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  • KX2-391 br fluorescence of each well was then read at the

    2020-08-04


    fluorescence of each well was then read at the coumarin fluorescence wavelength λexcitation / λemission: 390 / 500 nm to measure its release reflecting the ability of each cell line to produce ROS. This study was carried out in triplicate and the results of each cell line was compared to
    the cell line producing the least ROS (U87) results using a non-parametric Kruskall-Wallis test (p < 0.001) with GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA). A p value of < 0.0001 was accepted as statistically significant.
    In vitro cell-proliferation assay
    Chick chorioallantoic membrane assay (CAM assay)
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    Fertilized white leghorn chicken eggs (E.A.R.L. Morizeau, Dangers, France) were maintained at 24°C before hatching. Eggs were subsequently hatched in a humidified incubator (Sailnovo 56A incubator) at 37 °C and 70 % hygrometry. The initial day of incubation is considered day 0. At day 10, all eggs were mired and the air sac was visualized and delimited. The shell was cautiously cut near the air sac position to visualize the egg membrane. The egg membrane was homogeneously moisturized with NaCl 0.9 % solution and left 5 min. The solution was then aspirated and the egg membrane was carefully removed revealing the chorioallantoic membrane (CAM). Prior to implantation on the CAM, MiaPaCa-2 cell suspensions were prepared by detaching them with trypsin / EDTA followed by their counting. Cells were centrifuged at 1200 rpm for 5 min, washed twice using DMEM without FCS and suspended in a mix of Matrigel (ECM Matrigel, Sigma) and DMEM (75/25) at a final concentration of 5.107 cell per mL. 1.106 MiaPaCa-2 KX2-391 (20 µL) were deposited onto the CAM, without allowing the pipette tip to touch the CAM. The window was then sealed using adhesive plaster to prevent dehydration. The eggs were incubated (without shaking) awaiting the tumor to grow. After approximately three days, tumor were visible and the treatment could take place. The tumors were treated by intratumoral injections of DOX and 8a at different doses either as a single injection (184 nmol) or twice a day for two consecutive days (20 nmol/injection). In ovo tumors were photographed three days post-treatment using an Olympus digital camera 15.9 mega pixel using the macro feature (OM-D E-M5). Image treatment and tumor measurements were carried out using FIJI software.[42] The tumor were then taken out carefully and suspended in 1 mL of PBS before being sonicated to dissociate the tumor. After centrifugation at 1200 rpm for 5 min, the supernatant was filtered before being analyzed by HPLC.
    HPLC Detection of doxorubicin
    HPLC analysis was used to estimate the doxorubicin release within the tumor collected for the HET-CAM assay using a gradient method composed of acetonitrile as the phase A and water containing 0.1 % of formic acid as phase B. The HPLC separation was carried on a 1260 infinity HPLC system (Agilent Technologies, United Kingdom). The chromatographic separation was carried out on a Kinetex® 5µm C18 (250 x 4.6 mm) LC column at a flow rate of 1.0 mL/min. A gradient elution was used with initially 10 % acetonitrile which was increased linearly to 30 % over 4 min, then increased to 80 % over 2 min and remained at this condition for 6 min before being finally decreased to 10 % in 3 min and was held until the end of the 20 min run. All analyses were performed at 30 °C. The mobile phase were filtered and degassed in an ultrasonic bath before use. The retention times of DOX and 8a in these conditions of analysis were 9.03 and 9.85 min, respectively.
    Acknowledgements
    This work was funded by the “Comité Essonne de la Ligue Nationale Contre le Cancer” and the “Fondation ARC pour la recherche sur le cancer”. C. Skarbek is financed by a postdoctoral training grant from the “Fondation ARC pour la recherche sur le cancer”. S. Serra was a “Ligue Nationale Contre le Cancer” postdoctoral fellow. H. Maslah is supported by the “Ministère de l’Education Nationale de la Recherche et de la Technologie”.Pierre Milcendeau is acknowledged for technical assistance on organic synthesis.
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    References:
    [1] Cancer Research UK, https://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer#heading-One, Accessed in May 2019.
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    Arylboronate-doxorubicin conjugates as anticancer ROS-responsive prodrugs were synthesized.
    In vitro cytotoxicity of the prodrugs and controls was determined on a panel of cancer cells.
    MiaPaCa-2 pancreatic cancer cells were selected for their ability to efficiently produce ROS species.