1. Introduction
Early cancer detection represents the most promising way to reduce the growth of cancer [
1]. Additionally, about 1000 million people worldwide suffer from cancer every year, with an increase number of people with cancer around the world, the development of drugs for cancer diagnosis and treatment is an important part of the biological medicine. The most common form of cancer is a solid tumor of the lung, breast, prostate, colon, and rectum. If the cancer can be diagnosed at an early stage, most cancer patients after surgery, radiotherapy, chemotherapy, or combination therapy can survival for a long time. The five-year survival rate of for early-stage cancer is up to 90%.
Therefore, accurate early detection makes it possible to perform all kinds of treatment prior to tumor progression. The cancer currently can be detected by many imaging modalities, including computed tomography (CT), single-photon emission computed tomography (SPECT), positron emission tomography (PET), ultrasonography, and magnetic resonance imaging (MRI) [
2]. However, there are still many significant challenges in using molecular imaging for cancer detection, including that most of the specific receptors are not overexpressed in all types of cancer. Therefore, the development of a versatile molecular probe to detect the majority of cancer tissue has considerable benefits.
Near infrared fluorescent heptamethine cyanine dyes including indocyanine green (ICG), IR-783 and IR-780 iodide, has been shown to have abilities for noninvasive tumor imaging [
3,
4,
5]. It was reported that cancer cells have higher mitochondrial membrane potential (ΔΨm) than normal cells [
6] Because of mitochondrial membrane potential (ΔΨm) alteration in cancer cells, delocalized cationic compounds could accumulate in the hyperpolarized mitochondria as a tumor-targeting agent for imaging and therapy [
7,
8,
9]. For example, rhodamine-123, Acridinium, and cyanine dyes have been used for in vitro and in vivo fluorescent imaging of the altered mitochondrial membrane potential in tumor cells, and as radiotracers for nuclear imaging of tumors [
6,
10,
11,
12]. In addition, several radiolabeled triphenylphosphonium (TPP) cations were used as radiotracers for nuclear tumor imaging [
13,
14].
For the treatment of primary or metastatic tumors, although surgical resection is the standard treatment, the tumors are not easy to remove by surgery in many situations. Therefore, imaging-guided cancer surgery will offer more effective and safe modalities of cancer therapy [
15]. When the tumor in patients are really not suitable for surgery, tumor thermal ablation is another low-invasive treatment modality [
16]. Photothermal therapy (PTT) destroys cancer cells by generating heat within a tumor by absorbing specific light sources [
17,
18]. The heptamethine cyanine dyes also exhibits unique optical properties due to its strong absorption at NIR wavelengths, which causes photothermal effects that can trigger thermal injury and cell death both in vitro and in vivo [
6,
7,
8,
9].
Indium-111 (
111In, half-life = 2.83 days) only emits γ-ray (23, 171 and 245 keV), and is commonly used in nuclear imaging by radiolabeling targeted molecules [
19]. In this study, Indium-111 was used as a diagnostic radionuclide to label with DOTA-NIR790 for nuclear imaging of cancers. Lutetium-177 (
177Lu, half-life = 6.73 days) is an emerging, promising medium-energy beta-emitter (490 keV) with a low-abundance gamma emission (208 keV, 11% abundance) for biomedical use. This unique property makes
177Lu as a suitable radionuclide candidate for both therapeutic and diagnostic purposes.
In this study, we synthetized a cancer-targeted multimodal probe (DOTA-NIR790 as shown in
Figure 1) for cancer imaging and therapy by the mitochondrial potential difference between cancers and the surrounding normal tissues. Compared with other reported cyanine-based theranostic probes, the DOTA-NIR790 has the additional chelated capabilities of radioisotope for nuclear imaging and targeted radionuclide therapy of cancers. DOTA-NIR790 is a single molecule with multi-functions for cyanine-based near-infrared fluorescence imaging and photothermal therapy of cancers, and for nuclear imaging and targeted radionuclide therapy of cancers after radioisotope-labeling. The heptamethine cyanine-based dye allowed the DOTA-NIR790 to have dual functions in cancer NIR imaging and photothermal therapy (PTT). The DOTA-NIR790 will be labeled with lutetium-177 to detect tumors by using SPECT imaging and kill cancer cells by its beta-emission. The
177Lu-DOTA-NIR790 enabled imaging by NIR fluorescence and by nuclear imaging (SPECT) to monitor in real-time the tumor accumulation, intra-tumoral distribution, and the situation of cancer therapy, and to guide the surgery or the photothermal therapy of the cancer.
