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. 2014 Aug 24;4(11):1096-111.
doi: 10.7150/thno.9423. eCollection 2014.

Co-delivery of doxorubicin and Bmi1 siRNA by folate receptor _targeted liposomes exhibits enhanced anti-tumor effects in vitro and in vivo

Tan Yang  1 , Bin Li  1 , Shibo Qi  1 , Yong Liu  1 , Yongkang Gai  1 , Peng Ye  1 , Guang Yang  1 , Wendian Zhang  1 , Peng Zhang  2 , Xingxing He  3 , Weijie Li  4 , Zhiping Zhang  1 , Guangya Xiang  1 , Chuanrui Xu  1
Affiliations

Co-delivery of doxorubicin and Bmi1 siRNA by folate receptor _targeted liposomes exhibits enhanced anti-tumor effects in vitro and in vivo

Tan Yang et al. Theranostics. .

Abstract

Bmi1 gene overexpression is found in various human tumors and has been shown as a potential _target for gene treatment. However, siRNA-based treatments _targeting Bmi1 gene have been restricted to limited delivery, low bioavailability and hence relatively reduced efficacy. To overcome these barriers, we developed a folate receptor _targeted co-delivery system folate-doxorubicin/Bmi1 siRNA liposome (FA-DOX/siRNA-L). The FA-DOX/siRNA-L was prepared through electrostatic interaction between folate doxorubicin liposome (FA-DOX-L) and Bmi1 siRNA. In vitro and in vivo studies showed that FA-DOX/siRNA-L inhibited tumor growth by combinatory role of Bmi1 siRNA and doxorubicin (DOX). Co-delivery of Bmi1 siRNA and DOX by FA-DOX/siRNA-L showed significantly higher efficacy than sole delivery of either DOX or Bmi1 siRNA. Real-time PCR and western blot analysis showed that FA-DOX/siRNA-L silenced the expression of Bmi1 gene. In addition, higher accumulation of the siRNA and DOX in tumor cells indicated that folate ligand displayed tumor _targeting effect. These results suggest that Bmi1 is an effective therapeutic _target for siRNA based cancer treatment that can be further improved by co-delivery of DOX through _targeted liposomes.

Keywords: Bmi1 siRNA; doxorubicin; folate receptor; liposome; tumor _targeting..

