Iodine-Labeled Au Nanorods with High Radiochemical Stability for

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Iodine-Labeled Au Nanorods with High Radiochemical Stability for Imaging-Guided Radio- and Photothermal Therapy Peng Wang, Wenjing Sun, Qiang Wang, Jingwen Ma, Xinhui Su, Qing Jiang, and Xiaolian Sun ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02229 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on March 3, 2019

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ACS Applied Nano Materials

Iodine-Labeled

Au

Nanorods

with

High

Radiochemical Stability for Imaging-Guided Radioand Photothermal Therapy Peng Wang,1,2‡ Wenjing Sun,3‡ Qiang Wang,4 Jingwen Ma,1 Xinhui Su,4 Qing Jiang,2* Xiaolian Sun,1* 1

State Key Laboratory of Natural Medicines, Key Laboratory of Drug Quality Control and

Pharmacovigilance, Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China 2

Department of Sports Medicine and Adult Reconstructive Surgery, Drum Tower Hospital

affiliated to Medical School of Nanjing University, Zhongshan Road 321, Nanjing, 210008, P. R. China. 3

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for

Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 261005, China. 4

Department of Nuclear Medicine, Zhongshan Hospital Xiamen University, Xiamen 361004,

China KEYWORDS: Au nanorods, valent state, radioiodine labeling, pre-oxidization, ultrahigh radiochemical stability ABSTRACT:Radioactive iodine labeled gold nanomaterials (Au NMs) have been widely used in biomedical application such as cell tracking and disease theranostics. The radiolabeling can be accomplished via simply mixing Na131I and Au NMs ([131I(-1)] Au NRs) due to the specific absorption between iodide ions and Au NMs. However, it’s reported that the absorption strength 1 ACS Paragon Plus Environment

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is highly dependent on the incubation medium and the serious detachment of 131I from [131I(-1)] Au NRs-PEG in vivo inhibited their application. We found that adding a simple pre-oxidization treatment of Na131I could greatly improve their radiochemical stability both in vitro and in vivo. The [131I(0)] Au NRs-PEG we prepared barely showed iodine detachment in different medium and demonstrated six times higher cellular internalization of radioactivity than that of [131I(-1)] Au NRs-PEG since the detached radio-iodine of which could not enter the cells. The mechanism study pointed out that the pre-oxidized iodide could chemically react with Au NRs, mediating Au etching. But with trace amount of

131

I, both the morphology and the optical property of Au

NRs can be well-maintained. Our [131I(0)] Au NRs-PEG successfully retained in the tumor over 24 h after intra-tumoral injection and demonstrated synergistic radio- and photothermaltherapeutic effect. We believed that this simple and rigid labeling process improvement could promote the biomedical application and clinical translation of radioactive iodine labeled Au NRs. 1. INTRODUCTION Gold nanomaterials (Au NMs) have been widely used in cancer theranostics due to their superior biocompatibility and special optical properties.1, 2 Gold nanorods (Au NRs) with near infrared (NIR) absorption (600-900 nm) are especially attractive as photothermal conversion agents (PTCAs).3-5 Upon laser irradiation, Au NRs could generate localized heating to kill cancer cells, which known as photothermal therapy.6, 7 To effective eradicate tumor with minimized side effects to surrounding health tissues, it is significant to obtain the information of the in vivo location of Au NRs so that to individualize laser operation. Since nuclear imaging could quantitatively trace the distribution of radiotracers with high sensitivity, various radioisotopes have been labeled onto Au NRs to monitor their delivery.8, 9 2 ACS Paragon Plus Environment

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With the ability for both single-photon emission computed tomography (SPECT) imaging and radiotherapy,

