Highly Sensitive MoS2–Indocyanine Green Hybrid for Photoacoustic Imaging of Orthotopic Brain Glioma at Deep Site
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KeywordsMoS2–ICG hybrid Orthotopic brain glioma Photoacoustic imaging Molecular imaging
MoS2 nanosheets was covalently conjugated with indocyanine green (ICG) by facile mixing ICG-Sulfo-NHS and MoS2 nanosheets.
The 3.5 mm imaging depth demonstrated in this study is one of the deepest among all the glioma photoacoustic imaging research reported so far by using the nanoprobe in the NIR I spectral region.
The design and validation of the MoS2–ICG hybrid bring up an effective strategy for synthesizing highly sensitive photoacoustic nanoprobes, i.e., by covalently conjugating optical dyes with transition metal dichalcogenides.
Brain glioma is a highly invasive intracranial tumor that accounts for nearly one-third of all tumor cases in the central nervous system and shows a high incidence rate, high mortality rate, high recurrence rate, and low curing rate [1, 2]. The 5-year survival rate of glioma for adults is less than 5% . Invasive growth of glioma cells obscures the boundary between normal brain tissue and tumor tissue, making it extremely difficult to accurately diagnose this tumor and delineate the tumor boundary . Novel imaging methods with high sensitivity, specificity, and resolution at tumor-relative imaging depth are urgently needed for accurate diagnosis of glioma, based on which precise tumor surgery can be performed to achieve complete tumor removal and improve the prognosis of the patients [5, 6].
As a novel non-ionizing imaging method, photoacoustic imaging has been rapidly developed in recent years [7, 8]. This imaging technology acquires structural, functional, and molecular information for biological tissues by detecting the acoustic signals generated from the chromophores in the tissue [9, 10, 11]. As the contrast of photoacoustic imaging originates from the discrepancy of optical absorption, it retains the high sensitivity and specificity of conventional optical imaging methods [12, 13, 14]. Moreover, by detecting the significantly less scattered acoustic waves, photoacoustic imaging can achieve better resolution at higher imaging depth in biological tissues [15, 16, 17]. However, when used for brain glioma detection, the lack of endogenous contrast is a major limitation to the distinction between tumor and normal tissues through photoacoustic imaging.
The advent of molecular imaging has provided unprecedented opportunities for glioma detection with high sensitivity and specificity, as the tumor cells can be selectively labeled with exogenous contrast agents to achieve tumor-specific enhanced imaging. Hence, by combining photoacoustic imaging with molecular imaging (i.e., photoacoustic molecular imaging), brain glioma can be diagnosed with high sensitivity, specificity, and resolution at greater depth [18, 19, 20]. Nanomaterials such as gold nanoparticles and organic nanoparticles have been used as contrast agents for photoacoustic imaging of glioma [20, 21]. While these contrast agents have good photoacoustic imaging effects, the relatively low near-infrared (NIR) absorbance, narrow NIR absorption spectrum, and short absorbance–wavelength limit their potential for highly efficient in vivo photoacoustic imaging applications. Kircher et al.  first synthesized a photoacoustic-magnetic resonance imaging (MRI)-Raman triple-modality imaging nanoprobe and applied it to delineate brain glioma margins. The optical absorption peak of the nanoprobe is approximately 540 nm, which is very close to that of endogenous hemoglobin. Therefore, it is difficult to distinguish the photoacoustic signal of the nanoprobe from that of hemoglobin in in vivo imaging applications. Fan et al.  fabricated a perylene bisimide-based nanoparticle with a 675-nm absorbance peak for in situ photoacoustic imaging of mouse C6 brain glioma. The nanoparticle was shown to have excellent enhanced permeability and retention effects for tumor passive targeting. However, as an organic functional small molecule, perylene bisimide tends to be cleared from the blood circulation quickly. Moreover, the absorbance peak of perylene bisimide at 675 nm is close to visible light range, which may lead to high background photoacoustic signals in the blood. Therefore, it is of great needs to develop exogenous probe with long absorption wavelength and high sensitivity for photoacoustic imaging of orthotopic brain glioma.