3. Discussion
Globally, more than 10 million people are diagnosed with cancer every year. With a continuous rise in the number of cancer patients, the development of related drugs for cancer treatment has become an important area in the biotechnology and pharmaceutical industries worldwide. Early diagnosis and combined treatment, in particular, will be the trend for the future development of cancer treatment. The theranostic probes (DOTA-NIR790) for multimodality imaging, targeted radionuclide therapy, and photothermal therapy have been developed, as well as single-molecule probes that can be applied to tumors in various organs where drugs can accumulate in the targeted diagnostic or treated location.
This study has developed a multifunctional tumor diagnosis probe (DOTA-NIR790) that integrates near-infrared fluorescent and nuclear medical imaging, and at the same time achieves the goal of combined photothermal therapy. This study completed the synthesis, physicochemical properties, and the isotope labeling of a multifunctional tumor theranostic probe. The most suitable probe molecule was screened for DOTA-NIR790 and the establishment of its preparation method was completed. The target product DOTA-NIR790 was isolated by preparative HPLC purification, and the purity of HPLC analysis was about 95%. Mass analysis, molecular weight 1224 Da. DOTA-NIR790 was labeled with In-111 isotope and analyzed by Radio-HPLC; the radiochemical purity was about 90%. Further purification by RP-18 column could increase the radiochemical purity to more than 95%.
Radioisotope labeling of DOTA-NIR790 and SPECT imaging in the tumor-bearing animal model were performed. Near-infrared fluorescence (NIRF) imaging was performed using a non-invasive live imaging system and compared with SPECT imaging. A comparison of the results showed that 111In-DOTA-NIR790 began to exhibit a selective cumulative image of the tumor 24 h after administration, and showed significant tumor imaging ability by SPECT at 48 h. On the other hand, the NIRF imaging of the tumor showed that the probe could be effectively targeted to the tumor after 24 h of administration, and exhibit excellent tumor-imaging ability. The results of 111In-DOTA-NIR790 in the near-infrared image portion are consistent with single-photon computed tomography (SPECT). Compared with near-infrared fluorescence (NIRF) imaging, SPECT has a higher ability to detect deep tissue distribution. However, the near-infrared fluorescence (NIRF) imaging presents better tumor-targeted images in superficial tumors, which is due to the ability of the optics to penetrate the tissue.
This study also completed the tissue distribution of 111In-DOTA-NIR790, which showed that the probe had good tumor target. The anti-tumor effect evaluation verified that the photothermal therapy ability of the DOTA-NIR790 can effectively inhibit tumor growth. The multi-functional probe can also be labeled with the therapeutic isotope Lu-177 for targeted radionuclide therapy of cancer.
As verified by animal experiments, the application of DOTA-NIR790 in various types of cancer, such as breast cancer, colon cancer and lung cancer, shows a good tumor/muscle ratio. As shown separately in both the imaging of SPECT and NIRF, the tumor region is reflected as a thermal zone; the imaging of the tumor region presents a sharp contrast to that of non-cancerous tissue, indicating that this probe can effectively accumulate in the tumor location and generate good functional imaging. According to the previous review aimed to determine the overall sensitivity and specificity of indocyanine green (ICG) near-infrared (NIR) fluorescence in sentinel lymph node (SLN) detection in colorectal cancer (CRC) [
20]. The pooled sensitivity and specificity rates were 71% and 84.6%. The ICG-NIR fluorescence is a promising technique for detecting SLNs in CRC. Although those NIR fluorescent probes do not represent sufficient specificity for cancer targeting compared with molecular targeting probes, they had been widely used in clinical for detecting sentinel lymph node (SLN) in various types of cancer, such as non-small cell lung cancer [
21], gastric cancer [
22], metastatic colorectal cancer [
23], uterine, and cervical malignancies [
24]. Therefore, the future clinical application of DOTA-NIR 790 should have cancer specificity similar to that of ICG. Furthermore, the imaging generated during the process of photothermal therapy or targeted radionuclide therapy, which was carried out to monitor the treatment condition of tumors. It was conducted by combining the probe with different radioactive doses of Lu-177, clearly showing that the size of the tumor shows a trend of diminishing as the number of days in treatment increases, compared with the control group, which indicated the DOTA-NIR790 is a potential probe for cancer diagnosis and therapy.
For functional imaging used in cancer diagnosis, single photon emission computed tomography (SPECT) used for deep-tissue tumors and near-infrared fluorescence (NIRF) imaging used for subcutaneous-tissue tumors. Both have their own advantages. This probe can combine the above two imaging methods for cancer diagnosis, depending on imaging requirements. The DOTA-NIR790 can chelate a variety of radioisotopes, such as indium-111 (In-111), lutetium-177 (Lu-177), and gallium-67 (Ga-67) for single photon emission computed tomographic imaging, gallium-68 (Ga-68) and copper-64 (Cu-64) for positron emission tomographic (PET) imaging, or even gadolinium (Gd) in non-radioactive magnetic resonance imaging (MRI) in future. The combined usage of multiple imaging techniques can increase accuracy in tumor diagnosis.
