Overview
This video demonstrates a method for generating an mRNA vaccine candidate using polymeric nanoparticles. The cationic polymer interacts with negatively charged mRNA, forming polyplexes. The formation of polyplexes stabilizes the mRNA, making it a potential candidate for future mRNA vaccines in immunotherapeutic applications.
Protocol
1. Synthesis of poly(beta amino esters) (pBAE) polymer with end oligopeptides (OM-pBAE)
- Polymerization of C6-pBAE
- 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).
- 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.
- Reaction with peptides to obtain OM-pBAE
- 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.
- 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. - 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.
- 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.
- 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).
- 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.
- Thaw the polymers C6-peptide-pBAE and vortex the solution.
- 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.
- 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. - 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. - Mix the genetic material solution and C6-peptidepBAE solution (25x of the mRNA concentration) in a ratio of 1:1 (Vi = 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. - 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.
- Include the excipients. Add the same volume as the mixture of mRNA and pBAE (Vi) 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
- Instantaneously, freeze at -80 °C freezer of the previous polyplex solution for 1 h.
- 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.
- Store at -20 °C immediately to avoid rehydration until use.
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Materials
Name | Company | Catalog Number | Comments |
1,4-butanediol diacrylate | Sigma Aldrich | 123048 | |
1-hexylamine | Sigma Aldrich | 219703 | |
5-amino-1-pentanol | Sigma Aldrich | 411744 | |
Acetone | Panreac | 141007 | |
Chlor hydroxhyde | Panreac | 181023 | |
Chloroform-d | Sigma Aldrich | 151823 | |
Cys-His-His-His peptide | Ontores | Custom | |
Cys-Lys-Lys-Lys peptide | Ontores | Custom | |
D2O | Sigma Aldrich | 151882 | |
DEPC reagent for Rnase free water | Sigma Aldrich | D5758 | This reagent is important to treat MilliQ water to remove any RNases of the buffers |
Diethyl ether | Panreac | 212770 | |
Dimethyl sulfoxide | Sigma Aldrich | 276855 | |
HEPES | Sigma Aldrich | H3375 | |
mRNA EGFP | TriLink Technologies | L-7601 | |
RiboGreen kit | ThermoFisher | R11490 | |
Sodium acetate | Sigma Aldrich | 71196 | |
Sucrose | Sigma Aldrich | S0389 | |
Trifluoroacetic acid | Sigma Aldrich | 302031 |