Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated Photothermal Therapy

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1 Supporting Information Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated Photothermal Therapy Limin Pan, Jianan Liu, and Jianlin Shi* State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-Xi Road, Shanghai 5, China *Corresponding author: 1 Experimental Section 1.1 Applied Chemicals Sodium borohydride (NaBH 4 ), silver nitrate (AgNO 3 ), ascorbic acid,, cetyltrimethyl ammonium bromide (CTAB), tetrachloroauric acid (HAuCl 4 3H 2 O), propidium iodide (PI) and Calcein AM were purchased from Sigma-Aldrich. Nuclear localization signal NLS (TAT-Cys: YGRKKRRQRRRC) peptides were purchased from Chinese Peptide Company. Methoxypolyethylene glycol thiol (mpeg-sh, MW 5) was purchased from Jenkem Co., Ltd. PBS solution (ph 7.4), cell culture medium (RPMI 164), antibiotic solution, fetal bovine serum (FBS), and.25% trypsin were purchased from Shanghai Runcheng Biomedical Co., Ltd.. ELISA assay kit was provide by Shanghai Xin Ran Biological Co., Ltd.. All chemicals were used as received without any further purification. 1.2 Synthesis, Conjugate, and Characterization of GNRs GNRs were synthesized based on the seed-growth strategy. First of all, gold seeds capped with CTAB were synthesized by reduction reaction between HAuCl 4 1

2 and NaBH 4. 8 µl HAuCl 4 (.1 M) was added to 2.5 ml CTAB (.1 M), forming a homogeneous mixture. After that, gold seeds were obtained at once the injection of.2 ml ice-cold NaBH 4 (.1 M) to the above mixture quickly under vigorous stirring. The growth solution for GNRs contains CTAB (.1 M, 5 ml), AgNO 3 (.1 M,.5 ml), HAuCl 4 (.1 M, 2.5 ml), and ascorbic acid (.1 M,.375 ml). The initiation of GNRs growth was triggered by the injection of.4 ml gold seeds. The growth medium was stirred at 3 C in the whole procedure. After 3 hours, the solution was centrifuged (13 r/min, 15 minutes) for the collection of GNRs. It was redispersed in DI water, and had a longitudinal surface plasma resonance band centered at 83 nm. Then, GNRs were conjugated with NLS peptide. First, mpeg-sh was added to the GNRs solution overnight to achieve PEGylation of the nanorods which eliminate the cytotoxicity of CTAB. The PEGylated GNRs were treated with NLS (Cys-TAT) to achieve 1 5 molar excess. The solution was then shaken overnight at room temperature. And then the removal of excess ligands was carried out by centrifugation. TEM and STEM were used to measure the sizes and homogeneity of the nanorods. A UV-Vis spectrometer and zeta-sizer were used to test the conjugation. 1.3 Photothermal Performance of GNRs and GNRs-NLS Photothermal performance of GNRs and GNRs-NLS was measured and analyzed by irradiating 1.5 ml EP centrifuge tube containing 1 ml GNRs or GNRs-NLS dispersion with ppm. After irradiation by an 88 nm laser (.2 or 2 W/cm 2 ), the temperature and thermal images of the irradiated aqueous dispersion were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). 1.4 Cell Culture HeLa cells were traditionally cultured in RPMI 164 medium containing 1 % heat-inactivated fetal bovine serum and 1 % antibiotic solution. The temperature was kept constantly at 37 C in a humidified atmosphere with 5 % CO 2. For all of the experiments in this work, cells were detached by using.25 % trypsin (Sigma) and resuspended in fresh medium before plating. 2