131

I (t1/2=8.01 days) isotope attracts much attention for image

guided therapy. What’s more, gold nanorods are reported to enhance the sensitivity of radiotherapy.10, 11 There were mainly two approaches to radiolabel iodine onto Au NRs: introducing electron-rich aromatic groups for oxidative iodination12-14 and making use of the specific physical absorption between iodide ions and Au NMs.15-17 However, both methods face the challenges of unsatisfactory radiochemical stability in physiological conditions.18 The detached iodine has high affinity to thyroid glands, and the nonspecific radiation will damage the normal tissues. To prevent iodide detachment, Lee et al. formed another gold nanoshells on

131

I labeled Au NMs, resulting in dramatic change of their

original physicochemical properties.19 To maintain their capabilities as PTCAs, Zhang et al. coated a thin layer of polydopamine on Au NRs for iodine protection instead.15 Still, an easy-to-handle, highly efficient radioiodine labeling method with high radiochemical stability and minimal change to the original properties of Au NRs is highly desirable for potential clinical use. When many studies do the radiolabeling by physically mixing Na131I and Au NMs together ([131I(-1)] Au NMs),15-17 we found that adding a simple pre-oxidization step could greatly improve the radiochemical stability of

131

I labeled Au NRs even without conjugation of

electron-rich aromatic groups. A detailed comparison of [131I(-1)] Au NRs and [131I(0)] Au NRs has been carried out (Scheme 1). With a trace amount of radioactive iodine, no obvious morphology or optical properties change was observed. We confirmed that although both I(0) and I(-1) bond with Au as neutral iodine, I(-1) simply absorbed onto Au NRs20 while I(0) reacted with Au NRs, resulting in stronger bond21,22. Our [131I(0)] Au NRs demonstrated superior 3 ACS Paragon Plus Environment


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radiochemical stability over [131I(-1)] Au NRs both in vitro and in vivo. After PEGylated, the [131I(0)] Au NRs-PEG stayed in the tumor area over 24 h and proved to be suitable for SPECT imaging, radiotherapy as well as photothermal therapy. We believe that our study provided a more clear awareness that the valence state of iodine significantly affects its binding affinity to Au NMs. With the simplicity and effectiveness, we believe that the radiolabeling method is favorable for clinical translation and will greatly benefit the use of 131I in theranostic. Scheme 1 Schematic diagram of in vivo comparison of [131I(0)]Au NRs-PEG and [131I(-1)]Au NRs-PEG

2. EXPERIMENTAL SECTION 2.1 Materials. HAuCl4 was purchased from J&K Chemical Ltd. Poly(ethylene glycol)-thiol (SH-PEG, Mw 5 kDa) was purchased from Avanti Polar. Chloramine-T hydrate (95%) and Iodogen were obtained from Sigma-Aldrich Co. Ltd. (MO, USA). Na131I was provided by Zhongshan Hospital Affiliated of

Xiamen

University,

China.4-Nitrophenyl

chloroformate

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicilline-streptomycin solution were 4 ACS Paragon Plus Environment

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purchased from HyClone Inc. Deionized (DI) water with a resistivity of 18.2 MΩ was obtained from a Millipore Autopure system. All other chemicals were of analytical grade and used without further purification unless noted. 2.2 Characterization. Transmission electron microscopy (TEM) images were obtained by a JEOL 1200EX. The radiochemical stability was measured by mini-scan radio-TLC strip scanner (B-MS-1000F, BioScan, USA). The radioactivity was measured with automatic gamma-counters (WIZARD 2480, Perkin-Elmer, USA). SPECT/CT imaging was performed by a nanoScan-SPECT/CT preclinical scanner (Mediso, Hungary). NIR laser-induced PTT was carried out by NIR laser (808 nm) and real-time thermal imaging was acquired using a FLIR A×5 camera (FLIR Systems Inc., Wilsonville, OR). 2.3 Preparation of CTAB-Au NRs and Citric acid-Au NPs. Au NRs were synthesized using a seed-mediated growth method as previously reported using cetyltrimethylammonium bromide (CTAB) as ligands.23 Au NPs were synthesized by a reported method using citric acid as ligands as control.24 The resulting NRs or NPs were purified by centrifugation for at least three times before further use. 2.4 131I-labeling methods. Three 131I-labeling methods have been carried out in this study. 131

