Crushed Gold Shell Nanoparticles Labeled with Radioactive Iodine as a Theranostic Nanoplatform for Macrophage-Mediated Photothermal Therapy
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Crushed gold shell radionuclide nanoballs (124I-Au@AuCBs) were fabricated as unique photothermal therapeutics and nuclear medicine imaging nanoplatforms.
Macrophage-mediated delivery of 124I-Au@AuCBs and their potential photothermal therapy applications were demonstrated in mice with colon cancer.
KeywordsPhotothermal therapy Radionuclide crushed gold shell nanoballs Macrophage-mediated delivery
Gold nanomaterials have unique properties, such as strong localized surface plasmon resonance, biocompatibility, and easy surface modification via thiol–gold bonding [1, 2]. Numerous recent studies have attempted to develop several types of gold nanomaterials, such as Au nanoshells [3, 4, 5, 6], Au nanocages [7, 8, 9, 10], Au nanorods [1, 11, 12, 13, 14, 15], Au nanovesicles , Au nanostars [17, 18, 19], and Au nanohexapods , that are compatible with theranostic agents. In particular, plasmonic gold nanoparticles (AuNPs) have been widely studied as photothermal agents, owing to their excellent photothermal conversion induced by near-infrared (NIR) irradiation. AuNP-mediated photothermal therapy (PTT) has proven to be a promising treatment strategy for various cancers . However, several issues remain to be resolved for successful cancer therapy. In particular, the efficient delivery of PTT-compatible AuNPs to entire tumor lesions is required to ensure a cell death-inducing temperature.
Several approaches have been adopted to effectively deliver photothermal agents to tumor lesions, via passive or active targeting [1, 20, 21, 22, 23, 24, 25, 26]. For passive targeting, via enhanced permeability retention (EPR) effects, several types of coating materials (e.g., polyethylene glycol polymer, chitosan) have been introduced to the surface of AuNPs. To actively target tumor lesions, tumor-specific antibodies or ligands have been adopted to functionalize the AuNPs. Although these approaches have yielded promising results in delivering photothermal agents to tumors, nanoparticles can be entrapped in organs capable of EPR, such as the liver, kidney, and spleen, thereby inducing severe toxicity in normal organs. As an alternative delivery approach, intratumoral injection is useful to treat breast cancer and melanoma, owing to the accurate targeted delivery of PTT agents. However, most injected particles are retained in tumor lesions proximal to the site of injection and cannot penetrate deep into the tumors, thereby resulting in poor therapeutic outcomes. Thus, new approaches should be investigated for the effective delivery of photothermal agents to tumor lesions to elicit drastic therapeutic responses and to reduce toxicity in vital organs.
Macrophages are suitable transporters of various nanoparticles because they are present in the circulation and are easy to harvest from patients. Furthermore, owing to their unique properties, they easily engulf nanoparticles, such that each cell behaves as a “Trojan horse” delivery system, enabling the infiltration of otherwise inaccessible tumor lesions. Furthermore, several studies have reported the successful macrophage-mediated delivery of theranostic biomaterials to tumor lesions and resulting therapeutic effects in glioma, liver, and lung cancer models [27, 28, 29, 30, 31], revealing its great potential for cancer treatment.
We recently developed highly stable, biocompatible, and sensitive radioiodine-labeled AuNPs with crushed gold shells (124I-Au@AuCBs) as positron emission tomography/computed tomography (PET/CT) imaging agents for in vivo tumor imaging  and suggested their possible use in various biological applications. However, despite interesting findings regarding 124I-Au@AuCBs as useful biomaterials, we have not yet investigated the possibility of using 124I-Au@AuCBs as PTT agents. The generation of effective photothermally converted biomaterials is facilitated by the nanogap between the gold core and the gold shell, thus transforming these nanomaterials into promising theranostic biomaterials.
2 Materials and Methods
2.1 Materials and Instruments
All chemical reagents and tannic acid-capped AuNPs were purchased form Ted Pella, Inc. (Redding, CA, USA), and Na124I (half-life 4.2 days, emission type: high-energy γ and positron, energy 811 keV) was provided by KIRAMS (Seoul, South Korea). HAuCl4 was purchased from Sigma-Aldrich (St Louis, MO, USA).
