3.3.1 Gel
Retardation Assay
1. Mix 15 μL of the complexes with 3 μL of gel loading dye
solution and load MNP@PEI/dsRNA complexes on 1% agarose gel. As a control use the pure dsRNA.
2. Run electrophoresis in 1Â TBE buffer at a constant voltage
10 V/cm for 30 min.
3. Visualize the samples using gel imaging system.
4. Calculate the binding efficiency by comparing density data
obtained for complexes to those for the pure dsRNA.
3.3.2 UV-Vis
Spectrophotometry
1. Centrifuge MNP@PEI/dsRNA complexes: 9300 Â g, 10 min.
2. Measure the concentration of RNA contained in the supernatant at A260 on the spectrophotometer. Load 1 μL of the
supernatant on the measurement pedestal and measure the
absorbance.
3. Measure the concentration of pure dsRNA sample.
4. Calculate binding efficiency by comparing concentrations
obtained for complexes to those for the pure dsRNA.
3.4 Cell Culture
Maintenance
1. Grow adherent U-118 MG glioblastoma multiforme cell line in
T-75 flask. Use culture medium supplemented with 10% FBS
and 1% antibiotic solution, then replace it at intervals 24–36 h.
2. To passage the culture detach cells using trypsin. Carry out the
culture in CO 2 incubators at temperature 37
C, humidity
95%, CO 2 concentration 5% (see Note 3).
3.5 Visualization of
Transfection
Complexes in the Cells
3.5.1 Cellular Uptake of
Transfection Complexes
1. To obtain fluorescently labeled MNP@PEI, mix 2 mL of magnetic NPs suspension (2 mg Fe/mL) with 490 μL 0.1 M borate
buffer and 10 μL solution of Atto 550 NHS.
2. Incubate the suspension overnight at room temperature with
continuous stirring.
3. Dialyze the suspension against water using a Slide-A-Lyzer G2
cassette with a cut-off at 3500 MW. Determine the iron concentration in obtained sample on a spectrophotometer.
4. Seed U118-MG cells into 8-well Nunc Lab-Tek Chamber Slide
at 2.5 Â 10
4 cells/well (300 μL) and incubate under standard
tissue culture conditions for 24 h.
5. Prepare working dilution of transfection complexes containing
fluorescently labeled NPs with an iron-to-RNA ratio of 3:1
(w/w). Transfer 50 μL of the transfection complexes
corresponding to the ATN-RNA final concentration of
100 nM into the well of slide with the seeded cells. Incubate
the cells with complexes for 24 h.
Delivery of dsRNA to Glioblastoma Cells
75
Retardation Assay
1. Mix 15 μL of the complexes with 3 μL of gel loading dye
solution and load MNP@PEI/dsRNA complexes on 1% agarose gel. As a control use the pure dsRNA.
2. Run electrophoresis in 1Â TBE buffer at a constant voltage
10 V/cm for 30 min.
3. Visualize the samples using gel imaging system.
4. Calculate the binding efficiency by comparing density data
obtained for complexes to those for the pure dsRNA.
3.3.2 UV-Vis
Spectrophotometry
1. Centrifuge MNP@PEI/dsRNA complexes: 9300 Â g, 10 min.
2. Measure the concentration of RNA contained in the supernatant at A260 on the spectrophotometer. Load 1 μL of the
supernatant on the measurement pedestal and measure the
absorbance.
3. Measure the concentration of pure dsRNA sample.
4. Calculate binding efficiency by comparing concentrations
obtained for complexes to those for the pure dsRNA.
3.4 Cell Culture
Maintenance
1. Grow adherent U-118 MG glioblastoma multiforme cell line in
T-75 flask. Use culture medium supplemented with 10% FBS
and 1% antibiotic solution, then replace it at intervals 24–36 h.
2. To passage the culture detach cells using trypsin. Carry out the
culture in CO 2 incubators at temperature 37
C, humidity
95%, CO 2 concentration 5% (see Note 3).
3.5 Visualization of
Transfection
Complexes in the Cells
3.5.1 Cellular Uptake of
Transfection Complexes
1. To obtain fluorescently labeled MNP@PEI, mix 2 mL of magnetic NPs suspension (2 mg Fe/mL) with 490 μL 0.1 M borate
buffer and 10 μL solution of Atto 550 NHS.
2. Incubate the suspension overnight at room temperature with
continuous stirring.
3. Dialyze the suspension against water using a Slide-A-Lyzer G2
cassette with a cut-off at 3500 MW. Determine the iron concentration in obtained sample on a spectrophotometer.
4. Seed U118-MG cells into 8-well Nunc Lab-Tek Chamber Slide
at 2.5 Â 10
4 cells/well (300 μL) and incubate under standard
tissue culture conditions for 24 h.
5. Prepare working dilution of transfection complexes containing
fluorescently labeled NPs with an iron-to-RNA ratio of 3:1
(w/w). Transfer 50 μL of the transfection complexes
corresponding to the ATN-RNA final concentration of
100 nM into the well of slide with the seeded cells. Incubate
the cells with complexes for 24 h.
Delivery of dsRNA to Glioblastoma Cells
75