Figure 2.

Stability of CM-miR-143 lipoplexes in mouse blood and delivery to pelvic CRC tumors

(A) Scheme of in vitro experiments for measuring miR-143 levels using qRT-PCR. Naked miRNA was incubated in FBS. Retention rates of miR-143 were measured at 0, 10, 30, and 60 min after incubation. (B) Blue, purple, and orange lines indicate control (C) miRNA, Am-miR-143 (Am), and CM-miR-143 (CM), respectively. Values at 0 min for Am-miR-143 and CM-miR-143 are indicated as 1.0. After incubation for 10 min, Am-miR-143 levels decreased significantly. CM-miR-143 levels remained high until 60 min after incubation. Data are presented as mean ± SEM (∗p < 0.05; ∗∗∗p < 0.001; n = 3). (C) Scheme of in vivo experiments for measuring plasma miR-143 levels using qRT-PCR. Each miRNA lipoplex (330 μg/kg miRNA per administration) was injected into the tail vein once. Blood miR-143 levels were measured at 12, 24, 48, and 72 h after administration. (D) Purple and orange lines indicate miR-143 ratios in the Am-miR-143 and CM-miR-143 groups, respectively. miR-143 levels at 12 h are indicated as 1.0. At 24 h after administration, Am-miR-143 levels decreased, whereas CM-miR-143 levels remained high until 72 h. Data are presented as the mean ± SEM (∗p < 0.05; ∗∗p < 0.01; n = 7). (E) Scheme of in vivo experiments for in situ hybridization and qRT-PCR, aiming to characterize miR-143-143 distribution in pelvic CRC tumors (DLD-1 clone#1-Luc cells). In situ hybridization was performed using tissue samples from pelvic CRC tumors resected 10 min after miRNA lipoplex administration; qRT-PCR was performed 24 and 72 h after administration (330 μg/kg miRNA per administration). (F) Representative images from in situ hybridization and CT. Purple signals in situ hybridization indicate miR-143. The images illustrate 3 regions: the border area, tumor, and connective tissue in situ hybridization. The border area represents the region surrounding the tumor. The connective tissue, highlighted by an orange square in CT imaging, is a magnified image of the border area. CT imaging showed that blood flow was rich in connective tissue (scale bar: 5 mm). (G) Results from qRT-PCR revealed that miR-143 levels were significantly higher at 24 h in the CM-miR-143 group than in the control group. However, the 2 groups did not differ significantly in miR-143 levels by 72 h. Data are presented as mean ± SEM (∗p < 0.05; n = 4). ns, not significant; Adm, administration.