3 1.5 Intracellular Localization Profile of GNRs and GNRs-NLS in HeLa Cells by Confocal Laser Scanning Microscopic Images For CLSM demonstrations, HeLa cells (1 5 cells per dish) were seeded in Petri dishes, and then incubated with 6 µg/ml nanorods. The media were removed after 12 hours incubation, and the residual nanorods were removed by washing cells twice with PBS. Then, for nuclei staining, cells were treated with.5 ml mixed solution (1 % 4, 6-diamidino-2-phenylindole in methanol) for 15 minutes. After that, excessive DAPI was removed by softly washing cells twice. At last, after addition of 1 ml of PBS, the cells were observed under a multiphoton microscope under 6 oil-immersion objective. 1.6 Bio-TEM Observation HeLa cells were treated with 6 µg/ml GNRs and GNRs-NLS. After 12 hours incubation, cells were gently rinsed with PBS for two times and further incubated with.25% trypsin for 3 minutes. Afterwards, cells can be detached and then collected by centrifugation at 1 r/min for 5 minutes. The collected cells were fixed with 1 ml general fixative containing glutaraldehyde at 4 C for 24 hours. After that, cells were washed with PBS and dehydrated using ethanol with graded concentrations. Finally, the cell sample was treated with a standard procedure for TEM observation. 1.7 Cellular and nuclear quantification of GNRs and GNRs-NLS HeLa cells were cultured on 1-cm Petri dishes (5 1 6 cells in 1 ml culture medium). The cells were allowed to adhere for 24 hours and then treated with GNRs and GNRs-NLS (6 µg/ml) for 12 hours. After removing the culture medium and washing with PBS for three times, the cells were trypsinized and collected. In order to quantify the nuclear uptake of GNRs and GNRs-NLS in HeLa cells, cell nuclei were separated from cell cytosol by nuclei extraction. The nuclei extraction solution consisted 1 mm EDTA, 1% Triton X-1, 1 mm NaCl, and 1 mm Tris buffer (ph 7.4). The collected cells were treated with the nuclei solution at 4 C. After 1 minutes, cell nuclei were collected by centrifugation. To disrupt the cell nuclei, cell nuclei were further treated with cell lysis solution which containing 1 M NaOH and 3

4 .5% Triton X-1. The nuclear uptake of gold nanorods was measured through the detection of gold concentration by inductively coupled plasma atomic emission spectrometry (ICP-AES). The cellular uptakes of gold nanorods were detected following the above-mentioned procedure without nuclei extraction. Three independent experiments (n = 3) were performed for each data point. 1.8 In vitro PTT Effects of GNRs and GNRs-NLS Cells cultured in glass bottom dishes (φ mm, NEST) were incubated with GNRs and GNRs-NLS at the same final concentration (6 µg/ml) for 12 hours. After 88 nm laser irradiation (.2 or 2 W/cm 2, 5 minutes), the culture medium was replaced by 1 ml PBS mixed with 5 µm PI and 2 µm Calcein-AM. After incubation at 37 C (3 minutes), cells were washed with PBS twice, and further imaged by CLSM (OLYMPUS) (PI: λ ex : 535 nm, λ em : 617 nm; Calcein: λ ex :49 nm, λ em : 515 nm). Calcein-AM (green) and PI (red) were used to stain live cells and dead cells, respectively. MTT assay: HeLa cells seeded (1*1 4 cells per well) in 96 well plates were incubated with 6 µg/ml GNRs and GNRs-NLS for 12 hours. Cell viabilities without and with 88 nm laser irradiation (.2 or 2 W/cm 2, 5 minutes) were measured following standard MTT assay procedure. Flow cytometry analysis: HeLa cells seeded at 6 well plates were cultured with 6 µg/ml GNRs and GNRs-NLS for 12 hour. After irradiation with 88 nm laser (.2 or 2 W/cm 2, 5 minutes), cells were co-stained with PI and Annexin V-FITC. Finally, the collected cells were analysed by flow cytometry. 1.9 LDH Assay To evaluate the releasing level of LDH, a membrane integrity assay kit (LDH Release Assay Kit, Beyotime Biomedical Co., Ltd.) was used according to the instructions. In our research, HeLa cells were incubated with 6 µg/ml of GNRs and GNRs-NLS for 12 hours. Cells incubated with 1% Triton-X-1 for 3 min were served as positive control group. The fresh cell culture medium was served as background. The detection was carried out on a microplate reader (Bio-TekELx8). 4

