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  • br MTT cell viability assay br The cells

    2020-08-28


    2.5. MTT cell viability assay
    The cells were incubated for 24, 48 and 72 h with the different NPs/ substances. The cell medium was then aspirated and exchanged with 100 μl of medium containing a final concentration of 250 μg MTT/ml. The incubation was continued for 3 h at 37 °C for formation of the formazan-particles, which were dissolved in DMSO with 1% (v/v) NH4 Cl. The absorbance was read in a plate reader (Biosys Ltd., Essex, UK) at 570 nm, and background from absorbance at 650 nm was sub-tracted.
    2.6. Cell proliferation measured by [3H]thymidine incorporation
    Incorporation of [3H]thymidine into DNA was used to estimate cell proliferation. The cells were incubated for 24 h with the different NPs/ substances. The cell medium was then aspirated and substituted with serum free cell medium containing [3H]thymidine (3 μg/ml; 75 μCi/ ml). The incubation was continued for 30 min at 37 °C. The medium was removed and 5% (w/v) trichloroacetic TUDCA (TCA) was added. After 5 min the cells were washed twice with TCA and solubilized with 200 μl of 0.1 M KOH, before mixing with 3 ml scintillation fluid (Perkin Elmer,  Journal of Controlled Release 293 (2019) 183–192
    USA). The radioactivity was TUDCA counted for 1 min in a scintillation counter (Tri-Carb 2100TR, Packard Bioscience, USA).
    2.7. Protein synthesis measured by [3H]leucine incorporation
    To determine the impact of PEBCA-CBZ and CBZ on protein synthesis, the cells were incubated with these substances for 24 h. The cell medium was then aspirated, the cells washed once with leucine-free HEPES medium (28 mM HEPES, (4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid) in MEM) and further incubated with leucine-free HEPES medium containing [3 H]leucine (2 μCi/ml) for 30 min at 37 °C. The medium was removed and 5% (w/v) TCA was added to precipitate proteins. After 5 min the cells were washed again with 5% (w/v) TCA and solubilized with 200 μl of 0.1 M KOH, before mixing with 3 ml scintillation fluid and counting the radioactivity as described above.
    2.8. Cell viability estimated by measuring ATP
    Viability of the cells was tested measuring the ATP levels by using the CellTiter-Glo® (Promega,WI, USA) assay, as described by the sup-plier. Cells were incubated with PEBCA-CBZ or CBZ for 72 h, thereafter one half of the volume was removed, replaced with an equal volume of the ATP reagent and gently mixed. After incubation for 10 min the cell lysate was transferred to a light-protected 96-well plate and lumines-cence measured in a plate reader (Biosys Ltd., Essex, UK).
    2.9. Treatment efficacy evaluation in nude mice
    All animal experiments were approved and performed according to the Norwegian Animal Research Authority (Permit number 15-136041) and were conducted according to the regulations of the Federation of European Laboratory Animal Science Association (FELASA). The mice were kept under pathogen-free conditions, at constant temperature (21.5 ± 0.5 °C) and humidity (55 ± 5%); 15 air changes/h and a 12 h light/dark cycle. They had access to distilled water ad libitum, which was supplemented with 17-β-estradiol at a concentration of 4 mg/l [19]. All mice used in this study were female athymic nude foxn1nu mice (age 5–6 weeks and body weights of 18–20 g), locally bred at the Department of Comparative Medicine, Oslo University Hospital, Norway. In all procedures where anesthesia was required 4% sevo-fluran was used.
    The orthotopically growing basal-like xenograft MAS98.12 has been established in house [19] and was used as previously described [16]. When using the MDA-MB-231 cell line, 1.5 million cells were injected into the mammary fat pad and growing tumors were used for sequential implantation. As for the MAS98.12 model, 1–2 mm3 pieces of healthy tumor tissue were implanted bilaterally into the mammary fat pad of female athymic mice. After the tumors reached approximately 5 mm in diameter, the mice were randomly assigned to the different treatment groups (the average volume of each group was 49–57 mm3).
    All formulations were given twice (day 1 and day 4 after rando-mization) as i.v. tail vein injections. PEBCA-CBZ NPs were given with a dose of CBZ 15 mg/kg and comparable amount of empty PEBCA NPs (175 mg/kg) as control. Non-encapsulated cabazitaxel was prepared as a stock solution in Polysorbate 80 (40 mg/ml) and further diluted with 13% (v/v) ethanol to a working solution of 10 mg/ml CBZ. The injec-tion solution was prepared directly before the administration by dilu-tion of the working solution with 0.9% (w/v) NaCl. The same amount of ethanol (1.2–1.4% (v/v)) was used as vehicle control; injection volumes were in the range 200–290 μl. From the first day of treatment, tumor diameter and body weight were measured twice weekly. Mice were monitored daily for health status and were killed by cervical dislocation if they became moribund or if tumor reached 1500 mm3. The tumor was measured by calipers and the tumor volume was calculated according to the formula 0.5 x length x width2 and related to the tumor volume at