I(-1) labeling: 1 mL prepared Au NRs (0.025 mg/ml~0.5 mg/ml) were mixed with 10 μL

Na131I (0.5~1.0 mCi) under stirring at room temperature for 1 h. The [131I(-1)] labeled Au NRs were centrifuged at 8000 rpm and washed with DI water for three times. 131

I(0) labeling (chloramine T): 0.3 mg chloramine T aqueous solution was mixed with 10 μL

Na131I (0.5~1.0 mCi) by gentle vortex for about 40 s. 1 mL prepared Au NRs (0.025 mg/ml~0.5 5 ACS Paragon Plus Environment


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mg/ml) was added and the mixture reacted at room temperature for 1 h. Then, the [131I(0)] labeled Au NRs were collected by centrifugation at 8000 rpm for 10 min and then washed by DI water for three times. 131

I(0) labeling (Iodogen): Dispense 20 μg of Iodogen (in chloroform) in the glass tubes and

dry the solvent to make a uniform coating on the tubes. 1 mL prepared Au NRs (0.025 mg/ml~0.5 mg/ml) were mixed with 10 μL Na131I (0.5~1.0 mCi) in the tube. After shaking for 10 min, the solution was ready to use without further purification. 2.5 Surface PEGylation. 2 mg of SH-PEG was added to 1 mL of prepared Au NRs and stirred overnight. The obtained Au NRs-PEG were washed with DI water. 2.6 Radiochemical stability. In vitro 100 µL of Au NPs (citric acid) + [131I(0)], Au NRs (CTAB) + [131I(0)], Au NRs (PEG) + [131I(0)] and Au NRs (CTAB) + [131I(-1)] were incubated with 900 µL of PBS or FBS at 37 °C, respectively. The amount of detached 131I was quantified by radio-TLC. In vivoIn vivo stability test was conducted by SPECT/CT scanning after intravenous (i.v.) injection of [131I(-1)] Au NRs-PEG or [131I(0)] Au NRs-PEG (~ 150 μCi) into normal Balb/c mice. Detached 131I mainly targeted to the thyroid which can be easily monitored. 2.7 Cell experiment. The MCF-7 human breast cancer cells were cultured at 37 ℃ with 5% CO2 in DMEM medium containing 10% FBS and 1% penicillin/streptomycin in a humidified incubator. Cellular uptake After seeded in 24-well plates at a density of 105 cells per well and cultured for 24 h, the cells were incubated with [131I(0)] Au NRs-PEG, [131I(-1)] Au NRs-PEG and Na131I at 6 μCi/10 μg (131I/Au NRs). After incubation for some time, cells were washed with PBS buffer for 6 ACS Paragon Plus Environment

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three times and were detached using 0.1 mol·L-1 NaOH and collected. The radioactivity in pellet was counted using gamma counter. Cytotoxicity assay Cellular cytotoxicity was evaluated by a standard MTT assay. In brief, MCF-7 cells were seeded in 96-well cell culture plates at a density of 104 cells per well before [131I(0)] Au NRs-PEG, [131I(-1)] Au NRs-PEG and Na131I of different radioactivity were added. For photothermal therapy test, cells were irradiated by 808 nm laser (1 W·cm-2 for 5 min) after 24 h incubation. To test the cytotoxicity, 10 μL of cell culture medium containing MTT was added and incubated for another 4 h. The cell viabilities were determined by measuring UV absorption at 450 nm. 2.8 Animal experiment. Female nude mice (weight ~20 g) were obtained from Laboratory Animal Center of Xiamen University or Shanghai Slac Laboratory Animal (China). Animal studies were performed following the national laws related to the conduction of animal experiment. To construct MCF-7 tumor models, 5×106 cells suspension were subcutaneously injected onto the right rear flank of each mouse. The in vivo imaging and therapy were performed after the tumor volume reached around 100 mm3. SPECT/CT imaging of Balb/c mice bearing tumor were obtained at 1 h, 10 h, 18 h and 24 h after intratumorally injected [131I(0)] Au NRs-PEG using nanoScan SC (Mediso Medical Imaging System) equipped with pinhole collimator. The scanning parameters were as following (scan time: 30 min; frame: 45; FOV: 26 × 26 × 70 mm3; resolution: 0.4 mm). For the therapeutic study, tumor-bearing mice were randomly divided into six groups (n=5 per group) including control group and intra-tumoral injection with Na131I (50 µCi

