Formulating mRNA-Loaded Nanoparticles for Vaccination Purposes

Abstract

Source: Fornaguera, C. et al., Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes. J. Vis. Exp. (2021)

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 (~…

Materials

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