PET/CT imaging was performed with a PET/CT scanner (LabPET8; Gamma Medica-Ideas, Waukesha, WI, USA). Bioluminescence imaging (BLI) was performed using an IVIS Lumina III instrument (PerkinElmer, Waltham, MA, USA). Photothermal images were obtained using a digital thermometer (TES Electrical Electronic Corp, Taipei, Taiwan) and an NIR imaging camera (FLIR Systems, Wilsonville, OR, USA).
2.2 Animals and Cells
Specific pathogen-free immunocompetent 6-week-old BALB/c mice were obtained from SLC, Inc. (Shizuoka, Japan). All experimental procedures involving animals were performed in strict accordance with the appropriate institutional guidelines for animal research. This protocol was approved by the Committee on the Ethics of Animal Experiments of the Kyungpook National University (approval number: KNU 2012-43).
Murine colon cancer CT26 cells co-expressing firefly and mCherry genes (CT26/FM) were grown in RPMI medium 1640 supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Gibco, Waltham, MA, USA).
Murine macrophage Raw264.7 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 atmosphere.
2.3 Preparation of Crushed Gold Shell Radionuclide Nanoballs
We have previously reported 124I-Au@AuCBs synthesis methods . Briefly, to generate a crushed gold shell on 124I-AuNPs, 1.0 mL of 124I-AuNPs (1 nM) was mixed with 500 μL of 1.0% (w/v) poly(N-vinyl-2-pyrrolidone) (MW, 40 kDa) and 100 μL of 100 mM phosphate buffer (pH 7.5 and 12.0). The solution was mixed with 434 μL of hydroxylamine hydrochloride (10 mM) and 434 μL of HAuCl4 (5 mM), gently vortexed for 30 min at room temperature, and centrifuged twice at 6500 rpm for 15 min. The resulting supernatant was resuspended in 1.0 mL of distilled water.
2.4 Characterization of Crushed Gold Shell Radionuclide Nanoballs
UV–Vis spectroscopy was performed using a Cary 60 UV–Vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). Transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) mapping were performed using an FEI Tecnai F20 transmission electron microscope (FEI Company, Eindhoven, the Netherlands). The hydrodynamic size of the nanoparticles was measured using ζ-potentials (ELS-Z, Otsuka, Japan). Fourier transform infrared (FT-IR) spectra were recorded on a PerkinElmer spectrometer in the range between 4000 and 400 cm−1.
2.5 Photothermal Conversion In Vitro
The photothermal conversion was evaluated using an 808 nm NIR laser (LVI, Anyang, South Korea). 124I-Au@AuNPs and 124I-Au@AuCBs were suspended in glass vials (1 mL) at different concentrations (0.5, 1, 2, and 4 nM) and exposed to the NIR laser at different power settings (3, 6, and 9 W cm−2). The temperatures of the suspensions were recorded using a digital thermometer (TES Electrical Electronic Corp, Taipei, Taiwan), with an accuracy of ± 0.1 °C. To determine whether exposure to the NIR laser could damage the morphology of the gold nanomaterials, they were examined using TEM, before and after irradiation at 9 W cm−2 for 5 min.
2.6 In Vitro Photothermal Therapy
The following experiments were performed to evaluate the photothermal therapeutic effects of 124I-Au@AuCBs on cancer cells.
2.6.1 Study 1
Either CT26 or CT26/FM cells were seeded in 96-well plates and incubated with 124I-Au@AuCBs (2 nM) for 3 h, followed by irradiation with an NIR laser (808 nm, 6 W cm−2). After 2 days, cell morphology was determined by microscopic imaging (Leica, Wetzlar, Germany). Cell proliferation was evaluated using a Cell Counting Kit (CCK-8; Dojindo Laboratories, Tokyo, Japan), and in vitro bioluminescent imaging was performed using an IVIS Lumina III instrument.
For cell proliferation assays, 10 µL of CCK-8 solution was added to treated cells and the absorbance was measured at 450 nm using a microplate reader (BMG Labtech, Offenburg, Germany).