Molybdenum disulfide (MoS2), a kind of transition metal dichalcogenides with distinctive physical and chemical properties, has sparked an explosion of interest in biomedicine [22, 23, 24]. In our previous study, a MoS2 nanoplatform was synthesized and shown to have with good biocompatibility and excellent tumor targeting capability for orthotopic glioma imaging . In situ brain glioma sitting 1.8 mm beneath the mouse scalp was clearly visualized with the aid of MoS2 nanosheet-enhanced photoacoustic imaging. However, the imaging depth must be further increased for glioma detection at even deeper sites. Hence, contrast agents with higher photoacoustic imaging sensitivity (in specific, longer absorption wavelength and higher absorption coefficient) are needed. Because of its large specific surface area, MoS2 nanosheets have a high loading capability, providing an effective strategy for synthesizing highly sensitive photoacoustic contrast agents by loading the optical dyes onto its surface [23, 24, 25, 26, 27]. Once loaded onto the MoS2 nanosheets surface, the optical dye is stabilized, and its internal blood circulation time is significantly prolonged. This high loading capability of MoS2 nanosheets and synergetic absorbance of optical dye along with MoS2 nanosheets endow the agent with a high optical absorption characteristic, making it a suitable candidate for photoacoustic molecular imaging applications.
MoS2 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) was obtained from Biosharp (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA solution, and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Cell counting kit-8 (CCK-8) and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Dojindo (Tokyo, Japan). ICG-Sulfo-OSu was obtained from AAT Bioquest, Inc. (Sunnyvale, CA, USA).
3.2 BSA-Assisted Exfoliation of Monolayer MoS2 Nanosheets
Monolayer MoS2 nanosheets were exfoliated by ice-bath sonication in a solution of BSA and water. Briefly, 50 mg of MoS2 powder was added to 10 mL of an aqueous solution containing 10 mg of BSA. The mixed suspension was sonicated in an ice bath for 8 h. After centrifugation at 8000 rpm for 20 min, the supernatant was collected to obtain the monolayer MoS2 nanosheets.
3.3 Synthesis of ICG-Conjugated MoS2 Nanosheets (MoS2–ICG)
Monolayer MoS2 nanosheets were covalently conjugated to ICG-Sulfo-NHS, an ICG derivative. Briefly, 1 mg ICG-Sulfo-NHS powder was dissolved in DMSO, and then added to 1 mL MoS2 nanosheets solution (~ 1 mg mL−1) and stirred at 25 °C for 12 h. Unbound ICG-Sulfo-NHS was removed by dialysis (8000–12,000 molecular cutoff) in deionized water for 24 h. The MoS2–ICG hybrid was collected and stored at 4 °C. All procedures were carried out without any direct light exposure. To determine the ICG loading efficiency, the optical absorbance of the dialysate was measured with a UV–Vis spectrometer at 780 nm and compared against a calibration curve to calculate the amount of unbound ICG. Conjugated ICG in MoS2 nanosheets was determined as unbound ICG subtracted from the total ICG used. The loading efficiency was calculated as W1/W2 × 100%, where W1 represents the weight of the conjugated ICG in MoS2 nanosheets and W2 is the weight of MoS2.
Atomic force microscopy (AFM) images were captured with a Bruker microscope (Billerica, MA, USA). Fourier transform infrared (FTIR) spectra measurement was carried out using an FTIR spectrometer (Bruker Vertex 80 V). UV–Vis–NIR spectra were detected with a UV–Vis–NIR spectrophotometer (PerkinElmer Lambda 750, Waltham, MA, USA). Fluorescence spectra were measured with a Luminescence Spectrometer (LS 55, Perkin–Elmer). Cell viability was detected by a multimode reader (BioTek SynergyTM 4, Winooski, VT, USA). The concentration of MoS2 was measured by ICP-OES (PE ICP-OES Optima 7000DV, PerkinElmer, USA). Our custom-built acoustic resolution photoacoustic microscopy (AR-PAM) system was used for all photoacoustic measurements. The AR-PAM system consists of a tunable pulsed optical parametric oscillator (OPO) laser (Vibrant 355 II HE, Opotek, Carlsbad, USA), a focused ultrasound transducer (V315-SU, Olympus IMS, Waltham, USA; central frequency: 10 MHz; fractional bandwidth: 6 MHz; N.A.: 0.4), and a precision motorized 3D scanning stage (PSA2000-11, Zolix, Beijing, China).The lateral resolution of AR-PAM was measured to be 220 μm (theoretically calculated to be 210 μm) and the imaging depth reached ~ 10 mm as demonstrated in our previous study . Detailed information regarding the system is given in supplementary information (Fig. S1).
