Assembly and Characterization of Polyelectrolyte Complex Micelles

This method describes the full path of the design, assembly, and characterization of polyelectrolyte complex micelles which are nanoparticles that are formed from the self-assembly of oppositely charged polymers. Some major challenges with polyelectrolyte self-assembly are avoiding kinetic traps and characterizing the nanoparticles. The salt annealing technique that we describe allows for the repeatable assembly of micelles with low dispersity in both size and shape and we describe characterization methods including light scattering, small angle x-ray scattering, and electron microscopy.

Delivering therapeutic nucleic acids is a longstanding challenge for nanomedicine. These polyelectrolyte complex micelles take advantage of the strong negative charge of the nucleic acid to sequester them in the core of the micelle where the neutral polymer corona protects them from nucleases and immune response. The assembly method should be applicable to any type of charged polymers.

We've tested them with several polyanions and polycations and the characterization method should be applicable to any self-assembled nanoparticles including surfactant nanoparticles and other hydrophobically-driven systems. Begin by incubating the oligonucleotide solution at 70 degrees Celsius for five minutes. After the incubation, cool it for 15 minutes at room temperature to thermally anneal the nucleic acids, then add 40 microliters of 20 millimolar charged concentration diblock copolymer.

Vortex the solution immediately and incubate it for five minutes at room temperature. To perform the salt anneal, add sodium chloride solution to the oligonucleotide solution for a final concentration of one molar and vortex it for 10 seconds at maximum speed. Incubate the mixture for 10 minutes at room temperature, then proceed with loading it into the dialysis cartridge.

Prior to loading, label cartridges with permanent marker and soak them in buffer for at least two minutes to hydrate the membranes. Remove the cap by twisting counterclockwise and load the sample using a gel loading pipette tip. Gently squeeze the membrane to remove excess air and replace the cap.

Put the cartridges into 1X PBS 0.5 molar sodium chloride dialysis bath making sure that they are floating with both membranes exposed to the bath. After 24 hours, transfer the cartridges into 1X PBS and soak it for an additional 24 hours. After the final dialysis, recover the sample by removing the cartridges from the bath, removing the cap and removing the sample with a gel loading pipette tip.

Place the sample into a clean 1.5 milliliter microcentrifuge tube and refrigerate it until ready to use. Prepare the sample in DLS instrument according to manuscript directions, then acquire data for at least one minute making sure that the count rate is constant over the entire acquisition time. Examine the autocorrelation data.

The long time baseline should be flat and the autocorrelation curve should be smooth with minimal scatter. Noise in the data can be improved by acquiring more data. To perform data reduction and analysis using Irena, start by importing micelle in background data sets.

Plot the sample and background together on a log-log scale and compute the sample to background ratio and verify the high Q asymptote. Compute the average ratio over this Q range and use the data manipulation macro to scale the background with the calculated ratio. Then plot the background subtracted signal over Q and save the data with a new name making sure to not overwrite the original data.

Open the modeling macro, then load and plot the background subtracted data. To find an approximate model for the external surface of the polyelectrolyte complex micelle, or PCM, select flow to moderate Q range in the data controls making sure to include oscillations if they are present. In model controls, select the first scattering population and make sure it is the only one in use.

Select size distribution for model, choose the desired distribution type, and select the form factor. This example is for a flexible cylinder which must be added manually under the user form factor. Download and add the flexible cylinder form factor, then input the function names and initial values for parameters one and two which correspond to the length of the cylinder and the Kuhn length respectively.

These cylinders are longer than can be resolved by SAXS so the cylinder length parameter is fixed at a large value. Set initial parameters for the search by entering values in the scale, mean size, and width fields. Then click calculate model to draw the resulting form factor.

Once reasonable parameters have been found, click fit model to perform a nonlinear least squares fit to the data. Next, model the scattering of the individual polymers within the PCM core. Adjust the data controls to select the Q range where excess scattering occurs which is typically in the moderate to high range.

Add a second scattering population and make sure it is the only one in use. Select unified level for the model, adjust the GDA factors G and RG to ensure that the model does not predict excessive scattering at low Q and use the fit PB between cursors macro to obtain an initial guess for these parameters. As for the form factor, perform a nonlinear fit for the unified level model.

If a diffraction peak is present, add a third model for the diffraction peak in the Q range of interest. Once approximate fit values are obtained for the individual scattering populations, turn on all three together and optimize the combined fit. Finally, check that each value remains physically reasonable and save the fit by selecting store in folder.

The result of this procedure should be a composite model that describes the small angle x-ray scattering data well over a large range of size scales. This protocol was used to design, assemble, and characterize nucleic acid polyelectrolyte complex micelles or PCMs. Micelle core size is primarily driven by the length of the charged block of the block copolymer and is largely independent of the length of the homopolymer.

Dynamic light scattering data was acquired for spherical PCMs formed from relatively long block copolymers in short single-stranded oligonucleotides. The autocorrelation function decayed to a flat value with the single time scale resulting in the single size peak in the repIS size distribution. Complex small angel x-ray scattering or SAXS intensity spectra can be accurately fit by combining models for the multiple spatial correlations that are present and multiangle light scattering can be used to extend scattering measurements to longer length scales.

PCMs of varying morphology can also be imaged with electron microscopy to verify that core radii and shape are consistent with the values obtained from fitting SAXS data. When fitting SAXS data, it's important to account for each scattering feature and to use complimentary methods like TEM to make sure you're using the correct form factor.