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  • br The T and T cells were used to examine

    2022-05-04


    The 3T3 and 4T1 Lycopene were used to examine the cytotoxicity of CoNWs, CoNWs-GO, and CoNWs-GO-PEG, and the 4T1 cells were also used as the model in the photo-chem synergistic effect of materials. Cells were cultured in normal Dulbecco's Modified Eagle's Medium (DMEM, KGM12800-500, KeyGEN BioTECH, Nanjing, China) supple-mented with 10% fetal bovine serum (FBS, Zhejiang Tianhang Biotechnology, China) and 1% penicillin/streptomycin in a humidified incubator at 37 ℃ with 5% CO2. All the cells were cultured in 25 mL bottles before the dissociation for further use [56].
    2.10. Cytocompatibility evaluation
    The Cell Counting Kit-8 (CCK-8, KGA317, KeyGEN BioTECH, Nanjing, China) assay was conducted to quantitatively indicate the activity of 3T3 or 4T1 cells cultured with different samples. The cells were dissociated with Trypsin-EDTA Solution firstly and seeded into a 48-well plate at a density of 2 × 104 per well with 500 μL DMEM. When the cells almost filled the surface of each well, DMEM was removed, and the cells in each well were washed by PBS for three times before in-fusing 500 μL fresh DMEM containing samples at different
    concentrations or of various types, and pure DMEM was added as the blank control. After culturing for 9 h sequentially, each well was wa-shed with PBS to remove the samples and desorbed cells. Then the medium was replaced with 330 μL fresh DMEM containing 10% CCK-8 solution followed by incubation for another 2 h. After this, 100 μL of the supernatant was transferred into a 96-well plate, and the absorbance at 450 nm was read using a microplate reader (SN680 T1608203, Baiiu, Shanghai, China). The cell viability was calculated according to the formula from the specification.
    In order to intuitively observe the cytotoxicity of different materials to 4T1 cells under diverse treating conditions, live/dead staining was executed. Before dying, the cells in the pore plate which had been treated under diverse conditions were rinsed with PBS two times and then dyed with the working solution (2 μM AM, 8 μM PI in PBS) that was prepared in advance according to the specification of the dye kit. After incubation for 30 min, the working solution was removed. Soon afterward, the stained cells can be observed or pictured by fluorescence microscope (CKX53, OLYMPUS). The viable and dead 4T1 cells ap-peared green and red respectively under blue exciting light, and only the dead red cells can be observed under green exciting light.
    2.12. Statistical analysis
    The statistical analyses were processed by SPSS software (IBM, Chicago, USA) and statistically significant differences were set if the p-value was below 0.05.
    3. Result and discussion
    3.1. Surface characterization
    In order to know the surface morphologies of as-prepared cobalt nanowires and CoNWs-GO complex, and prove the successful loading of GO on nanowires, SEM and EDS are used to characterize the samples. As shown in Fig. 1, CoNWs presents rough surface morphology features, and distinguishable sheets can be observed on the nanowires of CoNWs-GO samples. EDS was further explored (Fig. 2b and c), and according to the EDS results (Fig. 2d), when the dose of GO is reduced by 50%, the weight percent of C and O atoms in the samples drops to approximately half of the value simultaneously, which proves the successful loading of GO on cobalt nanowires. We also characterized the X-ray diffraction (XRD) patterns of the as-prepared CoNWs. The result shows the suc-cessful synthesis of cobalt (Fig. 2e). Raman spectroscopy of CoNWs-GO was further performed and the well-designed peaks sited at 1320 cm−1, and 1600 cm-1 are corresponding to the D band and G band of GO sheets [57], substantiating that the compound consists of Co and GO (Fig. 2f). As we all know, nanocarriers with rough surface morphology features show better drug loading capacity compared to congeneric carriers with smooth surfaces. We speculate that the CoNWs-GO complex should display outstanding drug loading capacity.
    3.2. Magnetic targeting capability in vitro
    To explore the potential use of CoNWs-GO-PEG complex for tar-geting delivery of drugs to the tumorous site, CoNWs, CoNWs-GO and CoNWs-GO-PEG were dispersed into polyvinyl alcohol (PVA) solution (Fig. 2a). As shown in Fig. 3a, both CoNWs, CoNWs-GO, and CoNWs-GO-PEG are attracted to the side where an external magnet is placed within 1 min. Slight brownness can still be seen in the CoNWs-GO and CoNWs-GO-PEG complex solution in Fig. 3a, which is caused by in-finitesimal dispersing of GO into the solution. To further investigate the stability of CoNWs-GO-PEG in different solutions, the CoNWs-GO-PEG was dispersed in water, PBS, DMEM, and FBS respectively. Here we use  Colloids and Surfaces B: Biointerfaces 180 (2019) 401–410
    FBS instead of human blood. On the one hand, we have already tested the stability of the material with whole rabbit blood and have found that the material does not cause damage to the blood cells. On the other hand, blood coagulation will soon occur without anticoagulant. Even if we add anticoagulant into the blood, it is also hard for us to observe the changes of the material, because the solution is red but not transparent. So we used FBS instead of blood to test the stability of the prepared nanowires, which is similar to the serum form human blood. As shown in Fig. S1, there is no significant change in the color of the solution or the magnetism of the material after a 7-day-resting, indicating that CoNWs-GO-PEG is stable and can show its magnetic targeting capability steadily in different solutions including water, PBS, DMEM, and FBS media.