathy, and vividly enhancing solid component, which are features well described in numerous reports [
8,
9]. However, identification of the tumor tissues at an early stage with available imaging modalities still possesses a great challenge for both radiologists and clinicians.
Recent advances in nanoscience and nanotechnology have enabled the development of various contrast agents for MR imaging applications, such as Gd (III)- or Mn (II)-based T
1 MR contrast agents [
10,
11] and magnetic iron oxide nanoparticle (Fe
3O
4 NPs)-based T
2 MR contrast agents [
12‐
14]. The Fe
3O
4 NPs are the most commonly used magnetic materials for various biomedical applications [
15‐
18]. But, few reports on the application of Fe
3O
4 NPs for the diagnosis of ovarian cancer have been published.
Folic acid (FA) receptors as single-chain glycoproteins with high specific affinity for FA are highly overexpressed on various malignant tumors, including human ovarian cancer [
19]. The over-expression of FA receptors on malignant tumor tissues can be exploited as a specific targeting ligand since most healthy tissues have little FA receptors expression [
20]. This targeting strategy has the potential for diagnostic and therapeutic application in a wide variety of cancers [
21,
22].
In this research, we used FA-targeted Fe3O4 NPs as T2-negative contrast agents for in vivo MR imaging of ovarian cancer in an intraperitoneal xenograft tumor model. To the best of our knowledge, this is the first reported application of FA-targeted Fe3O4 NPs in MR imaging diagnosis of ovarian cancer.
Methods
Synthesis and characterization techniques
FA-targeted Fe
3O
4 NPs were synthesized and characterized according to our previous work [
23]. Non-targeted Fe
3O
4 NPs were synthesized by the same methods, except for the use of
mPEG-COOH in the PEGylation step instead of FA-PEG-COOH.
Branched polyethyleneimine (PEI, Mw = 25,000)-coated Fe3O4 NPs (Fe3O4@PEI NPs) were synthesized via a reduction route. FeCl3 · 6H2O (1.3 g) was dissolved in 20 mL water, and placed into a 250 mL three-necked flask. Under vigorous stirring, the solution was bubbled with nitrogen atmosphere for 15 min, then 10 mL freshly prepared sodium sulfite solution (0.2 g) was added slowly into the flask. 30 min later, 5 mL PEI (0.5 g) and 2 mL ammonia (25 %) was added into the flask successively. The reaction mixture was vigorously stirred for 30 min at 60 ~ 70 oC, and then at room temperature for another 1.5 h. The product (Fe3O4@PEI NPs) was magnetically collected and washed 3 times with water. Finally, the sample was centrifuged (8000 rpm, 10 min) to remove the aggregation and larger particles.
An aqueous solution of Fe3O4@PEI NPs (110 mg, 35 mL) was precipitated by virtue of an external magnet and re-dispersed in 20 mL DMSO. Another solution of 38.5 mg activated FA-PEG-COOH or mPEG-COOH in 2 mL DMSO was added dropwise into the above DMSO solution of Fe3O4@PEI NPs and kept shaking for 3 d. The formed products were collected by magnetic separation and washed with DMSO for 3 times to remove excess reactants. Finally, the amino groups on the surface of the particles were acetylated by reaction with acetic anhydride. Briefly, triethylamine (493 μL) was added into the aqueous solution of raw product of Fe3O4@PEI-PEG-FA NPs or Fe3O4@PEI-mPEG NPs under vigorous shaking using a shaker at room temperature. After 30 min, acetic anhydride (402 μL) was dropwise added into the above mixture solution and the reaction was continued for 1 d. After several times magnetic separation/washing/dispersion steps to remove excess reactants and by-products, the final products (FA-targeted Fe3O4 NPs and non-targeted Fe3O4 NPs) were obtained, re-dispersed in water and stored under 4 oC for further use.