131

I per mouse),

Au NRs-PEG (80 µg Au per mice) with laser irradiation (0.5 W∙cm-2, 10 min) and [131I (0)] 7 ACS Paragon Plus Environment


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AuNRs-PEG (50 µCi 131I, 80 µg Au per mice) with and without laser irradiation (0.5 W∙cm-2, 10 min). The laser is collimated and the power is measured via laser power meter. The

131

I dosage

used here is within safety dosage.25 The thermal images of tumors were taken with a FLIR A×5 camera and quantified by BM_IR software. The mouse tumor sizes were measured and calculated as the volume = width2×length×0.5. 3. RESULTS AND DISCUSSIONS With their excellent biocompatibilities, Au based NMs were frequently used as radioactive iodine cargoes. Table S1 summarized the studies on radioiodine labeled Au NMs, which can be mainly divided into two categories: grafting electron-rich phenol groups for iodination13,26,28 and making use of specific absorption between iodine ions and gold atoms.16,18,27 Compared with further modification of functional groups which might alter the surface properties of Au NMs, simply mixing Na[131I] and Au NMs is more favorable. However, the iodine absorption is highly dependent on the gold surface modification as well as the incubation medium,27 and is unstable in vivo.17,18 To enhance its radiochemical stability, many efforts have been made such as coating it with another layer15,19 or pre-injection of cold NaI to block the thyroid where the free iodine mainly target.29 We found that adding a simple pre-oxidation step of Na131I via traditional chloramine T or Iodogen method could greatly improve its radiochemical stability. 3.1. In vitro and In vivo radiochemical stability comparison of [131I(0)]Au NRs and [131I(-1)]Au NRs Au NRs with a length around 70.6±8.4 nm and width around 10.8±3.5 nm were prepared according to the reported method.30 Iodogen and chloramine-T, two of the most widely used chemical oxidants which could convert sodium iodide to iodine form for iodination of tyrosyl 8 ACS Paragon Plus Environment

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groups,31,32 have been used to pre-oxidize Na[131I(-1)] ahead of mixing with Au NRs, respectively. Since the two oxidants led to the similar result, we used [131I(0)]Au NRs to represent both the radiolabeled products. Both 131I(0) and 131I(-1) (simply mixing Na[131I] and Au NRs) treatment have the labeling yield over 90%, and no obvious morphology (Figure. 1a-b) or optical properties (Figure. 1c) change have been observed afterwards. The radiochemical stability of [131I(0)]Au NRs and [131I(-1)]Au NRs were systematically evaluated. Within 24 h, no detachment of

131

I happened for both samples in PBS buffer of different pH (pH= 5.0, 7.4 and

9.0) (Figure. 1d and Figure. S1). However, when incubated in cell culture medium (Dulbecco's modified Eagle's medium, DMEM), around 25% 131I(-1) detached from [131I(-1)]Au NRs within 8 h and after 24 h only 60% 131I(-1) was kept intact. Meanwhile, above 90% 131I(0) of [131I(0)]Au NRs retained intact even after 24 h incubation (Figure. 1e). The stronger binding of 131I(0) than 131

