■ In vitro 遺伝子導入効率 (α-CDE、GUG-β-CDE、Lac-α-CDE)
Transfection Efficiency of the Complexes of pDNA/carriers in NIH3T3 Cells
Cells were transfected with binary complexes for 24 h. The charge ratio of a-CDE/pDNA was 200/1. Each value represents the mean ± S.E. of 4-6 experiments. *p<0.05, compared with dendrimer.
Transfection Efficiencies of the pDNA Complexes with VariousCarriers in A549 Cells and RAW264.7 Cells
Transfection was performed with culture medium without FCS for 3 h. The amount of pDNA was 2.0 mg. The luciferase activity in cell lysates was determined 21 h after incubation. The charge ratio of carriers/pDNA was 100. Culture medium was supplemented with 10% FCS. Each value represents the mean±S.E. of 4 experiments.
*p<0.05, compared with a-CDE. †p<0.05, compared with b-CDE.
Transfection Efficiencies of the Complexes of pDNA/carriers in A549 cells, KB cells and HepG2 cells.
The luciferase activity in cell lysates was determined 24 h after incubation. The amount of pDNA was 2 μg. The charge ratio of carrier/pDNA was 100. Each value represents the mean ± SEM of 3-4 experiments. *p < 0.05, compared with a-CDE (G2).
Effects of Asialofetuin (AF) and BSA on tTransfection Efficiencies of pDNA Complexes with SAFETRANCE (Lac-a-CDE) in HepG2 cells.
The concentrations of AF and BSA were 0.5 mg/mL. The luciferase activity in cell lysates was determined 24 h after incubation. The amount of pDNA was 2 μg. The charge ratio of carrier/pDNA was 100. Each value represents the mean ± SEM of 3-4 experiments. *p < 0.05, compared with control. †p < 0.05, compared with BSA.
■ In vitro 細胞障害性 (α-CDE、GUG-β-CDE、Lac-α-CDE)
Cytotoxicity of Carrier Complexes with pSilencer in NIH3T3-luc Cells
Cells were transfected with carriers/pSilencer complexes for 24 h. Cell viability was assayed by the WST-1 method. The amount of pSilencer was 2.0 mg. The charge ratio of TF/pSilencer was 1/1. The volume to amount ratio of L2/pSilencer was 1/1. Each value represents the mean ± S.E. of 4-5 experiments. *p<0.05, compared with control.
Cytotoxicity of pDNA Complexes with Carriers in NIH3T3 Cells
Each point represents the mean ± S.E. of 4-6 experiments.
Cytotoxicity of pDNA complexes with various carriers in HepG2 cells.
Cells were incubated with carriers/pDNA complexes for 24 h. Cell viability was assayed by the WST-1 method. The amount of pDNA was 2.0 mg. Each point represents the mean ± SEM of 4 experiments.
*p < 0.05, compared with a-CDE (G2).
■ In vivo 遺伝子導入効率 (α-CDE、GUG-β-CDE、Lac-α-CDE)
In Vivo Gene Transfer Efficiencies of pDNA Complexes with SAFETRANS (a-CDE) after Intravenous Injection to BALB/c Mice
Twelve h after intravenous injections of suspensions (500 ml) containing pDNA complexes with various carriers (the charge ratio of 10/1), the mice were sacrificed, and then Renilla luciferase activity in various organs was determined using a luminometer.
The amount of pDNA was 50 mg. Each value represents the mean±S.E. of 9 mice.
*p<0.05, compared with pDNA.
In Vivo Gene Transfer Efficiencies of pDNA Complexes with Various Carriers after Intravenous Injection to BALB/c Mice
Twelve h after intravenous injections of suspensions (500 ml) containing pDNA complexes with various carriers (the charge ratio of 20), the mice were sacrificed, and then Renilla luciferase activity in various organs was determined using a luminometer. The amount of pDNA was 20 mg. Each value represents the mean±S.E. of 3 mice.
*p<0.05, compared with b-CDE.
In Vivo Gene Transfer Efficiencies of pDNA Complexes with Various Carriers to tail vein of BALB/c mice.
