Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo

Abstract

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3'-hydroxykinase p110-alpha-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-alpha levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA.

Fig. 1.

Monitoring luciferase inhibition in vivo with bioluminescent imaging. (A) Representative images of nude mice injected with 3 × 10⁶ PC-3M-luc-C6 cells suspended in 100 μl of sterile Dulbecco's PBS into the left ventricle of the heart. Four weeks after tumor injection, each animal was administered i.v. with 200 μl of 0.05% atelocollagen solution, 25 μg of luciferase GL3 siRNA, GL3 siRNA/atelocollagen complex, or nonspecific siRNA/atelocollagen complex. (B) Normalized fold change (1 day posttreatment/pretreatment) of bioluminescence emitted from whole body of mice. Data represent the mean ± SD (n = 4). ^*, P < 0.001 versus other experimental groups.

Fig. 2.

Distribution of siRNA delivered with atelocollagen in tumor tissues and normal tissues. The nude mice were inoculated s.c. with 3 × 10⁶ PC-3M-luc-C6 cells. Once tumors had reached 50-100 mm³, tumor-bearing mice were injected with 200 μl of 0.05% atelocollagen containing 25 μg of luciferase GL3 siRNA or siRNA alone by i.v. tail vein injection. The mice were killed 1 day after treatment with siRNA/atelocollagen complexes, and total RNA was extracted from a tumor (A) and selected tissues (B). Detection of luciferase GL3 siRNA was performed by RNase protection assay. GL3 siRNA levels were corrected for wet tissue weights.

Fig. 3.

The EZH2 and p110-α siRNA inhibit PC-3M-luc-C6 cell proliferation and suppress EZH2 and p110-α expression. (A) Inhibition of PC-3M-luc-C6 cell proliferation. For monitoring the inhibition of cell growth, cells were lysed on days 2, 4, and 6 and then analyzed for luciferase activity. (B) The effects of siRNA transfection on expression of EZH2 and p110-α mRNA. EZH2 and p110-α mRNA expression levels were measured by quantitative PCR. Data represent the mean ± SD (n = 3). ^*, P < 0.05 versus cells treated with atelocollagen alone.

Fig. 4.

Inhibition of metastatic tumor growth in bone tissues by the atelocollagen-mediated siRNA delivery system. Mice were inoculated with PC-3M-luc-C6 cells into the left cardiac ventricle on day 0. The EZH2, p110-α, and nonspecific control siRNAs (50 μg) with or without 0.05% atelocollagen in a 200-μl volume were injected into the mouse tail vein on days 3, 6, and 9 postinoculation. Each experimental regimen comprised eight animals. (A) Representative images of nude mice at the end of the experiment on day 28. (B) Normalized fold change (posttreatment/pretreatment) of bioluminescence emitted from whole body of mice. Data represent the mean ± SD (n = 8). ^*, P < 0.05 versus other experimental groups.

Fig. 5.

Confirmation of prostate cancer bone metastasis by ex vivo imaging and histopathology. (A) The isolated organs from mice (Fig. 4) were reimaged. The bioluminescent signals were detected from the bone region in the jaw and leg of mice treated with atelocollagen alone, EZH2 siRNA/atelocollagen, and p110-α siRNA/atelocollagen, respectively. (B) Hematoxylin/eosin-stained sections of the dental pulp from the same mice that were imaged in Fig. 4. Arrow marks the carcinomatous micrometastasis. (Scale bars, 100 μm.)

Fig. 6.

Absence of IFN response to the atelocollagen-mediated siRNA delivery system. Nude mice were injected with nonspecific control siRNA (50 μg) with 0.05% atelocollagen in a 200-μl volume by i.v. tail vein injection. The positive control group was injected with poly(I:C). Serum was collected 2 h postinjection; IFN-α and IL-12 levels were determined by ELISA. Data represent the mean ± SD (n = 4).