I(-1) to Au NRs were also proved in fetal bovine serum (FBS), with 98%

after 24 h, but more than 25%

131

131

I(0) stay intact

I(-1) detached from Au NRs (Figure. 1f). The Au NMs with

citric acid as surface ligands were also prepared24 and demonstrated the similar binding capability to

131

I(0) as Au NRs with cetyltrimethylammonium bromide (CTAB) as surface

ligands did (Figure. 1d-f). It is assumed that the

131

I(0) binds to Au atom, with few help of its

ligands. However, when we modified the Au NRs with poly (ethylene glycol) (Mw~ 5000) before

131

I(0) labeling, although no

131

I(0) detachment was observed in PBS, less than 25% and

60% 131I were intact in DMEM and FBS respectively. It seems that PEGylation ahead will hinder the interaction between iodine and Au surface, leading to dramatic leak of iodine in presence of cell culture medium or serum, the ingredients in which will compete for iodine.27 A more direct comparison of the in vivo radiochemical stability of [131I(0)]Au NRs and [131I(-1)]Au NRs was performed via SPECT/CT imaging (Figure. 1g). To ensure the biocompatibility, we PEGylated 9 ACS Paragon Plus Environment


the

131

I(0) and

131

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I(-1) labeled Au NRs before intravenous (i.v.) injection. It is well-known that

free iodine is preferentially uptaken by thyroid. The thyroid and bladder were lit up in the mice 1 h after post-injected with [131I(-1)] Au NRs-PEG. In comparison, no signal in the thyroid can be seen in the mice injected with

131

I(0)-AuNR-PEG even after 24 h injection

(Figure S2), confirming its excellent in vivo radiochemical stability.

Figure 1 (a-b) TEM image of Au NRs (a) and [131I(0)]Au NRs-PEG after decay (b). (c) UV-vis spectra of Au NRs and [131I(0)]Au NRs-PEG; (d-f) Radiochemical stability of Au NPs (citric acid) + [131I(0)], Au NRs (CTAB) + [131I(0)], Au NRs (PEG) + [131I(0)] and Au NRs (CTAB) + [131I(-1)] incubated in PBS (d), DMEM (e) and FBS (f). (g) Representative SPECT/CT imaging of the mice at 1 h after intravenous injection of [131I(-1)] Au NRs-PEG (left) and [131I(0)] Au NRs-PEG (right). Green arrow, thyroid area; red arrow, bladder.

10 ACS Paragon Plus Environment

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3.2. Interaction of Au NRs with I(0) and I(-1). We further investigated the interaction of Au NRs with I(0) and I(-1). Since trace amount of radioactive I(0) or I(-1) is difficult to detect, we used un-radioactive I(0) or I(-1) instead. Neither the morphology nor the UV absorption spectra of Au NRs was changed during the gradually addition of NaI with a molar ratio of NaI to Au increased from 1:2 to 4:1 (Figure. 2a, 2e, 2f and Figure. S3), except that Au NRs tended to aggregate at high concentration of NaI. However, when Au NRs were mixed with pre-oxidized NaI either via chloramine T or Idogen methods, its shape gradually changed from rod to sphere as the molar ratio of I to Au increased (Figure. 2g-h and Figure. S4). Correspondingly, the absorption peak shifted from 810 nm to 675 nm (Figure. 2b). When the ratio of I to Au is less than 2, the rod shape is partially maintained and iodine distributed throughout the Au NRs as the high-angle, annular dark-field scanning TEM (HAADF-STEM) images indicated (Figure. S4). When the ratio of I to Au over 4, nearly no nanoparticles could be found, indicating a complete etching process (Figure. S5c). XPS measurements were performed for Au NRs immersed in NaI aqueous solution, in pre-oxidized NaI aqueous solution as well as with I2, and the binding energy (EB) for Au and I at the interface are summarized in Figure. 2c-d and Table 1. After the three different treatment, EB for Au (4f7/2) are all within 83.9±0.5 eV, indicating the Au is in the zero valent state. The EB for I (3d5/2) was shifted from 618 eV to 618.94 eV for Au + NaI aqueous solution, 619.24 eV for Au + pre-oxidized NaI, and 619.20 eV for Au + I2. The results indicated that in all three cases, the iodine species absorbed onto Au NRs were iodine atoms with little ionic character.31 However, considering the different radiochemical stability (Figure. 1d-f) and morphology change (Figure. 2e-h) of I (0) and I(-1) on Au NRs, we assumed that although both I(0) and I(-1) bond with Au as neutral iodine, I(0) chemically reacted with Au NRs and resulting in a stronger binding.34 11 ACS Paragon Plus Environment


