Cationic solid lipid nanoparticles (SLN) complexed with plasmid DNA enhance prostate cancer cells (PC-3) migration

Figures & data

Table 1. Composition of each formulation tested.

	Stearic acid (mmol.L^-1)	DOTAP (mmol.L^-1)	Pluronic F68 (mmol.L^-1)
SLN	7.0	2.5	1.0
Stearic acid	7.0	–	–
DOTAP	–	2.5	–
Pluronic F68	–	–	1.0

Figure 1. SLN-mediated transfection in prostate cells. PNT1A and PC-3 cells were plated in a 12-well plate. After 24 h, cells were incubated with SLNplex (SLN:pEGFP-N1) for 4 h, and the transfection medium was replaced with a complete RPMI medium. After 24 h of transfection, cells were trypsinized and transfection efficiency was determined by flow cytometry using FACS calibur. (A) Transfection efficiency in PNT1A and PC-3 cells; (B) Fluorescence intensity in PNT1A and PC-3 cells. The Lipofectamine 2000 group was used as a positive control for transfection. (C) PNT1A cells transfected with SLN:pEGFP-N1 and cell morphology (phase contrast); (D) PC-3 cells transfected with SLN:pEGFP-N1 and cell morphology (phase contrast). Each value represents the average of three independent experiments (n = 2). (a and e) SLN vs. Lipofectamine 2000 (PNT1A); (b and f) DOTAP vs. Lipofectamine 2000 (PNT1A), (c and g) SLN vs. Lipofectamine 2000 (PC-3); (d and h) DOTAP vs. Lipofectamine 2000 (PC-3), p$<0.05$ significant difference by Two-way ANOVA, followed by sidak’s mutual comparison test. Scale bar 50 $\mathrm{\mu }$m.

Figure 2. Cell migration velocity of prostate cells in the presence of SLN. PNT1A and PC-3 cells were plated at a density of $5\times {10}^{}4$ cells/well. On the following day, cells were serum-starved for 24 h before being incubated for 4 h with SLN or TGF-$\beta$ (10 ng/mL). Then, the scratch was made and images were acquired every 15 minutes on Cytation 5 Hybrid Multidetection Reader (BioTek Instruments, Inc., Winooski, VT, USA). Cell migration velocity is in $\mathrm{\mu }\mathrm{m}$/min. Each value represents the mean $\pm$ standard deviation (SD). The points represents all the replicates of three independent experiments (n = 2). (i) SLN vs. Control (PC-3), p$<0.05$ significant difference by Two-way ANOVA, followed by Sidak’s mutual comparison test.

Figure 3. Cell migration velocity of prostate cells in the presence of SLN and its components. PC-3 cells were plated at a density of $5\times {10}^{}4$ cells/well. After 24 h, the cells were serum starved for 24 h. Afterward, the cells were treated with SLN, TGF-$\beta ,$ and the individual components stearic acid, DOTAP, and pluronic F68 for 4 h. Then, the scratch was made and images were acquired every 15 min in Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) for 48 h. Cell migration velocity in $\mathrm{\mu }\mathrm{m}$/min for PNT1A and PC-3 cells. Each value represents the mean $\pm$ the standard deviation of three independent experiments (n = 2). (i) SLN vs. Control (PC-3), p$<0.05$ significant difference by Two-way ANOVA, followed by Tukey’s multiple comparisons tests.

Figure 4. SLN induces the migration of PC-3 cells in a dose-dependent manner. PC-3 cells were plated at a density of $5\times {10}^{}4$ cells/well. After 24 h, the cells were serum starved for 24 h. The cells were then treated for 4 h with SLN in the following concentrations: 0 $\mathrm{\mu }\mathrm{g}$/mL (0×, control), 0.076 $\mathrm{\mu }\mathrm{g}$/mL (0.02×), 0.38 $\mathrm{\mu }\mathrm{g}$/mL (0.1×), 0.76 $\mathrm{\mu }\mathrm{g}$/mL (0.2×), and 3.8 $\mathrm{\mu }\mathrm{g}$/ml (1×). Then, the scratch was made and images were acquired every 15 min in Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) for 48 h. Cell migration velocity in $\mathrm{\mu }\mathrm{m}$/min. Each value represents the mean $\pm$ the standard deviation. The points represent all the replications of three independent experiments (n = 2). (i) Control (0x) vs. 0.76 $\mathrm{\mu }\mathrm{g}$/mL, (ii) Control (0x) vs. 3.8 $\mathrm{\mu }\mathrm{g}$/ml (1×), p$<0.05$ significant difference by Two-way ANOVA, followed by Tukey’s multiple comparisons test.

