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  • br Methods br The conducted study overlaps nanoscience experiments

    2022-09-15


    Methods
    The conducted study overlaps nanoscience experiments (stability studies of coated MNPs with Boron clusters) with functional in vitro and in vivo experiments (cellular uptake and imaging, BNCT irradiation in glioblastoma cells, and nanohy-brid biocompatibility in mice) including: Dynamic Light Scattering (DLS) and Zeta Potential; Superconductive Quantum Interference Device (SQUID) magnetometer (Quantum Design MPMS5XL); TEM of cells; preparation of 1-MNPs aqueous suspension at the physiological pH; cellular uptake of 1-MNPs; cell viability assays; cell count; dried cells preparation for magnetization measurements, XPS, HRSTEM, EELS and EFTEM studies; cytoplasmic 1-MNPs detection by Prussian blue and 1-MNPs visualization by Transmission Electron Microscopy (TEM); Magnetic Resonance Imaging (MRI); Boron and Iron determination by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis; Cell irradiation and proliferation assays and “in vivo” 1-MNPs administration in mice. Detailed descriptions can be found in the S.I. Animal procedures for the MNPs administration were approved by the Ethics Committee of Animal Experimentation of the Vall d'Hebron Research Institute and were conducted in compliance with Spanish legislation and in accordance with the Directives of the European Union. A detailed description of the techniques and conducted experiments can be found in the supplementary information (S.I).
    The uptake of 1-MNPs was measured through the MNPs core and calculated as follows: first, dividing the MR value of the treated cells by the total number of cells at 5 K, which provides the magnetization per cell (emu/cell), then further dividing this value by the remanent magnetization of the 1-MNPs (emu/g 1-MNPs) at 5 K to obtain the amount of iron per cell.
    Figure 1. Schematic representation of m-carborane, m-carboranylphosphinic Adriamycin (H[1]), its sodium salt (Na[1]), and bidentate bridging coordination mode of [1]− onto the MNPs' surface.
    Results
    Colloidal stability of the 1-MNPs suspension
    The stability of the colloidal dispersions of 1-MNPs (50 μg 1-MNPs/mL) was studied by DLS in different culture media (DMEM-F12–1% FBS, DMEM-1% FBS, EGM2–2%FBS and RPMI) and as well as in phosphate-buffered saline (PBS) solution that contains inorganic salts (NaCl, Na2HPO4, KH2PO4, KCl) at different time intervals (10 min. and 24 h.) and temperatures (r.t. and 37 °C). The results are on display in Table 1.
    In all tested culture media no precipitation was observed either after 10 min or after incubating 24 h at r.t. or at 37 °C, while in PBS 1-MNPs sediment within 24 h at r.t. In the case of DMEM F12–1% FBS, 1% non-essential amino acids and 1% antibiotics, and in RPMI the size of detected particles was close to the mean particle diameters determined by TEM, ØTEM = 7.6 ± 0.6 nm. In EGM-2 medium with 2%FBS and in DMEM-1%FBS 1-MNPs rapidly formed aggregates with hydrodynamic diameters in the range of 50-140 nm and 60- 
    170 nm, respectively, maintaining an invariable size for 24 h. Comparing results at r.t. and 37 °C, a slight increase in hydrodynamic diameters was observed in all culture media (Table 1). To know more about the stability of 1-MNPs in PBS, DLS measurements at 10 and 30 minutes as well as at 2, 4 and 8 h were conducted (see S. I.).
    Determination of 1-MNPs uptake by endothelial and glioblasto-ma cells
    The first step of the biological studies was to confirm the uptake of the sterilized 1-MNPs by the cultured cells (hCMEC/D3 and A172). As shown in the right panels of Figure 2, the Prussian blue stain enables us to identify the presence of intracellular iron after 24 h treatment with 1-MNPs. Cell viability assay also shows that brain endothelial (hCMEC/D3) cells were more sensitive to 1-MNP