Formulating mRNA-Loaded Nanoparticles for Vaccination Purposes

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)

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.

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