2.6.2 Study 2
Either CT26 or CT26/FM cells were seeded in 96-well plates at 24 h and incubated with 124I-Au@AuCB (2 nM)-labeled macrophages, followed by exposure to an NIR laser (808 nm, 6 W cm−2) for 5 min. Two days later, cell viability was determined by in vitro bioluminescent imaging using an IVIS Lumina III instrument.
2.7 Apoptosis Analysis
Treated cells were collected, stained with FITC-conjugated annexin V and propidium iodide (BD Biosciences, San Jose, CA, USA), and analyzed by flow cytometry using a BD Accuri C6 flow cytometer (BD Biosciences).
2.8 In Vivo Photothermal Therapy
2.8.1 Study 1
CT26 cells were injected subcutaneously into mice, and tumor-bearing mice were divided into the following two groups when tumor mass was detected by physical inspection and palpation: Group 1, free 124I-injected group; Group 2, 124I-Au@AuCB-injected group. Tumor lesions were exposed to an NIR laser (808 nm, 6 W cm−2, 5-min exposure), and temperature changes in the tumor lesion were monitored using a digital thermometer. Nine days after therapy, the tumor was excised and weighed.
2.8.2 Study 2
CT26/FM cells were injected subcutaneously into mice, and tumor-bearing mice were divided into the following five groups when tumor volume reached 100–120 mm3: Group 1, phosphate-buffered saline (PBS)-treated group; Group 2, NIR laser-treated group; Group 3, 124I-Au@AuCB-loaded macrophage group; Group 4, 124I-Au@AuCB-loaded macrophage + NIR laser-treated group; and Group 5, unlabeled macrophage + NIR laser-treated group.
After injection of 124I-Au@AuCB-labeled macrophages into each tumor, PET/CT imaging was performed to determine the successful delivery of the particles to the tumor site. After image acquisition (3 h after intratumoral injection of 124I-Au@AuCB-labeled macrophages), the tumor lesion was exposed to an NIR laser (808 nm, 6 W cm−2, 5-min exposure) and temperature changes in the tumor lesion were monitored using a digital thermometer. Therapeutic response was evaluated by in vivo fluorescent imaging of the mCherry reporter gene. At day 9 after therapy, tumors were excised and weighed.
2.9 PET/CT Imaging
For computed tomography imaging, a 20-min scan (tumor lesion imaging) was performed using a Triumph II PET/CT system (LabPET8; Gamma Medica-Ideas). For PET/CT imaging, a 15-min scan was performed using the same animal PET/CT system as described above. CT scans were performed with an X-ray detector (fly acquisition; number of projections 512; binning setting 2 × 2; frame number 1; X-ray tube voltage 75 kVp; focal spot size 50 μm; magnification factor 1.5; matrix size 512). CT images were reconstructed using filtered back-projections. All mice were anesthetized using 1–2% isoflurane gas during imaging. CT images were reconstructed using the 3D image visualization and analysis software, VIVID (Gamma Medica-Ideas).
2.10 In Vivo Fluorescent Imaging
For CT26/FM tumor imaging, in vivo fluorescent imaging (FLI) was performed at the indicated times after PTT, using an IVIS Lumina III instrument with filter settings for mCherry. Grayscale photographic images and fluorescent color images were superimposed using LIVING IMAGE (version 2.12, PerkinElmer) and IGOR Image Analysis FX software (WaveMetrics, Lake Oswego, OR, USA). FLI signals were expressed in units of photon per cm2 per second per steradian (P cm−2 s−1 sr−1).
2.11 Statistical Analysis
All data are expressed as the mean ± standard deviation (SD) from at least three representative experiments, and statistical significance was determined by unpaired Student’s tests using Prism 5 software (GraphPad, San Diego, CA, USA). Differences with P values less than 0.05 were considered statistically significant.
3 Results and Discussion
3.1 Characterization of 124I-Au@AuCBs as Photothermal Agents
We recently developed highly sensitive and stable PET/CT imaging agents, comprising a radioiodine-labeled gold core and a crushed gold shell (124I-Au@AuCBs)  or round gold shell (124I-Au@AuNPs) . Several studies have reported the importance of morphological modification of nanomaterials and intra-nanogaps for the induction of photothermal conversion. 124I-Au@AuCBs and 124I-Au@AuNPs exhibit a crushed or round gold shell shape, respectively, with an intra-nanogap of 0.21–0.25 nm. These results led us to further examine the possibility of their use as new photothermal nanomaterials.