5 1.1 ELISA assay for RPA 7 Expression ELISA assays were carried out to test the expressions of RPA 7 of HeLa cells. Cells were seeded (2 1 6 cells) in a 1-cm Petri dish. After 24 h attachment, cells were incubated with GNRs and GNRs-NLS. After different PTT treatment, cells were detached, and lysed with cell lysis solution. The expressions of RPA 7 were detected using ELISA Assay Kit (Shanghai Xin Ran Biological Co., Ltd., China) Animal Experiments Animals and Tumor Models. Specific pathogen-free female Balb/c nude mice at 6 weeks of ages, ~ g, were provided by Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS). To establish HeLa tumor model, HeLa cells (1 1 6 ) was first injected into the shoulder of a nude mouse subcutaneously. When the tumor reached to 1-2 cm, tumor tissue was collected and cut into pieces about 1 mm and injected to the shoulders of nude mice subcutaneously. The maximum length (L) and the maximum width (W) of each tumor were measured by a digital verniercaliper, and the tumor volume (V) was calculated as V = L W 2.5. The relative tumor volume was normalized to the initial tumor volume (V ). In Vivo Phototoxicity Study on Subcutaneous Tumor Model. When the size of tumors reached to about 5 mm 3, 4 mice were divided into 5 groups at random: group 1, intravenous injection of PBS (1 µl); group 2 and 4, intravenous injection of GNRs; group 3 and 5, intravenous injection of GNRs-NLS (with respect to gold: 5 mg/kg). 12 h later, mice in groups 2 ~ 5 were treated by exposing the tumor sites to an 88 nm laser for 5 minutes. The power densities of different group are.2 (group 4 and 5) and 2 (group 2 and 3) W/cm 2. The volumes of tumors and body weights of mice were measured every two days in 15 days post PTT treatment. For each group, three mice were sacrificed at 7 th day after PTT, and the pathological tissues sections of tumors were collected for pathological studies by typical hematoxylin-eosin (H&E) staining and immunohistochemical staining for antigen Ki67. Furthermore, after the nuclear targeted PTT treatment, major organs (liver, spleen, kidney, heart, lung) of a random mouse in fifth group were collected and also stained with hematoxylin-eosin. 5

6 IR Thermal Imaging: An IR thermal camera (FLIR TM A325SC camera, USA) was employed to obtain the infrared thermal images. HeLa tumor-bearing mice were treated with saline, GNRs or GNRs-NLS. During the irradiation with an 88 nm laser (.2 or 2 W/cm 2, 5 minutes), the temperatures of tumors were simultaneously recorded with n IR thermal camera. Biodistributions of nanorods: 24 HeLa tumor-bearing mice randomized into two groups and respectively i.v. injected with GNRs-NLS solution (with respect to gold: 5 mg/kg). At 3, 12 and 24 h post administration, mice were sacrificed for the systemic distribution analysis of GNRs-NLS (n = 8 at each time point). The main organs (heart, liver, spleen, lung and kidney) for each group were collected, wet-weighted, and dissolved in the digesting aqua regia solution. The concentrations of Au in organs of each group were measured by ICP-AES. The collected tumors for each group were further divided into 2 parts (total tumor accumulation and nuclear uptake, n = 4 for each part). The total tumor accumulation was quantified after digesting by ICP-AES. In order to detect the nuclear uptake of GNRs-NLS, tumor tissues were pretreated by Kit of Nucleus Extraction according to the specification. Then the contents of Au in the nuclei were also determined by ICP-AES. Same processes were carried out for the analysis of biodistribution of GNRs Characterization Transmission electron microscopy (TEM) images were performed on a JEM-21F electron microscope. Scanning transmission electron microscopy (STEM) images, scanning electron microscopy (SEM) images and EDX spectra were acquired with a field-emission Magellan 4 microscope. UV-vis spectra were obtained on a UV-311PC Shimadzu UV-vis-NIR spectrometer with QS-grade quartz cuvettes at room temperature. Dynamic light scattering (DLS) data was collected on Malvern Zetasizer Nanoseries (Nano ZS9). The Bio-TEM images were recorded on JEM-123 electron microscopy. The quantification of gold uptake was obtained on inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies, US). CLSM images were viewed on FV 1, Olympus, Japan. 6