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Figure 2 (a) UV-vis spectra of Au NRs, and Au NRs mixed with NaI at a molar ratio of NaI to Au from 1:2 to 4:1; (b) UV-vis spectra of Au NRs, Au NRs mixed with pre-oxidized NaI at a molar ratio of I:Au from 1:2 to 4:1; (c-d) Binding energy of Au (c) and I (d) in Au NRs mixed with NaI, pre-oxidized NaI and I2; (e-f) TEM image of Au NRs treated with NaI at a molar ratio of NaI to Au 1:1 (e) and 2:1 (f); (g-h) TEM imaging of Au NRs treated with pre-oxidized NaI at a molar ratio of NaI to Au 1:1 (g) and 2:1 (h). Table 1

X-ray photoelectron spectroscopic data analysis for gold (Au) and iodine (I). Binding Energy (eV)

Component

Au (4f7/2)

I (3d5/2)

Au

83.835

—

Au/I

83.9±0.533

618±0.533

Au NRs + NaI

84.04

618.94

Au NRs + pre-oxidized NaI

84.35

619.24

Au NRs + I2

84.34

619.20


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3.3. Cellular uptake and therapeutic effect comparison of [131I(0)]Au NRs and [131I(-1)]Au NRs. Cellular uptake of [131I(0)] Au NRs-PEG and [131I(-1)] Au NRs-PEG were investigated using MCF-7 cell as model. As shown in Figure. 3a, Na131I was barely internalized within 24 h, while only around 3-5% radioactivity of [131I(-1)] Au NRs-PEG was found inside cells. In comparison, the radioactivity in MCF-7 cells treated with [131I(0)] Au NRs-PEG demonstrated a time-dependent increase from 6.18±0.84% at 4 h, 13.08±0.83% at 12 h to 32.1±6.1% at 24 h, consistent with the cellular internalization behavior of Au NRs (Figure. S6). It seemed that [131I(0)] Au NRs-PEG could efficiently deliver

131

I into cells due to the strong interaction

between iodine and Au NRs. Meanwhile, [131I(-1)] Au NRs-PEG is unstable when incubated with cells, and the detached radio-iodine failed to enter the cells. The radio-therapeutic efficiency is highly related to the

131

I cellular internalization efficiency. As shown in Figure. 3b, Na131I did

not show obvious therapeutic effect at the radioactivity up to 20 μCi for 24 h. For [131I(-1)] Au NRs-PEG, around 90% cells keep alive with radioactivity up to 12 μCi. In comparison, [131I(0)] Au NRs-PEG demonstrated obvious radioactivity dependent cellular toxicity, with cellular viability decreased from 96.19±1.13%, 88.05±1.52%, 75.13±2.4% to 60.81±2.34% as the radioactivity increased from 0.6, 3, 6 to 12 μCi. The photothermal capability of Au NRs were then combined to synergistically enhance the therapy effectiveness. It is worth to mention that the trace amount of

131

I barely affect the optical property or photothermal capability of Au

NRs.15Under the NIR laser (808 nm, 1 W·cm-2) irradiation for 5 min, only 31.09±0.97% [131I(0)] Au NRs-PEG (100 μg/ml Au NRs, 60 μCi/ml