For cancer therapy, the photothermal property of cyanine-based dyes used in the probe serves to achieve the goal of tumor photothermal therapy. In addition, this probe can also apply isotopic labeling for targeted radionuclide therapy of cancer, such as lutetium-177 (Lu-177) and yttrium-90 (Y-90). In the future, the development of chemotherapy drugs with chemical bonds will serve to achieve the objective of single drugs combined with multiple treatment effects, whereby the cure rates for cancer can be improved.
4. Materials and Methods
4.1. General
ADS790WS is 2-[2-[2-(4-aminobenzenethio)-3-[(1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene)-ethylidene]-1-cycloxen-1-yl]-ethynyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, innersalt, monosodium, which was purchased from American Dye Source, Inc. (Montreal, QC, Canada). DOTA-NHS-ester (1,4,7,10-Tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester) was purchased from Macrocyclics (Dallas, TX, USA). The ESI (electrospray ionization) mass spectral data were collected on a AB Sciex 4000QTrap system (Concord, ON, Canada). 111InCl3 (indium chloride in 0.05 M HCl) was purchased from Institute of Nuclear Energy Research (INER), Taoyuan, Taiwan. 177LuCl3 was purchased from perkin elmer (Waltham, MA, USA).
Preparative reversed-phase high performance liquid chromatography (HPLC) was performed on a SHIMADZU Prominence Preparative HPLC System with a SHIMADZU SPD-20AV detector using YMC-Actus Triart C18 column (5 μm, 250 × 20 mm). Analytic reversed-phase HPLC was performed on a Waters 2695 Separations Module with a Waters 2487 Dual Wavelength Absorbance Detector plus a Bioscan radioisotope detector using YMC-Triart C18 column (5 μm, 250 × 4.6 mm). Waters Bridge column (5 μm, 200 ×4.6 mm). The flow rate was 20 mL/min for the preparative column and 1 mL/min for the analytic column running wth 60% ACN and 40% water with 0.1% TFA.
4.2. Synthesis of DOTA-NIR790
As shown in
Figure 1, ADS790WS (83.8 mg, 100 μmol) were dissolved in 5 mL dry DMF in the presence of triethylamine (20 mg, 200 μmol). Then, DOTA-NHS (153 mg, 200 μmol) dissolved in 5 mL DMF was added, and the mixture was stirred at room temperature for 3 days. The obtained crude product was purified by preparative HPLC with a C18 reversed-phase column using 40% ACN and 60% H
2O with 0.1% TFA as the mobile phase. The peak containing the desired product was collected (Rt = 9.2 min), and the collected solution was frozen and lyophilized overnight. ESI-MS: m/z = 1224.5 for [M + H]
+ (1224.3 calcd for [C60H79N7NaO13S3]
+).
4.3. Preparation of 111In-DOTA-NIR790 and 177Lu-DOTA-NIR790
111InCl3 (370 MBq) [111InCl3; Institute of Nuclear Energy Research (INER), Taoyuan, Taiwan, 16430 MBq/mL in 0.05N HCl; pH 1.5–1.9] or 177LuCl3 (370 MBq) were added to DOTA-NIR790 (10μg) diluted in 300 μL of 0.2 M sodium acetate buffer (pH 5.5), respectively. The reaction mixture was incubated for 1 h at 37 °C with constant shaking. The primary products were purified by RP-18 column using phosphate-buffered saline (PBS) as the washing buffer and ethanol as elution buffer. The radiochemical purity of 111In-DOTA-NIR790 was evaluated by radio-HPLC analysis. The radiochemical purity of 177Lu-DOTA-NIR790 was evaluated by the instant thin layer chromatography (ITLC) with 0.1M Na-citrate (pH 5.0) as the solvent (177LuCl3: Rf = 0.9~1.0, precursor-bound 177Lu: Rf = 0~0.1).
4.4. Cellular Uptake of DOTA-NIR790
For cellular uptake, clone9, HCT-15, FaDu, A549, CT26, 4T1, and HCT-116 cells were seeded in 24-well plates at a density of 105 cells per well 24 h before the assays. The medium was removed, and the 3.7 MBq of 111In-DOTA-NIR790 in 0.5 mL of medium containing 0, 2, 10, or 50 μM of carbonyl cyanine m-chlorophenylhydrazone (CCCP) were added per well (n = 3 per concentration of CCCP and cell). After 24 h incubation, the medium was removed, and the cells were washed twice with 1 mL of PBS and lysed by the addition of 0.2 mL of 0.1 M NaOH. Cell lysates were collected, and the radioactivity was measured by a γ-counter. The cellular uptake values were normalized to that of CCCP-untreated cells, and the normalized intensity (%) is calculated as the percentage of radioactivity obtained by CCCP-treated and CCCP-untreated cells.
