Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene

Figures & data

Figure 1. Agarose gel electrophoresis image of the recombinant c-Myc-siRNA-pDNAs: M, 100 bp DNA Ladder (100–1000 bp); L1, empty vector (269 bp); L2–L4, three recombinant c-Myc-siRNA-pDNAs (316 bp).

Table 1. A₂₆₀/A₂₈₀ ratios of siRNA-pDNAs.

pDNAs	c-Myc- siRNA1-pDNAs	c-Myc- siRNA2-pDNAs	c-Myc- siRNA3-pDNAs
A260/A280	1.83	1.82	1.85

Figure 2. Representative agarose gel electrophoresis image following restriction digestion of the recombinant c-Myc-siRNA-pDNAs: M, DNA marker; L1, c-Myc-siRNA3-pDNAs cut by BamHI; L2, c-Myc-siRNA3-pDNAs cut by HindIII; L3, empty vector; L4, c-Myc-siRNA3-pDNAs.

Figure 3. Fluorescence photomicrographs of green fluorescent protein expression of c-Myc-siRNA3-pDNAs after 24 h transfection: (A) c-Myc-siRNA1-pDNAs, (B) c-Myc-siRNA2-pDNAs, (C) c-Myc-siRNA3-pDNAs, and (D) c-Myc-siRNA3-pDNAs (detected with fluorescent and white light overlap).

Figure 4. Fluorescence photomicrographs of Hoechst33258 and propidium iodide double staining after 24 h transfection: (A) normal C6 cells, (B) the cells incubated with Lipofectamin 2000 only, (C) the cells transfected with empty siRNA vector, (D) the cells transfected with c-Myc-siRNA1-pDNAs, (E) the cells transfected with c-Myc-siRNA2-pDNAs, and (F) the cells transfected with c-Myc-siRNA3-pDNAs.

Figure 5. Apoptotic rate of C6 cells after transfection with c-Myc-siRNA-pDNAs: (A) normal C6 cells, (B) cells transfected with empty siRNA vector, (C) cells transfected with c-Myc-siRNA1-pDNAs, (D) cells transfected with c-Myc-siRNA2-pDNAs, and (E) cells transfected with c-Myc-siRNA 3-pDNAs.

Figure 6. Transmission electron micrograph of NPs-c-Myc-siRNA3-pDNAs negatively stained with phosphotungstic acid solution. The black bar in the photograph refers to 300 nm.

Table 2. Physicochemical properties of NPs-c-Myc-siRNA3-pDNAs.

Batch	Size (nm)^a	Zeta-potential (mV)	Encapsulation efficiency (%)	Loading capacity (%)
1	132.7	−18.29	70.6	1.85
2	128.6	−18.94	76.9	1.92
3	136.9	−17.68	81.3	1.97

^aThe particle size of NPs-c-Myc-siRNA3-pDNAs was expressed as the average number based diameter, which was measured by dynamic light scattering.

Table 3. Apoptotic rate of C6 cells after transfection with NPs loading c-Myc-siRNA3-pDNAs (n = 3).

Group^a	Normal control group	Test group I	Test group II
Apoptotic rate (mean ± SD) (%)	5.13 ± 1.55	5.08 ± 1.52	24.77 ± 5.39†

^aNormal control group: normal C6 cells; test group I: C6 cells directly incubated with c-Myc-siRNA3-pDNAs for 48 h without the help of transfection reagent; test group II: C6 cells transfected with NPs-c-Myc-siRNA3-pDNAs for 48 h. †p < 0.02, statistical significance with test group II versus normal control group.

Figure 7. Agarose gel electrophoresis image of c-Myc-siRNA3-pDNAs after preparation of NPs: L1, the original c-Myc-siRNA3-pDNAs; L2, c-Myc-siRNA3-pDNAs extracted from the supernatant; L3, c-Myc-siRNA3-pDNAs extracted from the NPs; M, marker.

Figure 8. Agarose gel electrophoresis image of c-Myc-siRNA3-pDNAs in acid condition: L1, original c-Myc-siRNA3-pDNAs; L2, c-Myc-siRNA3-pDNAs incubated in pH 4.0 PBS for 2 h; L3, c-Myc-siRNA3-pDNAs incubated in pH 7.0 PBS for 2 h; M, marker.

Figure 9. Agarose gel electrophoresis image of c-Myc-siRNA3-pDNAs on exposure to DNase I: L1, original c-Myc-siRNA3-pDNAs; L2, original c-Myc-siRNA3-pDNAs incubated with DNase I for 2 h; L3, NPs-c-Myc-siRNA3-pDNAs incubated with DNase I for 2 h; M, marker.

Figure 10. Fluorescence photomicrographs of green fluorescent protein expression of NPs-c-Myc-siRNA3-pDNAs after transfection for 12 h, 24 h, 48 h, and 72 h.

Figure 11. Fluorescence photomicrographs of C6 cells transfected with NPs-c-Myc-siRNA3-pDNAs after Hoechst33258 and propidium iodide double staining: (A) normal C6 cells and (B) C6 cells transfected with NPs-c-Myc-siRNA3-pDNAs for 48 h.