Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The current protocol is significant because it allows researchers to produce siRNA loaded nanoparticles, and to adequately characterize these nanoparticles to ensure their suitability for administration to animals prior to subsequent studies. The advantage of this approach is to produce nanoparticles that increase the bioavailability of siRNA and to produce nanoparticles that are neutrally charged and thus suitable for systemic administration. This technique is impactful because siRNA nanoparticles can be applied to treat a myriad of diseases necessitating systemic administration including cancer, cardiovascular disease, and autoimmune disorders, among others.

To begin, dissolve polymer in ethanol at a concentration of 33.3 milligrams per milliliter and stir to ensure dissolution. Then use a pipette to transfer polymer solution in 10 millimolar citric acid buffer to reach a concentration of 3.33 milligrams per milliliter. Add distilled water into a tube containing small interfering RNA to achieve a concentration of 50 micromolar.

Mix the polymer and small interfering RNA solutions thoroughly in a tube by pipetting up and down to achieve a nitrogen to phosphate ratio of 10. Place the tube at ambient temperature for 30 minutes. Then add one milliliter of 10 millimolar phosphate buffer at pH eight to the tube to achieve a concentration of 0.28 milligrams per milliliter and mix gently either by pipetting or inverting the tube.

To confirm that the final pH is neutral, around 7.2 to 7.5, pipette 10 microliters of the small interfering RNA nanoparticles solution onto the pH test strips. First, prepare a dynamic light scattering sample by filtering one milliliter of small interfering RNA nanoparticles at the concentration of 0.28 milligrams per milliliter through 0.45-micrometer pore size syringe filters into a square quartz or polystyrene cuvette. Then record size and surface charge measurements using a dynamic light scattering instrument according to the manufacturer's specifications.

To confirm the size and morphology of small interfering RNA nanoparticles using transmission electron microscopy first add five microliters of filtered small interfering RNA nanoparticle solution at 0.28 milligram per milliliter to each TEM grid. Place the grids at ambient temperature for 60 seconds. Blot dry with filter paper for three seconds.

Next, add five microliters of 3%uranyl acetate solution to each grid and incubate for 20 seconds. Blot dry for three seconds. Then place the grids in a desiccator to dry overnight before imaging under transmission electron microscopy.

To perform agarose gel retardation, first add two grams of electrophoresis-grade agarose powder to 100 milliliters of 1X Tris-acetate-EDTA buffer at pH eight in a beaker to produce 2%agarose gel. Stir to suspend the agarose. Place the beaker uncovered in a microwave to heat for one to three minutes until all agarose is dissolved.

Once cooled, add five microliters of ethidium bromide at the concentration of 10 milligrams per milliliter into the beaker and mix well. Then pour the agarose into a gel tray and place comb to produce wells. Let the gel dry for 30 minutes.

Carefully remove the comb to leave behind loading wells, and pour 1X Tris-acetate-EDTA buffer into the gel tray to fill to the maximum fill line. Then place two microliter aliquots of loading dye with no SDS or reducing agents on paraffin film for each small interfering RNA nanoparticle formulation at different nitrogen to phosphate ratios. Pipette to mix 10 microliters of small interfering RNA nanoparticle solution with loading dye on paraffin film.

Add the mixture to agarose gel wells. Plug the electrophoresis system to a power supply and run voltage source at 100 volts for 35 minutes or until samples have traversed 80%of gel length. Transfer the gel from the tray to a UV transilluminator and visualize the small interfering RNA bands on the UV transilluminator according to the manufacturer's specifications.

Determine the optimum nitrogen to phosphate ratio based on the disappearance of small interfering RNA bands at the bottom of the gel. To knock down model gene luciferase, first seed luciferase-expressing cells in a 96-well black walled plate with DMEM in 10%FBS at a density of 2, 000 cells per well. Place the plate into an incubator overnight to allow the cells to adhere.

In the morning remove media and add 10 microliters per well of small interfering RNA nanoparticles diluted into full serum media for a final small interfering RNA concentration of 100 nanomolar. Return the wells to the incubator for 24 hours to treat the cells with small interfering RNA nanoparticles. The next day, replace media with full serum media containing 150 micrograms per milliliter D-luciferin.

Incubate the cells at room temperature for five minutes before measuring luminescence on a plate reader. Then repeat the media refreshment twice with fresh full serum media free of D-luciferin, followed by incubation for 24 hours. Replace the media with full serum media containing 150 milligrams per milliliter D-luciferin and incubate at room temperature for five minutes.

Measure luminescence at the 48-hour time point. For longitudinal studies, leave lid on top of the well plate when outside of the biosafety cabinet to maintain cells under sterile conditions and continue the incubation after replacing luciferin-containing media with fresh full media. Dynamic light scattering measurements show that small interfering RNA nanoparticle one has an average diameter of 35 nanometers.

The presence of a single peak with narrow peak width indicates unimodal distribution and low polydispersity. TEM measurements confirm the size measurement from dynamic light scattering suggests the presence of a uniform population of small interfering RNA nanoparticles and revealed the spherical morphology of the small interfering RNA nanoparticles. Too high concentration of polymer in the preparation step resulted for small interfering RNA nanoparticle two in undesirable diameter of greater than 200 nanometers, a multimodal and polydispersed population, and aggregates in solution forming no distinct particle morphology.

Both small interfering RNA nanoparticles one and two display neutral surface charge indicated by near zero mean zeta potential values. Agarose gel retardation assay shows the disappearance of small interfering RNA bands at the gel bottom which indicates complexation of small interfering RNA to polymer. Polymer complexed small interfering RNA is unable to migrate through the gel and is thus visualized at the top of the gel nearby the loading wells.

As the nitrogen to phosphate ratio is increased small interfering RNA complexation increases as indicated by decreased intensity of the small interfering RNA band at the gel bottom. Bioactive luciferase small interfering RNA nanoparticle one exhibits significantly diminished luciferase activity when compared to scrambled control. In contrast, luciferase small interfering RNA nanoparticle two is not considered bioactive.

To produce consistent siRNA nanoparticles with desired physicochemical properties, one should use polymer solutions with appropriate concentrations and validate the pH of the buffer solutions at each step of the protocol. Using these methods in our research we've been able to produce siRNA nanoparticle-based therapies for previously undruggable cancer-causing genes and test the efficacy of these therapies in pre-clinical models of breast cancer.