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Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment.

Fujii H, Shin-Ya M, Takeda S, Hashimoto Y, Mukai SA, Sawada S, Adachi T, Akiyoshi K, Miki T, Mazda O - Cancer Sci. (2014)

Bottom Line: The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF.Intra-tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice.The treatment also inhibited induction of myeloid-derived suppressor cells, while it decreased interleukin-17A production.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

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Vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF)/CH-CA-Spe nanogel complex suppressed VEGF expression in vitro. (a) siVEGF/nanogel complex was prepared under the indicated conditions and added to Renca cell culture (0.8 μg of siRNA/10 μL/well). Twenty-four hours later, VEGF mRNA levels were evaluated by real-time RT-PCR analysis. (b) Transmission electron micrograph image of siVEGF/CH-CA-Spe nanogel complex formed at the cation/phosphate (C/P) ratio of 40 at 25°C for 30 min (concentration: 0.8 μg of siRNA/10 μL). The white small dots represent nanogel particles, and the white arrow indicates colloidal siRNA/nanogel complex with a diameter of 50–100 nm. (c and d) Renca cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at different doses, while control groups were transfected with control non-silencing siRNA (siCont)/nanogel (0.8 μg of siRNA/10 μL) or left untransfected (UT). Twenty-four hours later, real-time RT-PCR (c) and ELISA (d) analyses were performed. (e) VEGF mRNA was measured by real-time RT PCR at the indicated period after transfection with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL/well). (f and g) The indicated cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at doses of 15 (f) and 10 (g) μL/well, and real-time RT-PCR analysis was performed 24 h later. Data represent the means ± SD (n = 3). *P < 0.05 versus siCont; **P < 0.01 versus siCont; +P < 0.001 versus siCont; ++P < 0.005 versus siCont.
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fig02: Vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF)/CH-CA-Spe nanogel complex suppressed VEGF expression in vitro. (a) siVEGF/nanogel complex was prepared under the indicated conditions and added to Renca cell culture (0.8 μg of siRNA/10 μL/well). Twenty-four hours later, VEGF mRNA levels were evaluated by real-time RT-PCR analysis. (b) Transmission electron micrograph image of siVEGF/CH-CA-Spe nanogel complex formed at the cation/phosphate (C/P) ratio of 40 at 25°C for 30 min (concentration: 0.8 μg of siRNA/10 μL). The white small dots represent nanogel particles, and the white arrow indicates colloidal siRNA/nanogel complex with a diameter of 50–100 nm. (c and d) Renca cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at different doses, while control groups were transfected with control non-silencing siRNA (siCont)/nanogel (0.8 μg of siRNA/10 μL) or left untransfected (UT). Twenty-four hours later, real-time RT-PCR (c) and ELISA (d) analyses were performed. (e) VEGF mRNA was measured by real-time RT PCR at the indicated period after transfection with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL/well). (f and g) The indicated cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at doses of 15 (f) and 10 (g) μL/well, and real-time RT-PCR analysis was performed 24 h later. Data represent the means ± SD (n = 3). *P < 0.05 versus siCont; **P < 0.01 versus siCont; +P < 0.001 versus siCont; ++P < 0.005 versus siCont.

Mentions: CH-CA-Spe nanogel was mixed at various C/P ratios with a siVEGF duplex that had been selected as the most effective among the three different siVEGF duplexes tested (Fig. S1a,b). After incubation under various conditions, the mixtures were added to the culture of Renca cells. Efficient silencing of VEGF mRNA was obtained when the mixture was prepared at the C/P ratios of 10 and 15, regardless of the temperatures and incubation periods tested (Fig. 2a). Colloidal siRNAs/nanogel particles were formed under these conditions (Fig. 2b). The siVEGF/nanogel complex suppressed VEGF expression in Renca cells in a dose-dependent manner (Fig. 2c,d), and the silencing effect persisted for 2 days (Fig. 2e). A similar effect was obtained using B16 and MBT-2 (murine melanoma and bladder cancer cell lines, respectively) as well as 786-O and ACHN (human RCC cell lines) (Fig. 2f,g), suggesting effective nanogel-mediated delivery of siRNA into various tumor cells. The siVEGF/nanogel complex did not show any significant toxicity toward the cells (Fig. S1c).


Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment.