3.5 Cell Line and Animal Model
The human brain glioma cell line U87 was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM media supplemented with 10% FBS and 1% penicillin–streptomycin solution in a humidified incubator (5% CO2 at 37 °C). Balb/c nude mice (3–5 weeks old) were purchased from the Medical Experimental Animal Center of Guangdong Province (Guangzhou, China). For orthotopic glioma model establishment, 1 × 106 U87 tumor cells in 6 μL PBS were injected into the striatum: Bregma 2.0 mm, left lateral 2.0 mm, depth 3.4 mm. The tumor growth was monitored by MRI (3T Magnetom Trio, Erlangen, Germany). All animal handling and experimental procedures were approved by the Animal Study Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
3.6 Cellular Uptake of MoS2–ICG
Fluorescence imaging was applied to confirm the internalization of MoS2–ICG into tumor cells. U87 cells were first cultured in a confocal dish for 24 h, and then mixed with MoS2–ICG or free ICG (~ 0.2 mg mL−1) for 1, 3, and 8 h incubation. Free MoS2 nanosheets were removed from the cells by washing three times with PBS. The treated cells were fixed with paraformaldehyde solution (4%) for 8 min followed by DAPI (10 μg mL−1) for 3 min. Fluorescence images of the cells were captured with a Leica TCS SP5 confocal laser scanning microscope (Wetzlar, Germany; E x = 405 and 633 nm for DAPI and ICG, respectively).
3.7 In Vitro Biocompatibility
3.8 In Vitro Photoacoustic Measurement
3.9 In Vivo Photoacoustic Imaging of Orthotopic Glioma
Mice bearing orthotopic U87 glioma were anesthetized with a 2% isoflurane/oxygen mixture and placed in the prone position. Photoacoustic imaging of the tumor region was performed by AR-PAM under 800-nm laser irradiation before and at 1, 3, and 5 h post-intravenous injection of MoS2–ICG (100 μL, 1 mg mL−1). Ultrasound images of the tumor region were captured simultaneously with AR-PAM.
3.10 In Vivo Biocompatibility of MoS2–ICG
Healthy Balb/c mice (5 mice in total) were intravenously injected with 10 mg kg−1 MoS2–ICG (150 μL). At days 1 and 15 post-injection, mouse blood was collected via orbit for complete blood panel testing at Shenzhen Center for Disease and Prevention. Furthermore, major organs from the MoS2–ICG-treated mice were harvested, including the heart, liver, kidney, lung, and spleen. The organ tissues were stained with hematoxylin and eosin (H&E) and examined under a digital microscope (Olympus, CX31, Tokyo, Japan) after fixation in 10% neutral-buffered formalin, embedding into paraffin and sectioning at 5 mm thickness.