Twelve h after intravenous injections of the solutions containing pDNA complexes with carriers (α-CDE (G2), SAFETRANS (Lac-α-CDE), charge ratio of 20; commercial carrier B, charge ratio of 1), the mice were sacrificed, and then Renilla luciferase activity in various organs was determined using a luminometer. Each value represents the mean±SEM of 5–6 mice. *p<0.05, compared with α-CDE (G2).
# p<0.05, compared with commercial carrier B. †p<0.05, compared with SAFETRANS (Lac-α-CDE).
Confocal Laser Microscopes of NIH3T3 after Transfection of FITC Labeled pDNA
Cells were transfected with binary complexes for 24 h. The charge ratios of a-CDE/pDNA was 200/1. Scale bar = 25 mm.
Inhibitory Effects of Binary Complexes of Carriers/siRNA on Luciferase Activity in Cells Stably Expressing Luciferase Gene (NIH3T3-luc Cells)
Cells were transfected with binary complexes for 24 h. The charge ratios of a-CDE/siRNA and PEI/siRNA were 20/1 and 5/1, respectively. The volume to amount ratios of A reagent/siRNA and B reagent/siRNA were 3.75/1 and 6/1, respectively. The concentration of siRNA was 100 nM. Each value represents the mean ? S.E. of 4 experiments.
*p<0.05, compared with control. †p<0.05, compared with siGL2.
Inhibitory Effects of Binary Complexes of Carriers/siLamin on Lamin A/C Expression in Colon26-luc Cells
Colon26-luc cells were transfected with binary complexes of carriers/siLamin for 24 h. Lamin A/C expression was assayed by Western blot after adjusting the total protein content (50 mg/lane). The charge ratios of SAFETRANS (a-CDE)/siLamin and PEI/siLamin were 20/1 and 5/1, respectively. The volume to amount ratio of A reagent/siLamin was 3.75/1. The siRNA concentration was 100 nM.
*p<0.05, compared with control. Each value represents the mean ± S.E. of 3-5 experiments.
Inhibitory Effects of Binary Complexes of Carriers/siGL3 on Luciferase Activity in BALB/c Mice Bearing Tumor Cells Stably Expressing Luciferase Gene
Binary complexes of carriers/siRNA (100 mL) were directly injected to tumor of mice. After 24 h, luciferase activity was determined. The charge ratio of SAFETRANS (a-CDE)/siRNA was 20/1. The volume to amount ratio of L2/siRNA was 3.75/1. The amount of siRNA was 10 mg. *p<0.05, compared with control. †p<0.05, compared with siGL2.
Cellular Uptake of Binary Complexes of TRITC-SAFETRANS (a-CDE)/FlTC-siGL3 into NIH3T3-luc Cells after Transfection
The cells were transfected with binary complexes of SAFETRANS (a-CDE)/siRNA for 1 h. After two times washed with PBS, the cells were fixed with 100% MeOH for 5 min at 4?C. Two times washed with PBS, and then the cells were scanned with a CLSM. The charge ratio of SAFETRANS (a-CDE)/siRNA complexes was 20/1. The siRNA concentration was 100 nM.
Nuclear localization of the Alexa-pDNA complexes with TRITC-labeled SAFETRANS (Lac-a-CDE) in HepG2 cells.
The cells were observed by CLSM. Incubation time was 24 h. The charge ratio (carrier/pDNA) was 100. DIC represents a differential interference contrast microscopy.
Cellular Uptake of FlTC-siGL3 Complexes with Carriers into NIH3T3-luc Cells
Cells were transfected with binary complexes of carriers/siRNA for 1 h. After two times washed with PBS, the cells were fixed with 100% MeOH for 5 min at 4?C. The charge ratio of SAFETRANS(a-CDE)/siRNA was 20/1. The volume to amount ratio of LipofectaminTM2000/siRNA was 3.75/1. The concentration of siRNA was 100 nM.
Inhibitory Effects of pSilencer Complexes with Carriers on Luciferase Activity in Cells Stably Expressing Luciferase Gene (NIH3T3-luc Cells)
The cells were transfected with carrier/pSilencer complexes for 24 h. The amount of pSilencer was 2 mg. The charge ratio of carrier/pSilencer was 20/1. Each value represents the mean ±S.E. of 6-7 experiments. *p<0.01, compared with control. †p<0.01, compared with shGL2.