131

I) treated cells were alive, while the cell

viability after photothermal only is 67.53%±3.01% and after radiotherapy only is 73.96%±0.98% (Figure. 3c). 13 ACS Paragon Plus Environment


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Figure 3 (a) MCF-7 Cell uptake of radioactivity of Na131I, [131I(0)]Au NRs and [131I(-1)]Au NRs with the concentration of 6 µCi/10 µg (131I/Au NRs) at different time points. (b) Radio-therapeutic effect of Au NRs-PEG, [131I(0)]Au NRs-PEG and [131I(-1)]Au NRs-PEG at 100 µL of different concentration of

131

I and Au NRs. (c) Viability of MCF-7 cells treated with

laser only, [131I(0)]Au NRs-PEG (100 µg/mL Au NRs, 60 µCi/mL

131

I, 100 µL), Au NRs-PEG

with laser and [131I(0)]Au NRs-PEG with laser. 3.4. In vivo behavior of [131I(0)]Au NRs. We then tested the in vivo effectiveness of [131I(0)] Au NRs-PEG. In order to control the radioactive dosage of

131

I and reduce the damage of normal tissue, intra-tumoral injection was

employed. Both SPECT/CT imaging (Figure. 4a) and biodistribution studies (Figure. S7) proved that the radioactivity was well-retained in the tumor area for 24 h with no leaky to the normal tissues including thyroid. Female nude mice bearing MCF-7 tumors were randomly divided into five groups (PBS only, Free Na131I, Au NRs-PEG + laser, [131I(0)] Au NRs-PEG, [131I(0)] Au NRs-PEG + laser; 50 µCi

131

I, 0.5 mg Au NRs). Compared with free Na131I for

radiotherapy, [131I(0)] Au NRs-PEG inhibited the tumor growth more effectively due to the trap of radioactivity in the tumor area. Au NRs is well-known as photothermal therapeutic agents. Upon 808 nm laser irritation (0.5 W·cm-2), the tumor temperature of mice injected with Au NRs-PEG or [131I(0)]Au NRs-PEG increased to around 50 °C within 2 min and the tumor growth 14 ACS Paragon Plus Environment

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has been greatly delayed. [131I(0)] Au NRs-PEG demonstrated a similar photothermal effect in vivo since the radio-iodine labeling barely influence the optical properties of Au NRs-PEG (Figure. 4b-c, Fgiure S8). Combined the radiotherapy with photothermal therapy, the tumor has been completely suppressed and the mice survival time has been greatly extended. Within 28 days, no single death, tumor reoccurrence or significant body weight variation has been found (Figure. 4d-e, Figure. S9). The unnecessary accumulation of radiation in healthy tissue is one of the main challenge of internal radionuclide therapy. In clinical, the radioisotope is introduced to the target area via implant or catheter and will be removed after therapy to prevent the unnecessary radiation exposure. We first tested the safety of our iodine carrier via tail vein injection of 240 mg un-radioactive (cold) iodine labeled Au NRs (three times of therapeutic dose) prepared by the same labeling method but using cold iodine instead of 131I. No abnormal behavior was observed. The serum biochemical test (Table S2) further proved that the carriers are biocompatible. Then, we intratumorally injected the radioactive [131I(0)] Au NRs-PEG. With most

131

I isotope

immobilized stably at the injection location (Figure 4a), no obvious damage or inflammation in the healthy tissues were observed 7 days after treatment as indicated by the histology analysis (Figure S10). Together with the fact that Au NRs is a nanocarrier entering clinical trial36 and 131I is a clinically used isotope, we believe that [131I(0)] Au NRs-PEG has great potential in internal radionuclide therapy.



