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  • br In vitro cell migration after siRNA transfection

    2020-08-28


    5.6. In vitro cell migration after siRNA transfection
    GBM1A cells were seeded in laminin-coated 24-well plates (6 × 104 cells per well in 600 μL medium). After adhering overnight, they were transfected with 120 nM functional siRNA (single oligo or mixture of all five oligos) or 120 nM control scRNA. Three days after transfection, cells were collected using Accutase, counted, and seeded onto a CIM-Plate 16 (ACEA BioSciences, San Diego, CA) according to the manufacturer's instructions. Complete GBM1A culture medium supplemented with 10% FBS was used as the chemoattractant. The migration of GBM1A cells across the CIM-Plate membrane was mea-sured by the increase in impedance using an xCELLigence RTCA DP (ACEA BioSciences).
    Subcutaneous human GBM model and nanoparticle administration in vivo.
    All animal experiments were conducted following the guidelines and approval of the Johns Hopkins University Animal Care and Use Committee. Four-week-old athymic nu/nu mice were anesthetized by isofluorane inhalation. Two million human GBM 612 cells were injected subcutaneously into the flank of the mice to create tumors. After one month, when tumors had reached approximately 1 cm3, nanoparticles containing 27 μg siRNA were injected directly into the tumor. Gene knockdown was assessed via Western blotting as described below.  Biomaterials 209 (2019) 79–87
    5.7. Protein expression analysis
    Except where noted otherwise, cells were harvested three days after in vitro transfection experiments. Protein lysates were isolated using 5.2 μL/cm2 radioimmunoprecipitation assay (RIPA) buffer in tissue culture flasks. Cells were dissociated via cell scraping, lysates were kept on ice for 30 min with vortexing at 5 min intervals. Cell debris was removed by centrifugation at 19.2 × 103 RCF, and the supernatant was collected and stored at −80 °C.
    To measure in vivo protein expression, animals with subcutaneous tumors were treated with nanoparticles and then sacrificed after two days, and the tumors were cut from the surrounding tissue. The tumor tissue was homogenized in RIPA buffer and the debris removed via centrifugation as described above.
    All protein electrophoresis was carried out using 4–12% NuPAGE Novex Bis-Tris gels (Thermo Fisher Scientific) in 10% 3-(N-morpholino) propanesulfonic PF6700841 (MOPS) buffer (Thermo Fisher Scientific) at 135 V for 90 min. Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane using a Pierce G2 Fast Blotter (Thermo Fisher Scientific) according manufacturer's instructions. The membranes were blocked for 1 h with blocking buffer, 5% milk in 1 × Tris-buffered saline with Tween® 20 (TBST) at room temperature, and then incubated with primary antibodies overnight at 4 °C in blocking buffer. After washing with TBST, membranes were incubated with secondary anti-bodies for 1 h at room temperature in blocking buffer. The antibody dilutions and specifications are listed in Table S2.
    5.8. Orthotopic human GBM model and nanoparticle administration
    All animal experiments were conducted following the guidelines and approval of the Johns Hopkins University Animal Care and Use Committee. Experiments were conducted using 4-8 week-old athymic nu/nu mice. Transcranial cannulas (Plastics One Inc., Roanoke, VA) were implanted stereotactically into the right striatum of the brain. (X: 1.5 mm, Y: 1.34 mm, Z: -3.4 mm from bregma). A cyanoacrylate glue was used to attach the cannula to the skull, the surrounding skin was su-tured closed. Ketamine/xylazine was used as an anesthetic. Animals were inoculated with 500,000 luciferase-positive GBM1A cells using an internal cannula inserted within the transcranial cannula. A cell suspension (4 μL) was injected at a rate of 1 μL/min. Magnetic resonance imaging (MRI) acquisition and Tumor Size:
    Animals were monitored for tumor growth via small animal Bruker 9.4 T BiosSpin magnetic resonance imaging system with basic repetition time (TR) of 16 ms and echo time (TE) of 3000 ms. Rapid acquisition with refocused echoes (RARE) were taken with 3 averages; 128 × 128 voxels; 0.25 mm × 0.25 mm in plane resolution and 1.5 mm slice thickness.
    After 14 days, tumors approximately 1 mm in diameter were visible via MRI using T2-weighted images and confirmed with hematoxylin and eosin staining for an n = 4 mice. Tumor volumetric analysis was based on calculating the volume of a sphere in mm3 taken from the axial and coronal sections of MRI images. Corresponding H&E staining of the 14-day time-point confirmed radius of tumor.
    Intratumoral treatment with nanoparticles began and was repeated twice weekly for two weeks. For each treatment, 4 μL of lyophilized nanoparticles, prepared as previously described [51] using 30 mg/mL sucrose as a lyoprotectant, were injected at 1 μL/min through the cannulas. The total dose of siRNA per mouse was 0.6 μg.