4.5. Animal Model
Subcutaneous tumor model. Female nude mice that were 5 to 6 weeks old were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed with a 12-h light/dark cycle and allowed free access to water and standard diet. The cancer cells (1 × 106) were inoculated subcutaneously on both right and left flanks of 5 to 6 weeks old female nude mice, respectively. Tumor sizes and body weights were measured every three days for the duration of the experiment. Tumor volume was calculated as π/6ab2 where a is the length of the tumor, and b is the width of the tumor.
Brain metastasis model. Murine 4T1-luc2 breast cancer cells (ATCC® CRL-2539™) expressing firefly luciferase (luc2 vector) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL)/streptomycin (100 μg/mL) in 10 cm tissue culture plates in a 5% CO2-containing incubator at 37 °C. Eight-week-old female BALB/c mice were used in this study (n = 4 at each time point). During the tumor implantation, the mice were anesthetized by exposure to 1% to 3% isoflurane. A total of 2 × 104 of 4T1-luc2 tumor cells suspended in 2 μL of phosphate buffered saline (PBS) were slowly injected into the right caudate putamen 0.5 mm anterior and 2.0 mm lateral to the bregma at a depth of 3 mm from the dura over a 3-min duration. The needle was left in place for 5 min and then withdrawn slowly. The scalp wound was closed with 6-0 polydioxanone suture.
4.6. Biodistribution of Radioisotope-DOTA-NIR790
When the tumors reached a volume of 150 to 200 mm3 in the subcutaneous tumor model, mice received an intravenous injection of 111In-DOTA-NIR790 (equivalent to 10 MBq of 111In) or 177Lu-DOTA-NIR790 (equivalent to 37 MBq of 177Lu) (n = 3 at each time point). In brain metastasis model, the mice received an intravenous injection of 111In-DOTA-NIR790 at 10 days after tumor implantation. The distribution of 111In-DOTA-NIR790 or 177Lu-DOTA-NIR790 in the mice bearing tumors was evaluated by NanoSPECT/CT (Bioscan Inc., Washington, DC, USA) at 1, 4, 24, and 48 h after intravenous injection. The mice were sacrificed by cervical vertebra dislocation at 48 h after administration. The plasma, tumor, and normal tissue were collected, and the uptake of radioactivity was measured by a gamma counter. The distribution data obtained using radioactivity count methods are plotted as %ID/g.
4.7. Near-Infrared Fluorescence (NIRF) Imaging of 111In-DOTA-NIR790
The NIRF imaging of mice bearing tumors was studied at 1, 4, 24, 48, and 96 h after intravenous injection of 111In-DOTA-NIR790 (equivalent to 10 MBq of 111In) by the IVIS imaging system (Xenogen, Alameda, CA, USA) (n = 3 at each time point). The mice were anesthetized with a mixture of oxygen and isoflurane, and were placed on a 37 °C animal plate. The near-infrared fluorescence (NIRF) data were collected with a two second exposure time and an ICG filter set with excitation at 710–760 nm and emission at 810–875 nm. Quantitative analysis of fluorescence intensity in the images was performed using the region-of interest (ROI) function of the Living Image® software (Caliper Life Sciences Inc., Hopkinton, MA, USA).
4.8. Anti-Tumor Efficacy of 177Lu-DOTA-NIR790 with NIR Irradiation
After the subcutaneous tumors reached a volume of 150 to 200 mm3, mice received an intravenous injection of DOTA-NIR790 or 177Lu-DOTA-NIR790 (equivalent to 55.5 and 111 MBq of 177Lu). All mice were randomly divided into several groups (n = 6 per group), including normal saline, DOTA-NIR790 based photothermal therapy, radiotherapies with 55.5 and 111 MBq of 177Lu-DOTA-NIR790, and combination of radiotherapy and photothermal therapy, respectively. For photothermal therapy, 15 mg/kg of DOTA-NIR790 were administered via tail vein injections, and the tumors were exposed to the NIR laser with a spot size of 5mm at 1.8 W/cm2 for 5min at 24 h after administration. The size of subcutaneous tumors and body weight change of mice were recorded.
4.9. Immunohistochemical Analysis of Tumors with DOTA-NIR790-Mediated Photothermal Therapy
To evaluate the treatment effect, the mice with subcutaneous tumors were sacrificed, and tumor tissues were collected, after treatments of PBS (control), NIR irradiation alone, or DOTA-NIR790-mediated photothermal therapy. For the histopathological and immunohistochemical analysis, the section slides were stained with hematoxylin and eosin (H&E), proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Heat-shock proteins (HSPs) as described in previous reports [
25,
26,
27].