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All chemicals and solvents applied in this work were of analytical grade. DNA oligos and Cyanine 5 (Cy5), marked as single-stranded DNA (ssDNA), were synthesized by HITGEN (Chengdu, China). DOX was purchased from Beyotime (Shanghai, China). PEG-modified GNRs were synthesized in Beijing Zhongkeleiming Daojin Technology Co., LTD., through a two-step method. First, commercial gold seeds were synthesized via reducing HAuCl4 solution using AgNO3. Then, gold seeds were slowly crystallized into rod-shaped nanostructures in HAuCl4 solution, after which PEG was coated at the surfaces of obtained GNRs. Tris-HCl, MgCl2, and other chemicals were purchased from Aladdin (Shanghai, China).
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In synthesizing TNDs, four 1-µL specific single-stranded DNA (detailed sequences are listed in Table 1) were equally mixed with 96 µL of TM buffer (10 mmol/L Tris, 10 mmol/L MgCl2; pH = 8.0) and then heated to 95 °C. After being heated for 10 min at 95 °C, the solution containing DNA nanostructures was quickly cooled to 4 °C for 20 min. When preparing Cy5-labeled TDN, S1 strands were replaced by Cy5-S1 strands. Excessive DOX was incubated with the obtained TDNs (1 µmol/L) at 4 °C overnight to obtain TDN-DOX nanocomposites. Then, Tris-HCl, MgCl2, and excessive DOX were removed by ultrafiltration centrifugation using 30-kD ultrafiltration cubes. Based on the synthesis protocol in the previous report[31], individual designed TDN nanostructure following provided design can carry 20 DOX molecules, showing a high loading ability. Then the obtained GNR was mixed with TDN-DOX nanocomplex to form GNR@TDN-DOX nanocomposites with different concentrations such as 1, 2, 3, 4, and 5 nmol/L. Then, the product was stored at 4 °C for the following experiment.
SsDNA Direction Sequence S1 5′ to 3′ ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCAAGTCTGAA S2 5′ to 3′ ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG S3 5′ to 3′ ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCCG S4 5′ to 3′ ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG Cy5-S1 5′ to 3′ cy5-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTT GAGACGAACATTCCTAAGTCTGAA Table 1. Sequences of single-stranded DNA for the synthesis of TDNs
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Polyacrylamide gel electrophoresis (PAGE; 8%) was used to examine the synthesis of TDN, TDN@DOX nanocomposites, and GNR@TDN-DOX nanocomposites. Dynamic light scattering (DLS) was used to examine the hydrated particle size of the obtained nanocomposites. Transmission electron microscopy (TEM, FEI Tecnai F20 and Talos F200S G2, operated at 200 KV) was applied to investigate the specific structures and morphology of the obtained nanocomposites. TEM specimens of TDN and TDN-DOX were negatively stained by 5% phosphotungstic acid-staining solution for 4 min before loading into TEM. UV–vis absorption curves were measured to analyze the optical characteristics of the obtained nanostructures.
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GNRs and GNR@TDN-DOX nanocomposite solutions (1 nmol/L) were irradiated by 808-nm laser at a power density of 1.0, 1.5, and 2.0 W/cm2, respectively, for 10 min. The temperature difference was measured by using a thermocouple thermometer at time intervals of 30 s. The temperature difference in three cycles of heating and cooling was measured to test the photothermal stability of the obtained nanostructures.
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Cells were seeded in culture dishes and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin with an atmosphere of 5% CO2 at 37 °C.
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A total of 2 × 104 L929 cells and A375 cells were seeded in 96-well plates and cultured overnight. Then, the cells were incubated with GNRs, DOX, TDN-DOX, and GNR@TDN-DOX solutions. The concentrations of GNR and GNR@TDN-DOX solutions were set as 1 nmol/L, with identical concentrations of DOX were adopted in DOX, TDN-DOX and GNR@TDN-DOX experimental groups. All groups of A375 cells were either treated with 808-nm laser irradiation of 2.0 W/cm2 for 10 min or untreated. Then, the cells were cultured for 24 h, and their cell viability was analyzed by MTT assay.
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A375 cells were seeded in culture dishes as nanocomposites at 2 × 104 cells per well and cultivated overnight in DMEM with 10% FBS and 1% penicillin streptomycin at 37 °C with 5% CO2. Then, the cells were incubated with GNRs, DOX, Cy5-labeled TDN-DOX, and Cy5-labeled GNR@TDN-DOX nanoparticles for 4 h. The concentrations of GNR and GNR@TDN-DOX solutions were set as 1 nmol/L, with identical concentrations of DOX were adopted in DOX, TDN-DOX and GNR@TDN-DOX experimental groups. For the cellular uptake study, the cells were stained with Hoechst solution. Then, the cells were washed with PBS three times. For lysosomal escape experiments, the cells were stained by lyso-linkers and Hoechst, after which the cells were washed with PBS three times. The fluorescence of cells was observed by using a fluorescence microscope (CLSM).
Tetrahedral DNA Nanostructure-modified Gold Nanorod-based Anticancer Nanomaterials for Combined Photothermal Therapy and Chemotherapy
doi: 10.3967/bes2022.141
- Received Date: 2022-10-14
- Accepted Date: 2022-11-15
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Key words:
- Nanomaterials /
- DNA tetrahedron /
- Gold nanorod /
- Combination therapy
Abstract:
All authors declare no conflicts of interest.
Citation: | WU Hao, XU Jiang Shan, LI Yan Hong, WU Xing Han, HU Wei, LIU Meng Die, SUN Qiang, GUO Bin. Tetrahedral DNA Nanostructure-modified Gold Nanorod-based Anticancer Nanomaterials for Combined Photothermal Therapy and Chemotherapy[J]. Biomedical and Environmental Sciences, 2022, 35(12): 1115-1125. doi: 10.3967/bes2022.141 |