Formulating mRNA-Loaded Nanoparticles for Vaccination Purposes

1. Synthesis of poly(beta amino esters) (pBAE) polymer with end oligopeptides (OM-pBAE)

1. Polymerization of C6-pBAE
   1. Add 5-amino-1-pentanol (38 mmol; MW = 103.16 Da) 1-hexylamine (38 mmol; MW = 101.19 Da) into a round-bottom glass flask (100 mL). Then, add 1,4-butanediol diacrylate (82 mmol; MW = 198.22 Da).
   2. Pre-heat the silicone oil bath at 90 °C, place the round-bottom flask into the oil bath and stir the mixture with the aid of a magnetic stir bar overnight (~18 h). Then, take the product from the round-bottom flask and place it in the freezer at -20 °C.        
      NOTE: The product is in the form of a sticky powder and is taken out from the flask with the help of a spatula. It is essential to validate the structure of the obtained polymer through ¹H-NMR. NMR spectra were recorded in a 400 MHz instrument (see Table of Materials) using chloroform-d and D₂O as solvents. Around 10 mg of each poly(β-amino ester) were taken and dissolved in 1 mL of the deuterated solvent.
2. Reaction with peptides to obtain OM-pBAE
   1. Add 25 mL of 0.1 M HCl to selected peptides, e.g., peptide Cys-His-His-His with Trifluoroacetic acid (TFA, 200 mg), into a previously weighed 50 mL centrifuge tube that can stand freeze-drying and manually stir overnight to obtain a clear solution.
   2. Freeze the solution at -80 °C for 1 h and freeze-dry the resulting peptide hydrochloride.
      NOTE: Check whether the final weight corresponds to the theoretical value.
   3. Make a solution of C6-pBAE (0.031 mmol) in dimethyl sulfoxide. Also, make a solution of the peptide hydrochloride (0.078 mmol) in dimethyl sulfoxide.
   4. Mix the two solutions in a screw cap tube and screw on the cap. Stir the mixture solution in a water bath with a controlled temperature of 25 °C for 20 h with a magnetic stir bar.
   5. Add the mixture to 7:3 (v/v) diethyl ether/acetone. Centrifuge the resulting suspension at 25,000 x g at 4 °C to remove the solvent. Next, wash the solid with 7:3 (v/v) diethyl ether/acetone twice. Then, dry the product under a vacuum (<0.2 atm).
   6. Make a solution of 100 mg/mL of the product in dimethyl sulfoxide. The resulting product is named C6-peptide-pBAE. It is essential to validate the structure of the obtained polymer through ¹H-NMR to confirm the disappearance of the olefin signals associated with terminal acrylates. If not used, the polymer can be frozen at -20 °C.

2. Polyplexes formation

NOTE: All the procedures should be performed inside a conditioned room to maintain a constant temperature.

1. Thaw the polymers C6-peptide-pBAE and vortex the solution.
2. Pipette the polymer mix up and down and prepare a solution of 12.5 mM (V₁) in sodium acetate (NaAc). Then, vortex the mixture and wait for 10 min.
3. Prepare the messenger ribonucleic acid (mRNA) at 0.5 mg/mL and mix by pipetting (V₂).
   NOTE: It is crucial to avoid vortexing the mRNA.
4. Vortex the polymer mixture at the final concentration to achieve a homogeneous solution between the polymer stock in dimethyl sulfoxide (DMSO) and the acetate buffer.      
   NOTE: The final polymer concentration depends on the N/P (nitrogen to phosphate groups) ratio selected. The N/P ratio depends on each specific mRNA to be used. For enhanced green fluorescent protein (eGFP) encapsulation, for example, a 25:1 ratio was used, as previously reported.
5. Mix the genetic material solution and C6-peptidepBAE solution (25x of the mRNA concentration) in a ratio of 1:1 (V_i = V₁ + V₂).     
   NOTE: C6-peptide-pBAE is loaded in a microcentrifuge tube where the mRNA is added by pipetting up and down for mixing. Once prepared, the polyplex, the nucleic acid, and C6-peptide-pBAE concentrations are half diluted.
6. Incubate at 25 °C for 30 min in a thermal block. Precipitate with 1:2 RNase free water by adding the sample to a pre-loaded microcentrifuge tube with water.
7. Include the excipients. Add the same volume as the mixture of mRNA and pBAE (V_i) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20 mM and sucrose 4% by pipetting up and down. At this point, the sample has been diluted 3x.

3. Polyplexes lyophilization

1. Instantaneously, freeze at -80 °C freezer of the previous polyplex solution for 1 h.
2. Perform the primary drying by following the steps: (1) 1 h at -60 °C and 0.001 hPa; (2) 1 h at -40 °C and 0.0001 hPa; (3) 4 h at -20 °C and 0.0001 hPa; (4) 12 h at 5 °C and 0.0001 hPa.
3. Store at -20 °C immediately to avoid rehydration until use.