In addition, we investigated various characteristics of 124I-Au@AuCBs. The 124I-Au@AuCBs had bumpy surfaces, as evident from HR-TEM results. Subsequently, Au and I distributions were determined around the AuCBs by X-ray energy distribution mapping analysis (Fig. S2). Zeta-potential analysis results revealed that the surface charges of the particles were − 44.73 ± 2.18, − 50.73 ± 5.49, and − 31.66 ± 3.2 mV for AuNPs, 124I-AuNPs, and 124I-Au@AuCBs, respectively (Fig. S3a). FT-IR spectroscopy analysis revealed peaks at 1213 cm−1 for CH2, 1651 cm−1 for C=O stretching, 2858 cm−1 for C–H stretching, and 3408 cm−1 for O–H stretching. The FT-IR spectrum of AuCB products was the same as the spectra of the products of chemical reactions of their respective gold nanomaterials (Fig. S3b).
3.2 Photothermal Therapeutic Effect of 124I-Au@AuCBs on Colon Cancer In Vitro and In Vivo
3.3 Effects of PTT with 124I-Au@AuCB-Labeled Macrophages on Colon Cancer In Vitro and In Vivo
The development of nanoparticle systems with a high uptake efficiency is vital for successful cell-mediated photothermal therapy. Macrophages can be easily labeled with nanoparticles owing to their unique phagocytic activity . Thus, several studies have attempted to load therapeutic or imaging particles into macrophages for cell tracking and effective in vivo delivery of cytotoxic agents (doxorubicin) throughout tumor lesions [29, 31, 36, 37].
Thereafter, we evaluated whether 124I-Au@AuNP-labeled macrophages resulted in the death of colon cancer cells in the following groups: control group, NIR laser group, 124I-Au@AuNP-labeled macrophages group, 124I-Au@AuNP-labeled macrophages + NIR laser treatment group, and unlabeled macrophages + NIR laser treatment group. To visualize cell viability in vitro and in vivo, we engineered CT26 cells to co-express firefly luciferase and mCherry as optical reporter genes (CT26/FM cells). Briefly, CT26/FM cells were co-incubated with 124I-Au@AuNP-labeled macrophages and their respective cells were irradiated. After an additional incubation for 12 h, the in vitro effects of PTT were confirmed using BLI and apoptosis analysis. In vitro BLI (Fig. 5d) and apoptosis assays (Fig. 5e) showed the lowest BLI signals and the highest number of annexin V/PI-positive cells in the 124I-Au@AuNP-labeled macrophages + NIR laser treatment group. These findings indicate that 124I-Au@AuCBs engulfed by macrophages retain the potential for photothermal conversion, thereby effectively eliminating colon cancer cells surrounding the 124I-Au@AuNP-labeled macrophages.
This study evaluated the possibility of using 124I-Au@AuCBs as a novel theranostic nanoplatform for in vivo PTT. 124I-Au@AuCBs exhibited great photothermal conversion ability, and macrophage labeling was achieved via a simple incubation, with no associated cytotoxicity. Furthermore, the effects of 124I-Au@AuCBs on PTT were retained upon macrophage uptake, thereby inducing discernible cell death in tumors in vitro. More importantly, it is possible to visualize the successful delivery of 124I-Au@AuCBs to tumor lesions by using macrophages as Trojan horses, owing to the presence of 124I, which can be detected by PET imaging. Finally, 124I-Au@AuCB-labeled macrophages resulted in significant tumor ablation, suggesting that this is a potentially effective photothermal approach for optimizing nanoplatform delivery in vivo. Further studies are required to examine the tracking of 124I-Au@AuNP-loaded macrophages to tumor lesions after intravenous injection and to assess the potential for translation of our experimental findings to clinical applications in cancer therapy.
This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korea Government (MSIP), a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1501), a grant from the Medical Cluster R&D Support Project through the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) funded by the Ministry of Health and Welfare (HT16C0001, HT16C0002, HT17C0009), a National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (2014R1A1A1003323, 2017R1D1A1B03028340, 2018R1D1AB07047417).
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