4 Results and Discussion
4.1 Synthesis and Characterization of MoS2–ICG Hybrid
The ICG loading efficacy of MoS2–ICG was calculated to be 23.5%, which is higher than that of human serum albumin encapsulated nanoparticles (11%) , demonstrating that MoS2 with unique layer nanostructure and high large specific surface area favored the absorption of small optical dyes. The optical properties of MoS2 nanosheets were significantly altered after the loading of ICG. As shown in Fig. 1e, the optical absorbance of MoS2–ICG at the peak wavelength (800 nm) was significantly higher than that of MoS2 (Fig. 1e) because of the conjugation of ICG. (ICG loading efficiency was calculated to be 23.5%.) The absorbance intensity of MoS2–ICG was 22.6-fold (vertical dashed line in Fig. 1e) higher than that of MoS2 nanosheets under the same concentration. In addition, the NIR absorption spectrum of MoS2–ICG was expanded compared to that of ICG, with a redshift of the absorbance peak by 20 nm (from 780 to 800 nm) (Fig. S8). The broadened NIR spectrum and redshift of the absorbance peak occurred presumably because of the covalent conjugation of ICG onto the MoS2 surface, resulting in local aggregation of ICG molecules into oligomers [33, 34, 35]. After conjugating ICG to MoS2 nanosheets, the fluorescence of MoS2–ICG decreased by nearly 50% compared to free ICG at the same concentration of ICG (Fig. 1f), likely due to the fluorescence quenching effect induced by ICG aggregation and FRET (fluorescence/Förster resonance energy transfer) photoacoustic effect as reported previously [29, 36, 37, 38, 39, 40]. The decreased fluorescence intensity leads to greater photothermal conversion. Therefore, the photothermal/photoacoustic conversion efficiency of MoS2–ICG was enhanced compared to that of free ICG.
4.2 Photoacoustic Properties of MoS2–ICG Hybrid
4.3 Cellular Uptake and in Vitro Biocompatibility of MoS2–ICG Hybrid
4.4 In Vivo Photoacoustic Imaging of MoS2–ICG in Orthotopic Glioma Model
4.5 In Vivo Biocompatibility of MoS2–ICG
In summary, a MoS2–ICG hybrid was successfully prepared and applied for in vivo photoacoustic imaging of deep-sitting orthotopic brain glioma. Covalent conjugation of ICG and MoS2 is facile by mixing ICG-Sulfo-NHS and monolayer MoS2 nanosheets. The high imaging sensitivity of MoS2–ICG was validated, and potential causes were investigated and found to be: (1) strong optical absorbance across a broad NIR spectrum, enabling high photoacoustic signal generation; (2) redshifting of the MoS2–ICG absorption peak, enabling deeper penetration and lower background for in vivo imaging applications and (3) reduced ICG fluorescence due to the aggregation induced fluorescence quenching and FRET photoacoustic effect, enabling more energy to be converted to photoacoustic signal emission. Cellular uptake experiments showed that MoS2–ICG was internalized into the cytoplasm of U87 glioma cells with high efficiency. Both in vitro and in vivo studies showed that MoS2–ICG has excellent biocompatibility. In vivo photoacoustic imaging of orthotopic brain glioma demonstrated that the tumor mass sitting 3.5 mm below the scalp can be clearly identified through the enhancement by MoS2–ICG, which is nearly twofold deeper than that in our previous report using MoS2 nanosheets and to the best of our knowledge, is one of the deepest among all the glioma photoacoustic imaging studies reported so far by using the nanoprobe in the NIR I spectral region. Notably, the depth of photoacoustic molecular imaging depends on both the sensitivity of the imaging probes and the performance of the imaging system. While the effort for pursuing novel imaging probes with higher sensitivity should be a sustained ongoing process, photoacoustic imaging implementation with centimeter penetration depth capability such as photoacoustic computed tomography system should also be employed to translate the current study further on bigger animal models or even human beings. To conclude, the distinctive performance of MoS2–ICG, combined with the unique capability of photoacoustic imaging, reveals its potential for highly sensitive and accurate glioma detection in future translational medicine.
The authors gratefully acknowledge the following Grants support: National Natural Science Foundation of China (NSFC) Grants 91739117, 81522024, 81427804, 61405234, 81430038 and 61475182; National Key Basic Research (973) Program of China Grant 2014CB744503 and 2015CB755500; Guangdong Natural Science Foundation Grant 2014B050505013 and 2014A030312006; Shenzhen Science and Technology Innovation Grant JCYJ20170413153129570, JCYJ20160531175040976, JCYJ20150521144321005, JCYJ20160608214524052, JCYJ20160422153149834; JCYJ20150731154850923; SIAT Innovation Program for Excellent Young Researchers 201510.
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