A JEOL 2010 F transmission electron microscopy (TEM, JEOL, Tokyo, Japan) was used to characterize the morphology of the FA-targeted Fe3O4 NPs and non-targeted Fe3O4 NPs at an operating voltage of 200 kV. A dilute particle suspension of the sample in water (10 μL) was deposited onto a carbon-coated copper grid and dried in air before measurements. The effect of MR imaging for FA-targeted and non-targeted Fe3O4 NPs was evaluated with a 1.5 Tesla MR imaging machine (Siemens Avanto, Erlangen, Germany). Samples were diluted with water to have different Fe concentrations in the range of 0.005–0.08 mM before measurements. The T2-weighted imaging parameters with turbo spin echo sequence were set as follows: point resolution = 156 mm × 156 mm, section thickness = 1.5 mm, TR = 4000 ms, TE = 85 ms, bandwidth (Hz) = 260, number of excitation = 1, and voxel size = 1.1 × 1.1 × 4.0 mm.
Cell culture
Skov-3 cells was obtained from the Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases (Shanghai, China). Skov-3 cells were grown in FA-free RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C and 5 % CO2.
Cytotoxicity of FA-targeted Fe3O4 NPs and non-targeted Fe3O4 NPs
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay was carried out to evaluate the cytotoxicity of the FA-targeted Fe3O4 NPs and non-targeted Fe3O4 NPs. Briefly, 1 × 104 Skov-3 cells were seeded into each well of 96-well cell culture plates with 200 μL regular RPMI-1640 medium and cultured at 37 °C and 5 % CO2 overnight to bring the cells to confluence. Next, the medium in each well was discarded carefully and 200 μL of fresh medium containing phosphate-buffered saline (PBS), FA-targeted Fe3O4 NPs or non-targeted Fe3O4 NPs at the Fe concentration of 0.5 to 1.0 mM was added. After 24 h incubation at 37 °C and 5 % CO2, 20 μL MTT solution (5 mg/mL in PBS buffer) were added to each well to reveal the viable cells. After further incubation for 4 h at 37 °C and 5 % CO2, the medium was carefully removed, and DMSO (200 μL) was added to dissolve the formazan grains. The absorbance value of each well was measured with a microplate reader at 450 nm wavelength.
Cellular uptake of FA-targeted Fe3O4 NPs and non-targeted Fe3O4 NPs
To qualitatively confirm the cellular uptake of Fe3O4 NPs by Skov-3 cells, the cells was stained with Prussian blue. In brief, 5 × 105 cells were seeded into each well of 24-well cell culture plates. After overnight incubation at 37 °C and 5 % CO2 to bring the cells to 80 % confluence, the medium was replaced with fresh medium containing PBS buffer (control), FA-targeted Fe3O4 NPs, or non-targeted Fe3O4 NPs at the Fe concentrations of 0.2 and 0.4 mM. The cells were continuously incubated for another 4 h. The cells were then washed three times with PBS, fixed with p-formaldehyde solution at 4 °C for 15 min, and stained with Prussian blue reagent (potassium ferrocyanide [1 g] dissolved in water [9 mL] mixed with 36–38 % HCl [1 mL]) at 37 °C for 30 min. The cells were imaged with a Leica DMIL LED inverted-phase contrast microscope.
The Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-OES, Hudson, NH, USA) also was used to quantify the cellular uptake of the Fe3O4 NPs by Skov-3 cells. The Skov-3 cells were seeded into 12-well plates with a density of 1 × 106 cells/well. After overnight incubation to bring the cells to confluence, the medium was discarded carefully, and 1 mL fresh medium containing PBS buffer (control), FA-targeted Fe3O4 NPs or non-targeted Fe3O4 NPs at Fe concentrations of 0.2 and 0.4 mM was added. The cells were further incubated at 37 °C and 5 % CO2 for 4 h. The medium was then removed. The cells were washed with PBS buffer four times, trypsinsized, collected, and suspended in 1 mL PBS buffer. The cell numbers in each sample were estimated with a hemocytometer. For the cellular uptake assay, the cells were centrifuged (1000 rpm, 5 min), collected, and lysed with an aqua regia solution (0.5 mL) for 12 h. The Fe content was determined by ICP-OES after the samples were diluted 2 times with PBS.
