This article describes a method for preparing mRNA-polymer complexes, or polyplexes, which can be used as mRNA vaccine candidates. The process involves the interaction of positively charged polymers with negatively charged mRNA, leading to the formation of nanoparticles.
Begin with a frozen polymer solution containing positively charged polymers.
Thaw the polymer solution until it reaches room temperature.
Add a buffer and vortex for uniform mixing, generating a homogeneous solution.
Introduce target mRNA to the polymer solution in an equal ratio for optimum reaction.
Incubate to allow the positively charged polymers to interact with the negatively charged phosphate groups in the mRNA via electrostatic interactions.
This facilitates the incorporation of the mRNA within the polymer structure, forming nanoparticles of mRNA-polymer complexes or polyplexes.
Transfer these polyplexes to a tube containing RNase-free water.
Mix them and allow the precipitation of the polyplexes.
Introduce a buffer containing sucrose and freeze it. Sucrose acts as a cryoprotectant that maintains the structural integrity of the polyplexes.
Then, perform freeze-drying, to remove any traces of water.
Store it at a low temperature as an mRNA vaccine candidate for future use.
For polyplexes formation, thaw the previously prepared polymers C-6 peptide poly-beta amino esters and vortex the solution. After pipetting the polymer mix up and down, prepare a 12.5-millimolar solution in sodium acetate. Vortex the solution and wait for 10 minutes.
Next, prepare mRNA at 0.5 milligrams per milliliter and mix by pipetting. Vortex the polymer solution again. Then, mix the genetic material solution in the polymer solution in a one-to-one ratio in a microcentrifuge tube, and incubate the tube at 25 degrees Celsius for 30 minutes in a thermal block. After incubation, precipitate the mixture with one to two RNase-free water by adding the sample to a microcentrifuge tube containing the water.
Then, to include the excipients, add 20-millimolar HEPES and 4 percent sucrose solution in the same volume as the mixture of mRNA and poly-beta amino esters and mix by pipetting. For lyophilization, after freezing the polyplex solution at minus 80 degrees Celsius for one hour, perform the primary drying, then immediately store the polyplexes at minus 20 degrees Celsius to avoid rehydration.
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