T2-mapping image of the Bel-7402 cells.

<p>(A) T₂-mapping image of the Bel-7402 cells incubated with PEG-<i>g</i>-PEI-SPION or RGD-PEG-<i>g</i>-PEI-SPION at different Fe concentrations. (B) T₂ values of the cells incubated with various complexes at different Fe concentration. (means±SD; n = 3).</p

<i>In vitro</i> cell transfection efficiency analysis and cell uptake analysis.

<p>(A) The percentage of FITC-positive cells incubated with RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA or RGD-PEG-<i>g</i>-PEI-SPION/siRNA in the presence of free RGD at various N/P ratios. (means±SD, n = 3; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA at the same N/P ratio). (B) The laser confocal microscope images of Bel-7402 cells transfected with RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA or RGD-PEG-<i>g</i>-PEI-SPION/siRNA in the presence of free RGD at a N/P ratio of 10.(×630; scale bar:10 µm).</p

Tumor growth inhibition analysis.

<p>The tumor growth inhibition analysis in nude mice bearing Bel-7402 tumors after injection of various complexes formed at a N/P ratio of 10. (means±SD; n = 10; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA; Control: the mice injected with PBS).</p

The apoptotic response to different treatments in Bel-7402 cells.

<p>(A) Images show Bel-7402 cells incubated with: RGD-PEG-<i>g</i>-PEI-SPION/siRNA, PEG-<i>g</i>-PEI-SPION/siRNA, RGD-PEG-<i>g</i>-PEI-SPION/siNC or PEG-<i>g</i>-PEI-SPION/siNC (×200; scale bar:100 µm) (B) Percentage of apoptotic cells induced by various complexes at N/P ratio of 10 as quantified by TUNEL analysis (means±SD; n = 3; *<i>p</i><0.05, compared with RGD-PEG-<i>g</i>-PEI-SPION/siRNA; Control: the cells without transfection).</p

<i>In vivo</i> tumor targeting evaluation.

<p>(A) T₂WI and T₂-mapping images of mice bearing Bel-7402 subcutaneous tumors before and after tail vein administration of RGD-PEG-<i>g</i>-PEI-SPION or PEG-<i>g</i>-PEI-SPION. (B) The normalized MR signal intensity of the tumors before and after tail vein administration of the various complexes. (C) T₂ values of the tumors before and after tail vein administration of the various complexes. (means±SD; n = 5).</p

Characterization of the complexes.

<p>A, B) Particle size and zeta potential of complexes PEG-g-PEI-SPION/pDNA and scAb_GD2-PEG-g-PEI-SPION/pDNA. Both PEG-g-PEI-SPION and scAb_GD2-PEG-g-PEI-SPION condensed pDNA to form stable nanoparticles 80-100 nm in diameter at an N/P ratio of 20. The surface charge of the complexes increased with increasing N/P ratio. C) Electrophoretic migration of complexes scAb_GD2-PEG-g-PEI-SPION/pDNA and PEG-g-PEI-SPION/pDNA at various N/P ratios. PEG-g-PEI-SPION and scAb_GD2-PEG-g-PEI-SPION completely bound pDNA at N/P ratios of 2.3 and 2.4, respectively. D) In vitro cytotoxicity of complexes in hBMSCs determined by CCK-8 assay. The CCK-8 assay was used to evaluate the cytotoxicities of the complexes PEG-g-PEI-SPION/pDNA and scAb_GD2-PEG-g-PEI-SPION/pDNA in hBMSCs. The complexes were formed at various N/P values, and the pDNA mass in each well was set to 0.15 µg. No statistical difference was found between the two groups (<i>P</i>>0.05). The results were presented as the mean ± standard deviation (n=3).</p

<i>In vitro</i> MRI scan.

<p>The cells were scanned with a 1.5 T MRI scanner at room temperature (A). All complexes were formed at an N/P ratio of 20. B shows the normalized MR signal intensity of different complexes at various Fe concentrations (n=3).</p

Schematic diagram of targeted gene delivery to hBMSCs.

<p>Schematic diagram of targeted gene delivery to hBMSCs.</p

Cellular internalization of the complexes.

<p>Images assignment: green: delivery agent, blue: nucleus, Red: pDNA. The image on the right is an overlay of the three fluorescent colors. Cells incubated with targeting complex displayed clearly stronger delivery agent (green) and pDNA (red) fluorescence compared with cells incubated with nontargeting and targeting/GD2 complexes.</p

Prussian blue staining.

<p>Compared with the cells incubated with nontargeting complex (B), cells incubated with targeting complex displayed much more intensive blue color (A), indicating much more intracellular SPION.</p