Figure 5. Cell proliferation in the presence of SLN in PC-3 and PNT1A cells. Cells were plated at a density of $5\times {10}^{}4$ cells/well onto coverslips. After overnight incubation, the cells were serum starved for 24 h. Next, the cells were incubated with SLN:pET28A for 4 h. After that, the medium was replaced by RPMI without FBS but containing EdU, and after 5, 24, and 48 h, the cells were fixed with 4% PFA and prepared for Click-iT Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647. The images were acquired in a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) with a 20× objective. Cell proliferation rate is based on the ratio of cells labeled with EdU (in red) to the total cells (DAPI, in blue) at 5, 24, and 48 h for PNT1A (A) and PC-3 (B) cells; Representative images of the control cells and treated with SLN after 5 h, 24 h, and 48 h of PNT1A cells (C) and PC-3 cells (D). Each value represents the median (central line in the boxplot) $\pm$ the lower quartile, the upper quartile, and outliers represented by points above or below the boxplot, from two independent experiments (n = 2) p$<0.05$ significant difference by One Way ANOVA. About 7,000 cells were counted in each treatment. Scale bar 100 $\mathrm{\mu }\mathrm{m}.$

Figure 6. Vimentin expression in PC-3 cells at the migratory front in the presence of SLN after 12 hours of migration. PC-3 cells were deprived of FBS for 24 h, then were treated with SLN:pET28A or TGF-$\beta$ (10 ng/mL) for 4 h. After treatment, the culture medium was replaced with RPMI without SFB and vertically scratched using a p200 pipette tip. The cells were fixed after 12 h of migration and immunostained for vimentin. Images were acquired on Cytation 5 cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) using 20× objective. Quantification of the intensity and texture in single-cells was made using CellProfiler. Cells with mesenchymal-like phenotypes are indicated with a yellow arrow. Scale bar represents 50 $\mathrm{\mu }\mathrm{m}.$ (A) Cells_texture_InfoMeas1_vimentin_20_03_256 feature,(B) Cells_intensity_IntegratedIntensity_vimentin, and (C) Representative images of PC-3 cells. Cells with mesenchymal-like phenotypes are pointed by yellow arrows. Scale bar 50 µm. All points represent the single-cells of two independent experiments (n = 1). Z-test (comparison of means with all other means) was performed using statannotations and statmodels package: ****for p< =1.00e-04.

Figure 7. Nuclear translocation of Smad2 in PNT1A cells. PNT1A cells were plated on coverslips previously treated with poly-L-lysine and plated at a density of $5x{10}^{}4$ cells/well. After 24 hours, cells were treated with SLN:pET28A or TGF-$\beta$ (10 ng/mL) for 45 min, 1 h, 2 h, and 4 h. After the treatment periods, the cells were fixed with 4% PFA and prepared for immunofluorescence of Smad2, and then labeled with Alexa Fluor 488. After immunostaining, the images were acquired using a 10× objective with Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). (A) Single-cells nuclei integrated intensity of Smad2 after 45 min, 1 h, 2 h, and 4 h; (B) Intensity ratio between the integrated intensity of the nuclei/integrated intensity of the cells; (C) Representative images of Smad2 in grayscale. Each value represents the median (central line in the boxplot) $\pm$ the lower and upper quartile, and outliers are represented by points from three independent experiments (n = 2). Z-test (comparison of means with all other means) was performed using statannotations and statmodels package: ****For p< = 1.00e-04. Scale bar 50 $\mathrm{\mu }\mathrm{m}.$

Figure 8. Nuclear translocation of Smad2 in PC3 cells. PC-3 cells were plated on coverslips previously treated with poly-L-lysine and plated at a density of $5\times {10}^{}4$ cells/well. After 24 hours, cells were treated with SLN:pET28A or TGF-$\beta$ 10 ng/mL for 45 min, 1 h, 2 h, and 4 h. After the treatment periods, the cells were fixed with 4% PFA and prepared for immunofluorescence of Smad2, and then labeled with Alexa Fluor 488. After immunostaining, the images were acquired using a 10x objective with Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). (A) Single-cells nuclei integrated intensity of Smad2 after 45 min, 1 h, 2 h, and 4 h; (B) Intensity ratio between the integrated intensity of the nuclei/integrated intensity of the cells; (C) Representative images of Smad2 in grayscale. Each value represents the median (central line in the boxplot) $\pm$ the lower quartile, the upper quartile, and outliers represented by points from three independent experiments (n = 2), Z-test (comparison of means with all other means) was performed using statannotations and statmodels package: ****For p< =1.00e-04. Scale bar 50 $\mathrm{\mu }\mathrm{m}.$

Figure 9. ZEB1 protein in PC3 cells after 24 h of treatment. PC-3 cells were treated with SLN:pET28A and TGF-$\beta$ (10 ng/mL) for 4 h. After 4 h, the culture medium was replaced by RPMI without FBS. The cells were fixed at 4, 7, and 24 h after transfection and prepared for immunofluorescence for ZEB1 and SNAIL. The images were acquired in Cytation 5 cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) using a 10× objective. On average, $5\times {10}^{}4$ cells were counted in each treatment. (A) Intensity ratio between the integrated intensity of the nuclei/integrated intensity of the cells for ZEB1; (B) Integrated intensity of ZEB1 within the cells, and (C) Representative images of the PC-3 cells for ZEB1 marker. Each value represents the median (central line in the boxplot) $\pm$ the lower quartile, the upper quartile, and outliers represented by points from three independent experiments (n = 1) Z-test (comparison of means with all other means) was performed using statannotations and statmodels package: ****For p< =1.00e-04. Scale bar 50 $\mathrm{\mu }\mathrm{m}.$

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.