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  • br Preparation of the nanoprobe CTMR

    2019-10-07


    2.2. Preparation of the nanoprobe [email protected]
    The reaction pathway for preparing the nanoprobe [email protected] BHHBCB-Eu-FA is illustrated in Scheme S1. The experimental details are described as follows.
    stirred at 25 °C for 3 h, 54 μL aqueous solution of EuCl3 (0.0025 mM) was added dropwise, and the mixture was stirred for another 1 h. Then 50 μL DMSO (dimethyl sulfoxide) was added into the mixture to make the solution homogeneously. The obtained solution containing APTES-BHHBCB-Eu3+ was used in the next process without further purifica-tion.
    Preparation of [email protected] nanoparticles. After a water-in-oil reverse microemulsion was prepared by mixing Triton X-100 (1.77 g), cyclohexane (5.80 g) and octanol (1.33 g) in water (300 μL), APTES-CTMR (140 μL) and TEOS (100 μL) were added into the solution, and the solution was stirred at 25 °C for 40 min. Then 100 μL con-centrated aqueous ammonia was added to initiate the copolymerization reaction between APTES-CTMR and TEOS. The mixture was stirred at room temperature for 24 h to afford the CTMR-doped silica nano-particles (silica core). Then APS-BHHBCB-Eu3+ (100 μL), TEOS (100 μL) and APTES (5 μL) were sequentially added, and the mixture was further stirred at room temperature for 24 h to afford the core–shell nanoparticles [email protected] By adding 15 mL acetone, the na-noparticles were precipitated, and then collected via centrifugation at 10000 rpm for 5 min. The nanoparticles were washed with ethanol and water each for three times, and then dispersed in water with a con-centration of 20 mg/mL.
    2.3. Cytotoxicity of the nanoprobe to HeLa cells
    The cytotoxicity of [email protected] to HeLa and L-02 Haloperidol was determined by the MTT assay method [46,47]. HeLa and L-02 cells were seeded at a density of 5 × 104 cells per well in a 96-well culture plate. After cells were incubated in RPMI-1640 medium (Sigma-Al-drich) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin at 37 °C in a 5% CO2/95% air incubator for 12 h, the culture medium was replaced by freshly prepared culture medium containing different concentrations of the nanoprobe (0, 15, 30, 60, and 100 µg/mL). The wells added the culture medium without cells were used as blanks, and wells added free culture medium (no nanoprobe) were used as controls. After incubation for 24 h in dark, the culture medium was removed, and cells were washed three times with PBS. Then 100 μL PBS solution of MTT (0.5 mg/mL, pH 7.4) was added into each well, and cells were incubated for another 4 h. The excess MTT solution was carefully removed from each well, and the formed for-mazan was dissolved in 150 μL DMSO. The absorbance of each well at 540 nm was then measured on Infinite M200 Pro Microplate Reader. The results from five individual experiments were averaged. The for-mula, vialibity (%) = (mean of absorbance value of treatment group-blank)/(mean absorbance value of control-blank) × 100, was used to calculate the viability of cell growth.
    2.4. Luminescence imaging of live cells
    For luminescence imaging of live cells, HeLa (FR+, FR-positive cell line) and L-02 (FR-, FR-negative cell line) cells were seeded in cover glass bottom culture dishes (ϕ 20 mm) with the density of 5 × 104 cells/mL. After the cells were incubated at 37 °C for 24 h, the culture medium was replaced by freshly prepared culture medium containing [email protected] (30 μg/mL), and then the cells were incubated at 37 °C for another 1 h. Followed by removing the excess nanoprobe,  Methods xxx (xxxx) xxx–xxx
    the cells were washed with PBS for three times, and then subjected to the luminescence imaging measurements. As controls, HeLa cells pre-treated with FA (1.0 mM) and then incubated with [email protected], HeLa cells incubated with [email protected], and L-02 cells incubated with [email protected], were also imaged.