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Figure 4 (a) SPECT/CT imaging of MCF-7 tumor-bearing mice after 1 h, 10 h, 18 h and 24 h intra-tumor injection of [131I(0)]Au NRs-PEG; (b-c) Thermal imaging (b) and corresponding quantitative analysis of temperature (c) of MCF-7 tumor bearing mice after intra-tumoral injection of PBS, Au NRs-PEG and [131I(0)] Au NRs-PEG. (d) The tumor growth rate after the different treatments. Tumor volumes were normalized to their initial size. The error bars represent the standard deviation of 5 mice per group. (e) Body weight curves of tumor-bearing mice for each group. 4. CONCLUSIONS With their great potential in clinical translation, radioactive iodine (131I,

125

I,

124

I) labeled Au

NRs have been widely used in biomedical application such as cell tracking, disease imaging and therapy. However, although iodine can be chemisorbed onto the Au surface when simply mixing Na131I and Au NRs together, the radiochemical stability is unsatisfactory and the serious 16 ACS Paragon Plus Environment

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detachment of 131I inhibited their application. We found that adding a simple pre-oxidization step of Na131I either via chloramine T or Iodogen (both of which are in common use and easy-to-handle) could prevent the iodine detachment. We assumed that the stronger binding is due to the chemical reaction between trace amount pre-oxidized 131I and Au. After radiolabeling, the [131I(0)] Au NRs-PEG still maintained their NIR triggered photothermal properties. Compared with previous [131I(-1)] Au NRs-PEG, our [131I(0)] Au NRs-PEG demonstrated excellent radiochemical stability both in vitro and in vivo. The radioactivity delivered into MCF-7 cells by [131I(0)] Au NRs-PEG is six times than that of [131I(-1)] Au NRs-PEG, greatly benefiting the radio-therapy. The [131I(0)] Au NRs-PEG could retain in the tumor over 24 h and demonstrated synergistic radio- and photothermaltherapeutic effect. We believe this simple and rigid labeling process could promote the biomedical application of iodine labeled Au NMs. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Summary of previous radio-iodine labeled Au NMs. Radiochemical stability of [131I(0)] Au NRs and [131I(-1)] Au NRs incubated in different medium. High-angle, annular dark-field scanning TEM (HAADF-STEM) images and elemental mapping of iodide treated Au NRs. TEM images of Au NRs before and after incubation with different concentrations of 131I(0) and 131I(-1). Biodistribution of [131I(0)] Au NRs. Survival rate and H&E staining of different tissues after treatment. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] 17 ACS Paragon Plus Environment


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Author Contributions ‡

P. Wang and W.J. Sun contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We acknowledge the National Key Research and Development Program of China (2016YFA0203600), National Natural Science Foundation of China (51502251, 81571743, and 81571707), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZRC05), the International Cooperation and Exchange of National Natural Science Foundation (NSFC 81420108021), Key Program of NSFC (81730067), Notes and references. REFERENCES (1) Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; El-Sayed, M. A., The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev.2012, 41, 2740-2779. (2) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A., Gold Nanoparticles for Biology and Medicine. Angew. Chem., Int. Ed. 2010, 49, 3280-3294. (3) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A., Cancer Cell Imaging and Photothermal Therapy in the Near-infrared Region by Using Gold Nanorods. J. Am. Chem. Soc.2006, 128, 2115-2120. (4) Dickerson, E. B.; Dreaden, E. C.; Huang, X.; El-Sayed, I. H.; Chu, H.; Pushpanketh, S.; McDonald, J. F.; El-Sayed, M. A., Gold Nanorod Assisted Near-infrared Plasmonic Photothermal Therapy (PPTT) of Squamous Cell Carcinoma in Mice. Cancer Lett.2008, 269, 57-66. (5) Shen, S.; Tang, H.; Zhang, X.; Ren, J.; Pang, Z.; Wang, D.; Gao, H.; Qian, Y.; Jiang, X.; Yang, W., Targeting Mesoporous Silica-encapsulated Gold Nanorods for Chemo-photothermal Therapy with Near-infrared Radiation. Biomaterials2013, 34, 3150-3158. 18 ACS Paragon Plus Environment

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Table of Contents


Iodine-Labeled Au Nanorods with High Radiochemical Stability for

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