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
The structure and anti-tumor mechanisms of FA-DOX/siRNA-L. First, long circulating FA-DOX/siRNA-L is bound to and internalized into tumor cells via folate receptors. Then FA-DOX/siRNA-L is transported through the endosome and lysosome. DOX and siRNA are released to the cytoplasm. Finally, the Bmi1 siRNA binds to Bmi1 mRNA and the DOX binds to chromosomal DNA.
Figure 2
Figure 2
Complexation of siRNA with folate DOX liposomes (FA-DOX-L) and characterization of liposomes. A. A representative agarose gel electrophoresis image illustrating siRNA loading efficiency at the following w/w ratio (FA-DOX-L/siRNA): (1) 0:1 (siRNA only, control); (2) 40:1; (3) 80:1; (4) 120:1; (5) 160:1; (6) 200:1, and (7) FA-DOX-L only. SiRNA combined to FA-DOX-L has limited migration in the agarose gel, near the loading wells. B. Key parameters of DOX-L, FA-DOX-L, DOX/siRNA-L and FA-DOX/siRNA-L liposomes. Data are expressed as mean ± SD of three independent samples.
Figure 3
Figure 3
The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P < 0.01.
Figure 4
Figure 4
Cellular uptake and distribution of DOX in KB and HeLa cells. KB, HeLa and Hep3B cells (FR negative) grown in a monolayer were incubated with FA-DOX-L, DOX-L, and free DOX for 1 h at 37°C. A. Fluorescence detection and localization of DOX in the KB cells. B. Cellular uptake of DOX delivered by DOX-L and FA-DOX-L in KB, HeLa and Hep3B cells. Hep3B cell was used as a negative control. Data are expressed as mean ± SD of 3 independent samples. **: P < 0.01.
Figure 5
Figure 5
Co-localization of Bmi1 siRNA and DOX in KB cells. KB cells grown in a monolayer were incubated with free DOX, DOX/siRNA-L, FA-DOX/siRNA-L and for 1 h at 37°C. For folate blocking group, 1 mM free folate was added to the incubation media prior to the addition of FA-DOX/siRNA-L. Bmi1 siRNA was labeled with FAM (Green) and DOX emits red fluorescence by itself. The nucleus was counterstained with DAPI (Blue).
Figure 6
Figure 6
Cytotoxic effects of FA-DOX/siRNA-L in KB, HeLa and Hep3B cells. Cells were treated with liposomes with the final siRNA concentration of 100 nM and DOX concentration of 1 mg/mL. The Hep3B cells were used as a negative control. The multiple groups were compared by one-way ANOVA with Dunnett's t-test. Data are expressed as mean ± SD (n = 3). *: P < 0.05; **: P < 0.01.
Figure 7
Figure 7
Stability test of the siRNA of the FA-DOX/siRNA-L. The FA-DOX/siRNA-L liposomes were cultured with mouse serum for 1, 3 and 6 h at 37 ºC. Then the liposomes solutions with serum were added to KB cell culture for 1 h under 37 ºC. The siRNA was labeled with FAM (green) and the nucleus was counterstained with DAPI (blue). Note the color of the nucleus was merged from blue (DAPI) and red (DOX).
Figure 8
Figure 8
The pharmacokinetic analysis of DOX in mouse plasma. A. Pharmacokinetic curves of DOX, DOX/siRNA-L, FA-DOX/siRNA-L in the mouse plasma. The mice were injected intravenously by a single dose of drugs at a DOX concentration of 5 mg/kg in female Kunming mice and the blood samples were collected at 8 time points (n = 4). B. Pharmacokinetic parameters of DOX calculated from the same experiment by a non-compartment model (n = 4 animals for each group). AUC: area under curve; CLz: clearance; t1/2z: plasma half-life; Vz: distribution volume during elimination phase; Cmax: maximum plasma drug concentration.
Figure 9
Figure 9
DOX distribution and Bmi1 expression in normal and tumor tissues in xenograft nude mice. For panel A and B, the mice were injected via tail vein at a single dose of saline, free DOX (5 mg/kg), DOX/siRNA-L (170 μg/kg siRNA, 5 mg/kg DOX) and FA-DOX/siRNA-L (170 μg/kg siRNA, 5 mg/kg DOX) (n = 3) when the tumors grew to about 500 mm3. The tissues were collected at the 3 h after the injections. A. Quantitative analysis DOX distribution by the microplate reader in the normal tissues of xenograft nude mice. B. DOX distribution by fluorescence detection in tumor tissues of mice. C. Relative Bmi1 mRNA levels in tumor tissues treated with saline, free DOX, DOX-L, DOX/siRNA-L, FA-DOX-L and FA-DOX/siRNA-L by a single dose tail vein injection (170 μg/kg siRNA, 5 mg/kg DOX) (n = 3). Total mRNA was extracted from the isolated tumor tissues 24 h after the drug injections. Data are expressed as mean ± SD. *: P < 0.05.
Figure 10
Figure 10
Anti-tumor effects of FA-DOX/siRNA-L in xenograft mouse model. A. Excised tumors at the end point of the experiment (day 24 after the drug injection). B. Growth curves of xenograft tumors treated with saline, free DOX, DOX-L, FA-siRNA-L, FA-DOX-L and FA-DOX/siRNA-L by a single dose tail vein injection (170 μg/kg siRNA, 8 mg/kg DOX). The curves present the changes of tumor sizes from the day of injection (day 0). The comparisons were performed versus the FA-DOX-L group by the Student's test. Data are expressed as mean ± SD (n = 3). *: P < 0.05. C. Body weight changes of mice treated with saline, free DOX, DOX-L, FA-siRNA-L, FA-DOX-L and FA-DOX/siRNA-L. Saline injection was used as the control. Data are expressed as mean ± SD (n = 3).
Figure 11
Figure 11
TUNEL staining in biopsies of KB tumors treated with a single dose injection of siRNA and/or DOX liposomes (170 μg/kg siRNA, 8 mg/kg DOX). The tumors were removed from the mice after the efficacy study (day 24 after the drug injection) and biopsies were made after 4% PFA fixation. TUNEL signal was colored by diaminobenzidine (DAB, brown color) and cell nucleus was counterstained by hematoxylin. A. Saline; B. FA-siRNA-L; C. FA-DOX-L; D. FA-DOX/siRNA-L.
Figure 12
Figure 12
The expression of Bmi1 _target genes in tumor tissues when Bmi1 was knockdown. Tumor tissues were resected from the mice treated with a single dose injection of FA-DOX-L, FA-DOX/siRNA-L liposomes (170 μg/kg siRNA, 8 mg/kg DOX) and saline (NC group) after the efficacy study (day 24 after the drug injection). Total mRNA was extracted from the frozen tumor tissue samples (n = 3).
Figure 13
Figure 13
Determination of the liver and kidney toxicity of FA-DOX/siRNA-L. Female Kunming mice were injected intravenously with a single dose of saline, FA-L, and FA-DOX/siRNA-L at a blank liposome concentration of 310 mg/kg (n = 3). Blood samples were collected 24 h after drug injections. A. Liver toxicity determined by AST, ALT level and AST/ALT ratio. B. Kidney toxicity determination by BUN and CR level in plasma. *: P < 0.05.
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