br The morphology of nanoparticles was also
The morphology of nanoparticles was also visualized by transmis-sion electron microscopy (TEM). Nanoparticles were formed as de-scribed above at final concentrations of 800 nM scRNA and 1.8 mg/mL R646 in NaAc. A 30 μL solution of nanoparticles were added to 400-square mesh carbon coated TEM grids and allowed to coat grids for 20 min. Grids were rinsed with ultra pure water, allowed to dry fully, and imaged with a Philips CM120 TEM.
Primary fetal neural progenitor cells (NPCs) were utilized as a healthy 1883299-62-4 tissue control in order to assess the cell type specificity of siRNA delivery via R646 [16,17,49]. NPCs are obtained as described previously following procedures approved by the Johns Hopkins Uni-versity Institutional Review Board . NPCs 34, 54, and 61, with each number indicating diﬀerent tissue samples, were used to provide three separate cell source samples. NPCs were grown in a 2:1 mixture of high glucose DMEM to Ham's F-12 nutrient mixture (Gibco®) supplemented with 1 × B-27® Serum-Free Supplement, 1% Antibiotic-Antimycotic (Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF, Roche Applied Science), 10 ng/mL epidermal growth factor (EGF, Sigma), 5 ng/mL leukemia inhibitory factor (LIF, Millipore), and 2.5 μg/mL heparin (Sigma).
Primary human GBM cell samples 276, 612, 854, and 965, each number indicating samples harvested from a diﬀerent patient tumor, were isolated from intraoperative samples by the Quiñones laboratory . GBM cells were grown in 1:1 DMEM/F-12 (Gibco®) supplemented with 1% Antibiotic-Antimycotic (Invitrogen), 1 × B-27® Serum-Free Supplement, and 20 ng/mL each of bFGF and EGF for all experiments. Tissue culture plates were coated with 5 μg/mL laminin (Sigma) to allow GBM and NPC cells to adhere for all experiments. Primary human neurospheres (GBM1A, used for orthotopic tumors) were grown in suspension culture using the same medium as that used for other GBM cells, and GBM1A cells were only plated as adherent culture on laminin for in vitro experiments. For all in vitro experiments, cells were plated at a density of 4.7 × 104 cells/cm2 and allowed to adhere overnight.
5.5. In vitro siRNA delivery
For all transfections, polymer R646 and siRNA were separately di-luted in NaAc, mixed in a 1:1 v/v ratio, and allowed to self-assemble into nanoparticles for 10 min before being added to cells. Nanoparticles were incubated with cells for 2 h at 37 °C, and then the media and re-maining nanoparticles were removed and replaced with fresh media. For all transfections, the total concentrations of R646 and siRNA were 270 μg/mL and 120 nM, respectively. For experiments in which more than one siRNA sequence was used in a nanoparticle formulation, siRNA oligos were blended in 25 mM NaAc before being mixed with the polymer. For functional siRNA oligos, the best time point for assessing knockdown was measured via Western blotting (Supplementary Fig. S1), and the most eﬀective isoform was selected by comparing the protein knockdown using Western blotting (Supplementary Fig. S2); the sequences of siRNA oligos are listed in Supplementary Table S1.
For cellular uptake experiments, scrambled control RNA (scRNA) was first labeled with a Cy5 fluorophore using the MirusBio Label IT® Nucleic Acid labeling kit according to the manufacturer's instructions and then mixed with unlabeled scRNA (1:4 labeled to unlabeled scRNA). Nanoparticles on the external surface of cells were removed by washing with 50 μg/mL heparin sulfate prior to flow cytometry. The cells were then detached from the plate using Accutase® (Life Technologies) and analyzed using a high-throughput BD Accuri™ C6 flow cytometer equipped with a Hypercyt autosampler. The percentage of cells that had taken up particles was calculated using the Cy5 signal from the FL4 detector (excitation: 640 nm, emission: 675/25 nm). The data were analyzed using FlowJo 7 software. Quantification of siRNA-induced cell killing in vitro.
For initial experiments, cells were transfected with either siDeath or scRNA. Five days later, cells were stained with 5 μg/mL propidium io-dide (PI), fixed with 10% formalin, and stained with 750 nM 4′,6-dia-midino-2-phenylindole (DAPI). Microscopic images were captured at 5× magnification using a Zeiss Axio observer A1 microscope with a Zeiss Axiocam MRm camera and AxioVision software. Live and dead cells were quantified using ImageJ v1.47 software, and dead cells were subtracted from the live cell count to yield the total cell count for each well. To measure the eﬀect of the functional siRNAs, cells were trans-fected with scRNA, siYAP1, siNKCC1, siRobo1, siEGFR, siSurvivin, or a combination of all five functional sequences. For all groups except the scRNA control, cells were administered 120 nM total functional siRNA. Cell death was measured and quantified as above.