Fujii H, Shin-Ya M, Takeda S, Hashimoto Y, Mukai SA, Sawada S, Adachi T, Akiyoshi K, Miki T, Mazda O - Cancer Sci. (2014)

Vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF)/CH-CA-Spe nanogel complex suppressed VEGF expression in vitro. (a) siVEGF/nanogel complex was prepared under the indicated conditions and added to Renca cell culture (0.8 μg of siRNA/10 μL/well). Twenty-four hours later, VEGF mRNA levels were evaluated by real-time RT-PCR analysis. (b) Transmission electron micrograph image of siVEGF/CH-CA-Spe nanogel complex formed at the cation/phosphate (C/P) ratio of 40 at 25°C for 30 min (concentration: 0.8 μg of siRNA/10 μL). The white small dots represent nanogel particles, and the white arrow indicates colloidal siRNA/nanogel complex with a diameter of 50–100 nm. (c and d) Renca cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at different doses, while control groups were transfected with control non-silencing siRNA (siCont)/nanogel (0.8 μg of siRNA/10 μL) or left untransfected (UT). Twenty-four hours later, real-time RT-PCR (c) and ELISA (d) analyses were performed. (e) VEGF mRNA was measured by real-time RT PCR at the indicated period after transfection with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL/well). (f and g) The indicated cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at doses of 15 (f) and 10 (g) μL/well, and real-time RT-PCR analysis was performed 24 h later. Data represent the means ± SD (n = 3). *P < 0.05 versus siCont; **P < 0.01 versus siCont; +P < 0.001 versus siCont; ++P < 0.005 versus siCont.
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fig02: Vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF)/CH-CA-Spe nanogel complex suppressed VEGF expression in vitro. (a) siVEGF/nanogel complex was prepared under the indicated conditions and added to Renca cell culture (0.8 μg of siRNA/10 μL/well). Twenty-four hours later, VEGF mRNA levels were evaluated by real-time RT-PCR analysis. (b) Transmission electron micrograph image of siVEGF/CH-CA-Spe nanogel complex formed at the cation/phosphate (C/P) ratio of 40 at 25°C for 30 min (concentration: 0.8 μg of siRNA/10 μL). The white small dots represent nanogel particles, and the white arrow indicates colloidal siRNA/nanogel complex with a diameter of 50–100 nm. (c and d) Renca cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at different doses, while control groups were transfected with control non-silencing siRNA (siCont)/nanogel (0.8 μg of siRNA/10 μL) or left untransfected (UT). Twenty-four hours later, real-time RT-PCR (c) and ELISA (d) analyses were performed. (e) VEGF mRNA was measured by real-time RT PCR at the indicated period after transfection with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL/well). (f and g) The indicated cells were transfected with siVEGF/nanogel complex (0.8 μg of siRNA/10 μL) at doses of 15 (f) and 10 (g) μL/well, and real-time RT-PCR analysis was performed 24 h later. Data represent the means ± SD (n = 3). *P < 0.05 versus siCont; **P < 0.01 versus siCont; +P < 0.001 versus siCont; ++P < 0.005 versus siCont.
Mentions: CH-CA-Spe nanogel was mixed at various C/P ratios with a siVEGF duplex that had been selected as the most effective among the three different siVEGF duplexes tested (Fig. S1a,b). After incubation under various conditions, the mixtures were added to the culture of Renca cells. Efficient silencing of VEGF mRNA was obtained when the mixture was prepared at the C/P ratios of 10 and 15, regardless of the temperatures and incubation periods tested (Fig. 2a). Colloidal siRNAs/nanogel particles were formed under these conditions (Fig. 2b). The siVEGF/nanogel complex suppressed VEGF expression in Renca cells in a dose-dependent manner (Fig. 2c,d), and the silencing effect persisted for 2 days (Fig. 2e). A similar effect was obtained using B16 and MBT-2 (murine melanoma and bladder cancer cell lines, respectively) as well as 786-O and ACHN (human RCC cell lines) (Fig. 2f,g), suggesting effective nanogel-mediated delivery of siRNA into various tumor cells. The siVEGF/nanogel complex did not show any significant toxicity toward the cells (Fig. S1c).

Bottom Line: The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF.Intra-tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice.The treatment also inhibited induction of myeloid-derived suppressor cells, while it decreased interleukin-17A production.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Show MeSH
Related in: MedlinePlus