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  • In order to investigate the

    2020-08-12

    In order to investigate the uptake of EBNPs in cancer Salinosporamide A and macrophages, HCT-116 and RAW264.7 cells were suspended in culture medium and seeded in six-well plates at a density of ˜1 × 106 cells/ well. After the cells were incubated for 24 h, the medium was removed and replaced with fresh medium containing EBNPs and SNPs (at an SN38 equivalent concentration of 100 μg/mL). The cells were incubated for another 1, 2, 4, 6, and 12 h. At each time point, cells were washed three times with cold PBS, digestion with trypsin and then cells pellet was harvested by centrifugation [16]. RIPA lysis buffer (100 μL) was added to each cell pellet, which was incubated for 10 min at room temperature. Subsequently, acetonitrile (300 μL) was added and the cell lysate vortexed to extract free SN38 and LA-SN38. The concentration of drugs in the supernatant was measured by HPLC after centrifugation at 13,000 rpm for 10 min. The results were presented as dose per 106 cells.
    2.6. Cytotoxicity study by CCK8 assay
    The cytotoxicity of EBNPs in human colon carcinoma cells was determined by CCK8 assay. HCT-116 and HT-29 were seeded at a density of 1.0 × 104 cells/well in 96-well plates and incubated in a cellular incubator for 24 h. The medium was replaced with fresh medium containing various concentrations of EBNPs. Cell viability was determined using Cell Counting Kit-8 (CCK8) assays after incubation for 48 h. Briefly, CCK8 solution (10 μL) was added to each well and the cells were incubated for another 2 h at 37 °C. OD value of each well was recorded at 450 nm by a spectrophotometer. Untreated cells were set as controls, and the cell viability of which was defined as 100%.
    2.7. Apoptosis analysis
    HCT-116 cells were seeded at a density of 1 × 106 cells/well in 6-well plates and incubated for overnight. Then the culture medium was replaced with fresh medium with SNPs and EBNPs (5 μM SN-38 equivalent) and the cells were incubated for 24 h in the incubator. Cells that were untreated or treated with CPT-11 (5 μM) were used as ne-gative or positive controls, respectively [17]. Then the medium was removed and the cells were digested it with trypsin and washed with cold PBS three times. An Alexa Fluor® 488 annexin V/propidium iodide (PI) apoptosis assay kit was used to assay cell apoptosis according to the manufacturer's protocol. Briefly, treated and untreated cells (1 × 105) were suspended in 1×annexin V binding buffer (200 μL). Then, 5 μL Alexa Fluor® 488 annexin V was added to each sample, which was in-cubated for 15 min at room temperature. After then, and 5 μL PI (100 μg/mL) was added and 300 μL binding buffer was added under gently mixing. The cells were stained and then analyzed directly by the flow cytometer.
    2.8. Animal experiments
    Male Sprague Dawley rats (250 ± 20 g), specific pathogen-free male ICR mice, and male BALB/c nude mice (20 ± 2 g) were supplied by the Animal Experiment Center of Zhejiang Academy of Medical Sciences (Hangzhou, China) and kept under specific pathogen-free conditions. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The animals with free access to rodent diet and sterile water and were maintained under a 12/12 h light/dark cycle at a temperature of 22 ± 2 °C [18]. The entire animal protocol was reviewed and approved
    by the ethics committee of the Animal Experiment Center (approval ZJAMS20180409) prior to conducting the experiments and adhered to the aforementioned guidelines.
    HCT-116 tumor-bearing mice were established as described pre-viously. Briefly, ˜ 5 × 106 HCT-116 cells in 100 μL PBS were sub-cutaneously inoculated in the right flank of the BALB/c nude mice. Tumors were allowed to grow to the needed volume before the ex-periment.
    2.9. Pharmacokinetic studies
    Ten male Sprague-Dawley rats (200 ± 20 g)were divided into 2 groups at random (n = 5), the pharmacokinetics of LA-SN38 were in-vestigated after intravenous administration. Rats were given a single 8 mg/kg dose (SN-38 equivalent) of formulations by tail vein injection. Blood samples (0.5 mL) were collected at designed time points (5, 15, 30 min, 1, 2, 4, 8, 12 and 24 h) and stored in heparinized tubes. Blood was immediately centrifuged at 4000 rpm for 10 min, 200 μL of upper plasma separated and stored at −80℃ prior to analysis. LA-SN38 and its derivative in the plasma were extracted by acetonitrile. Briefly, 50 μL of the internal standard (diazepam, 10 μg/mL acetonitrile) was added to 200 μL of plasma sample, which was vortexed. Then, 750 μL of acetonitrile was added and the sample was vigorously vortexed for 2 min. The mixture was centrifuged at 13,000 rpm for 10 min, and then 500 μL upper layer solution was transferred to the injection bottle for HPLC assay.
    To investigate the tumor accumulation and the in vivo distribution EBNPs, DiR-labeled EBNPs were fabricated. The DiR-labeled EBNPs were prepared by the method as described in Section 2.2, except re-placement of LA-38 with DiR. Nude mice with tumors of ˜500 mm3