In vivo targeted MR imaging of tumors
Four-week-old female BALB/c nude mice (Shanghai Cancer Institute, Shanghai, China) were treated according to protocols approved by the Ethical Committee of Obstetrics and Gynecology Hospital ([2007]-No. 6), Fudan University. The nude mice (three mice in each group) were injected intraperitoneally with 1 × 106 Skov-3 cells/mouse at a site 1 cm left of the midline. Two weeks later, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg). After that, 200 μL of FA-targeted Fe3O4 NPs or non-targeted Fe3O4 NPs (0.6 mg Fe) were delivered into the mice via the tail vein. MR scans were performed before injection and 0.5, 1, 2, and 4 h after injection of the particles. A 1.5 T clinical MR system was used with a custom-built rodent receiver coil (Chenguang Med Tech, Shanghai, China). The sequence parameters were set as following: Axial fat-suppressed T2WI (FS T2WI), point resolution = 156 mm × 156 mm, TR/TE: 8000/83 ms, thickness: 2 mm, field of view: 50 mm, voxel size: 1.4 × 1.4 × 1.9 mm, flip angles: 150 °. Signal intensity in the tumors at each time point was measured and recorded.
Statistical analysis
Quantitative data were expressed as mean ± standard deviation (SD). Means were compared by use of unpaired two-sided Student’s t-test. The data are indicated with (*) for p < 0.05, (**) for p < 0.01 and (***) for p < 0.001.
Discussion
In this study, we report our preliminary experience in imaging human ovarian cancer in the xenograft tumor model by using FA-targeted Fe3O4 NPs as contrast agents. Owing to the good contrast enhancement and low cytotoxicity, the FA-targeted Fe3O4 NPs can detect the ovarian cancer tissues planted in the abdominal cavity of nude mice at in vivo levels. Our results indicated that FA-targeted Fe3O4 NPs hold promise for being effective magnetic molecular probes for detecting tumor tissues in gynecologic cancer.
Ovarian cancer is the most malignant gynecological tumor and therefore deserves extensive basic and clinical research in the quest for early diagnostic tests and effective treatments [
24‐
27]. Fe
3O
4 NPs are low-toxic and eventually biodegrade to form blood hemoglobin [
14], and they have been used for liver imaging since the 1900s [
28]. With recent advances in nanotechnology and nanoscience [
29‐
32], various polymers have been coated onto the surface of Fe
3O
4 NPs to improve their stability and decrease their uptake by the reticuloendothelial system [
16,
33,
34]. Numerous studies on application of NPs in biomedical imaging have been reported in recent decades [
35‐
40], but few have examined application of the particles in ovarian cancer.
In our previous work, we demonstrated that FA-targeted Fe
3O
4 NPs have good water-dispersibility, colloidal stability and fairly high relaxivity [
23]. In addition, the particles have excellent hemocompatibility and cytocompatibility in the studied range of concentrations. We found that FA-targeted Fe
3O
4 NPs had excellent binding specificity to a human cervical cancer cell line (HeLa cells) overexpressing FA receptors via an active FA targeting pathway. In the present study, we found excellent lesion targeting ability of the FA-targeted Fe
3O
4 NPs to ovarian cancer in the T
2-weighted MR imaging, which may be attributed to the following aspects: First, although the mean size of the FA-targeted Fe
3O
4 NPs was small (9.2 ± 1.7 nm), the particles had a very high r
2 relaxivity coefficients (475.92 mM
−1s
−1), which is much higher than those of other reported Fe
3O
4 NPs [
33,
40]. This feature made the particles more sensitive to magnetic susceptibility effects. Second, the presence of FA on the surface of the Fe
3O
4 NPs increased their ability to target tumor tissues. Third, the passive enhanced permeability and retention effect into solid tumors may also facilitate the specific MR imaging of tumors [
36].
