Table 1.
Primer sequences for qRT-PCR.
Fig 1.
Schematic diagram of preparation of Mag@PEI /ATN-RNA complexes and its application in RNAi therapy of GBM cells.
Fig 2.
A. TEM picture of Mag@PEI NPs B. FTIR spectra of Mag@PEI NPs C. Hysteresis loop recorded for Mag@PEI NPs by means of SQUID D. Zeta potential of synthesized Mag@PEI NPs.
Fig 3.
Contrast properties of Mag@PEI.
Relaxation rates R2 as well as relaxivity obtained from MRI experiment for Mag@PEI NPs in agarose gel 2 wt %.
Fig 4.
Binding of ATN-RNA to Mag@PEI NPs.
A. Agarose gel electrophoresis of Mag@PEI/ATN-RNA complexes at the different mass ratio. B. Binding capability of Mag@PEI NPs towards ATN-RNA recorded using Nanodrop. C. Zeta potential of complexes obtained between Mag@PEI NPs and ATN-RNA at ratio 3:1. D. DLS size distribution for Mag@PEI NPs and Mag@PEI/ATN-RNA complexes at ratio 3:1.
Fig 5.
Cellular uptake of complexes containing fluorescently labelled Mag@PEI NPs into U-118 cells.
The representatives of the colors are blue (Hoechst 33342) for nuclei, green (Concanavalin A-FITC) for cell membranes, and red (ATTO550) for Mag@PEI nanoparticles.
Fig 6.
Colocalization of Mag@PEI NPs and ATN-RNA in U-118 cells.
The representatives of the colours are blue (Hoechst 33342) for nuclei, green (FITC) for ATN-RNA, and red (ATTO550) for Mag@PEI nanoparticles.
Fig 7.
Representative high-content images of U-118 cells exposed to Mag@PEI/ATN-RNA complexes (6.25–100 nM).
10% DMSO was used as a positive control. Images were obtained using different filters to detect nuclei (DAPI), live cells (FITC), and dead cells (TexasRed). The scale bars denote 100 μm. Cell viability of U118 cells exposed to Mag@PEI/ATN-RNA complexes (6.25–100 nM) for 24 h. The value at X-axis in Fig 7 corresponds to concertation of free ATN-RNA on the carrier.
Fig 8.
The expression level of immune response genes after lipo- and nano-mediated ATN-RNA delivery to U-118 cell line.
The relative expression level of the expression of OAS1, OAS3, RIG1, INFγ and TLR3 established by qRT-PCR calculated wit the –ΔΔCp method. Statistical evaluation of ATN-RNA versus control (Clipo or Cnano, respectively) cells was performed using one-way ANOVA followed by Tukey’s posthoc test. Significance value: * p< 0.05, ** p< 0.01, *** p< 0.001 compared to untreated cells (Clipo or Cnano, respectively). Legend: Mag@PEI- nano-mediated ATN-RNA delivery; Lipofectamine—lipo mediated ATN-RNA delivery.
Fig 9.
The expression level of TN-C after lipo- and nano-mediated ATN-RNA delivery to U-118 cell line.
A. The relative expression level of the expression of TN-C established by qRT-PCR calculated with the ΔΔCp method. B. The protein expression level measured by Western blot with densitometric analysis (C.) Statistical evaluation of ATN-RNA versus control (Clipo or Cnano, respectively) cells was performed using one-way ANOVA followed by Tukey’s posthoc test. Significance value: ** p< 0.01, *** p< 0.001. Legend: Mag@PEI- nano-mediated ATN-RNA delivery; Lipofectamine- lipo mediated ATN-RNA delivery.
Fig 10.
Anti-proliferative activity of ATN-RNA, after nano- (A) and lipo-mediated (B) delivery. Proliferation was monitored in real-time using the xCELLigence system. Differences between CI values for ATN-RNA treated and control cells were statistically evaluated using one-way ANOVA followed by Tukey’s posthoc test. Dose-dependent effects of ATN-RNA on proliferation was evaluated using non-linear regression by fitting experimental values to sigmoidal, bell-shaped equation. Legend: Mag@PEI- nano-mediated ATN-RNA delivery; Lipofectamine—lipo mediated ATN-RNA delivery.
Fig 11.
The effect of nano-mediated delivery of ATN-RNA on the migration processes.
(A) Migration of U-118 GBM cancer cells was studied using the xCELLigence system. Serum-depleted cells were transfected with increasing concentrations of ATN-RNA (from 10 to 100 nM) or vehicle (Opti-MEM, C-control). Impedance (CI values) of each experimental condition was recorded over time, plotted against time, fitted to four-parameter logistic non-linear regression model and ET50 was calculated for each ATN-RNA concentration to generate dose-response curves. The ET50 value was normalized to the data obtained for cells treated with native Mag@PEI and plotted as normalized half maximal effective time (ET50) of cell migration against ATN-RNA concentrations. (B) The scratch assay analysis. U-118 GBM cells were transfected with Mag@PEI/ATN-RNA complexes. Images were captured after 24 and 48 h (Figure D in S1 File). The rate of migration was measured by quantifying the total distance that the U-118 cells moved from the edge of the scratch toward the centre of the scratch.