7 Figure S1. (a, b) Bright/dark field scanning transmission electron microscopy (STEM), and (c) high angle annular dark field (HAADF) images of GNRs-NLS. 3 Zeta potential (mv) 1-1 GNRs GNRs-PEG GNRs-NLS - -3 Figure S2. Zeta-potential of GNRs, GNRs-PEG and GNRs-NLS. 7

8 Figure S3. Energy-dispersive X-ray analysis (EDX) of the selected areas in Bio-TEM images (Figure 2b) of GNRs (a) and GNRs-NLS (b) in HeLa cells. The red circle in (a) and (b) show the existence of gold. 8

9 1 Relative cell viabilities (%) Control Laser only -.2 W/cm 2 Laser only -2 W/cm 2 Figure S4. Relative viability of cells treated by 88 nm laser irradiation at.2 and 2 W/cm 2 for 5 min. 9

10 Percentage of LDH release (%) Positive control Laser only -2 W/cm 2 Laser only -.2 W/cm 2 GNRs only GNRs-NLS only Figure S5. LDH releasing levels from HeLa cells after exposure to 88 nm laser only, GNRs and GNRs-NLS only. 1

11 1 Relative RPA7 content (1%) Untreated control Laser only -2 W/cm 2 Laser only -.2 W/cm 2 GNRs only GNRs-NLS only Figure S6. Levels of RPA 7 after exposure to 88 nm laser only, GNRs and GNRs-NLS only. 11

12 (a) Au concentration (% ID/g) h 12 h 24 h Heart Liver Spleen Lung Kidney Tumor (b) Au concentration (% ID/g) h 12 h 24 h Heart Liver Spleen Lung Kidney Tumor (c) Au concentration (µg/g) 3. GNRs GNRs-NLS h 12 h 24 h Figure S7. Biodistribution of (a) GNRs and (b) GNRs-NLS in 3, 12 and 24 h post intravenous injection into HeLa tumor-bearing mice, as determined by measuring Au concentrations in tissue lysates with ICP-AES. (c) The quantification of Au in the nuclei of the tumor tissues in 3, 12 and 24 h after intravenous injection of GNRs and GNRs-NLS. 12

13 24 22 Weight (g) Control GNRs-.2W/cm 2 GNRs-2W/cm 2 GNRs-NLS-.2W/cm 2 GNRs-NLS-2W/cm Time (days) Figure S8. Effects of GNRs and GNRs-NLS on body weight of mice bearing HeLa xenografts under 88 nm laser irradiation at varied power densities. Body weights were measured at the indicated time points. 13

14 1 8 Survival (%) 6 4 Control GNRs-.2 W/cm 2 GNRs-2 W/cm 2 GNRs-NLS-.2 W/cm 2 GNRs-NLS-2 W/cm Days after treatment Figure S9. Survival curves of mice bearing HeLa tumors of control group and the differently treated groups with GNRs and GNRs-NLS under 88 nm laser irradiation at varied power densities. 14

15 Figure S1. Histological images of H&E-stained tissue sections of liver, spleen, heart, kidney, and lung from mice received nuclear targeted PTT at the ultralow power density (GNRs-NLS,.2 W/cm 2 ) followed by dissections in 7 and 15 days post-injection. Scale bar: 1 µm. 15