Human ovarian cancers are located deep in the pelvic space [
41]. An ideal humanized xenograft mouse model of ovarian cancer would simulate the true microenvironment for tumor angiogenesis [
24,
42‐
44]. Thus, in the present study, we implanted the tumor cells in the abdomen rather than in subcutaneous sites, believing that the intraperitoneal location would reflect the hemodynamic condition of ovarian cancer in humans-at least more accurately than would a subcutaneous site, as has been often used [
10‐
12,
25]. Our results corroborated this point: both targeted and non-targeted particles were evident by T
2-weithted MR imaging at 2 h after injection in abdominal tumors compared with 1 h in subcutaneous tumors [
23], perhaps because more time was needed for Fe
3O
4 NPs to reach the deep abdominal tumors in sufficient concentration to be evident on T
2-enhanced imaging. We must confess that the T
2 signal intensity in MR images also achieve the lowest point at 2 h post injection of non-targeted Fe
3O
4 NPs, which may be due to the enhanced permeability and retention (EPR) effect (passive uptake) as well documented in solid tumors [
14,
36,
37]. However, both in vitro and in vivo imaging results (as shown in Figs.
5 and.
7) proved FA-targeted ligands can enable the tumor uptake through more active pathway, thus making the tumors look like more dark compared with non-FA targeted group.
Further, we also found that, after injection of Fe
3O
4 NPs in the nude mice, the tumor T
2 signal intensity had reverted to pre-injection intensity after 4 h, a little earlier than that we had found previously in subcutaneous tumors [
23]. We also acknowledge that the injection of suspensions of tumor cells into the mice is different from the formation of tumors in the natural environment.
Other methods for imaging detection of ovarian cancers have been described. Hensley, et al [
45] described a dual MR-fluorescence molecular tomography approach, with commercially available fluorescent molecular imaging probes for the detection and quantification of tumor-associated metabolites in ovarian carcinomas in a transgenic mouse model of epithelial ovarian cancer. The authors concluded that the combination of in vivo molecular and MR imaging can effectively detect orthotopic ovarian tumors and their response to therapy [
25]. In another study, Satpathy, et al [
24] reported that in an orthotopic human ovarian tumor xenograft model, HER-2- targeted magnetic NPs labeled with a near infrared dye (NIR-830) were specifically delivered into primary and disseminated ovarian tumors, enabling optical and MR imaging of tumors as small as 1 mm in the peritoneal cavity. The authors designed the non-conjugated magnetic NPs with 14 ± 3.4 nm diameter and targeted-conjugated magnetic NPs with 22.9 ± 4.8 nm diameter, respectively [
24]. However, they did not report the exact MR acquisition time point, which we believe is crucial for tumor imaging, especially for magnetic NPs.
Our study also has some limitations. First, by 2 weeks after injection of ovarian cancer cells into the peritoneal cavity, the tumors often had become large (average diameter about 5 mm) with isointensity signals on T2-weighted MR imaging making them easily detectable and distinct from surrounding tissues, which had mostly hyperintensity signals. However, tumors at an earlier stage or smaller might not be detected because of overlapping neighboring organs (such as gut, kidney, or bladder) and background tissues. Perhaps the specificity and sensitivity could be improved by the use of bimodal magnetic nanoprobes with fluorescent materials incorporated into Fe3O4 NPs. Second, since FA receptors are overexpressed in most malignant tumors, the FA targeting ligand we used may not be specific for detecting ovarian cancer. Further studies should be conducted to image ovarian cancer with targeting motifs that may be more specific.
Competing interest
The authors declare that they have no competing interest.
Authors’ contributions
XS, MS and GZ designed of the whole study; HZ, JL and YH performed the experiments and analyzed the data; HZ, JL wrote and revised the manuscript finally. All authors read and approved the final manuscript.