The overall goal of these methods is to unable the prediction of small interfering RNA mediated gene silencing efficiencies and facilitate control over the resulting protein expression levels, in a spatial temporal manner. This method can elucidate structure function relationships and stimuli responsive delivery vehicles. Gaining better control over binding versus release, can unlock translatable platforms and drug discovery, as well as regenerative medicine technologies.
The main advantages of this technique are the changes in gene expression can be controlled and predicted on the base as a simple, siRNA release assays and kinetic modeling. Though these methods employ novel photo-responsive polymers, this set of assays can be readily adapted to test the wide variety of other stimuli responsive systems. To begin, prepare a 32 micrograms per milliliter siRNA solution, by adding siRNA to a 20 micromolar HEPES solution at pH six.
Then, prepare a polymer solution by adding dissolved mPEG block polyAPN BMA polymers to a 20 millimolar HEPES solution at pH six. To achieve a concentration of 220 micrograms per milliliter, this concentration will result in an N to P ratio of four, meaning there will be four times as many amine groups on the polymer as phosphate groups on siRNA. Next, add the polymer solution drop-wise to an equal volume of the siRNA solution, while gently mixing on a vortex machine.
Continue to vortex for 30 seconds following polymer addition. Then, incubate the samples in the dark at room temperature for 30 minutes. Add the STS solution to each nanocarrier solution drop-wise while gently mixing on a vortex machine.
Continue to vortex for 30 seconds following SDS edition. Centrifuge the samples at 3, 000 Gs for five seconds, then, incubate the samples in the dark at room temperature for 30 minutes. Calibrate and set a UV laser with a 365 nanometer filter to an intensity of 200 watts per square meter.
Next, cut a hole into a rubber gasket, place the rubber gasket onto a washed and dried glass slide. Pipette the nanocarrier SDS solution onto the glass slide inside the hole of the rubber gasket. Load an excess of solution onto the glass slide, while avoiding contact with the rubber gasket.
Place the second glass slide on top of the slide gasket. To avoid air bubble generation, place one end of the slide down first and then, slowly lower the other end. Attach binder clips to each side of the glass chamber to hold it close.
Irradiate the samples for the desired length of time, using the UV laser with the 365 nanometer filter at an intensity of 200 watts per square meter. Remove the binder clips and open the chamber. Next, pipette 25 microliters of the irradiated nanocarrier SDS samples onto micro-centrifuge tubes.
Incubate the solutions in the dark at room temperature for 30 minutes. Add five microliters of the loading buffer solution to 25 microliters of each nanocarrier SDS sample. Incubate the samples in the dark at room temperature for 10 minutes.
Then, load 30 microliters of each nanocarrier SDS sample into a 2%agarose gel, pre-stained with ethidium bromide. Run the gel in the dark at 100 volts for 30 minutes. Image the gel, using a gel imaging system with ethidium bromide filters.
Save the gel image files and proceed to bend intensity quantification as described in the text protocol. Remove the pieces of paper from a double sided adhesive spacer to expose the adhesive surface. Attach the spacer to a glass cover slip.
Pipette the nanocarrier SDS solution onto the cover slip. Then, place a pre-washed glass slide on top of the cover slip with the hole from the adhesive spacer, centered around the sample. Push on the glass slide to ensure that the glass slide and cover slip are well attached and form a seal.
Use a confocal microscope with a 40 times water immersive, aperchromal objective, having a numerical aperture of 1.2 for FCS measurements. Use the appropriate excitation laser channel to collect at least 30 measurements of 10 seconds each, per sample. Then, input the siRNA release data from FCS into a kinetic model to predict gene silencing responses, as detailed in the text protocol.
To prepare in-vitro siRNA delivery, first add two milliliters of cell suspension to each well of the six well plate. Let the cells adhere and recover for 24 hours in the incubator. After washing the cells with phosphate buffered saline, prepare them for transvection by adding 1.5 milliliters of serum and antibiotic free transvection medium.
Next, add 25 microliters of nanocarrier solution, containing 30 picomoles of siRNA tp each well. Gently pipette the medium up and down to mix. Place the cells in the incubator for three hours.
Remove the transvection medium and wash each well with PBS. Then, add one milliliter of supplemented growth medium and place the cells in the incubator to recover for 30 minutes. To prepare the cells for treatment with a photo stimulus, remove the supplemented growth medium.
Then, add one milliliter of transvection medium to each well. After calibrating the UV laser as described in the text protocol, place the cells on a hot plate set to 37 degree celsius. Remove the cover from the six well plate.
Irradiate the cells from above the plate for the desired time, using the UV laser with a 365 nanometer filter at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium. Place the cells in incubator until further analysis.
To prepare a photo mask that completely blocks 365 nanometer light and minimizes reflections, first cut, punch and/or machine the desired shape into the photo mask. Glue the photo mask to the bottom of the six well plate, with the pattern centered onto the well containing the cells and the anti-reflective side facing the plate. Set up two ring stands approximately 25 meters apart and detach a platform to each ring stand, so that the platforms are of equal height.
Suspend the cell plate between the two stands by resting the plate on top of the platforms. Irradiate the cells from below the sample for the desired time, using a UV laser with a 365 nanometer filter, at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium.
Then, place the cells in incubator to recover for at least 24 hours. Image the cells using fluorescence microscopy as described previously. Shown here, is an example of simple kinetic modeling of gene silencing dynamics.
The model was run to predict the concentrations of mRNA, protein and siRNA, as a function of time following photo-induced siRNA release. The relative amounts of siRNA released following different dosages of light, were inputted as the initial siRNA concentration in the kinetic model. The resulting model predictions for the protein expression levels were an agreement with the experimentally determined expression levels obtained through western blotting.
For visualization, GFP expressing cells were treated with GFP targeting siRNA and an anular photo mask was placed on the plate prior to irradiation to generate a circular pattern. Cells that were protected from the light exhibited fluorescence intensity that were indistinguishable from control samples not treated with siRNA and light. However, nearly all cells within the circular pattern exhibited no GFP expression, indicating efficient siRNA release and gene knockdown.
Moreover, the use of light is a trigger enabled gene expression, to be controlled on cellular length scales. In summary, this method enables the rapid determination of irradiation conditions to spatio-temporally control siRNA release and predict gene silencing a priori. Employing this protocol will help elucidate the structure function relationships governing nanocarrier stability and efficacy.
This technique may enable applications in regenerative medicine that requires spatio-temporal control over gene expression. These formulations also are well suited to the treatment of localized disorders such as skin cancer, as well as dermatological disorders.
