Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Summary

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

Abstract

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Introduction

Vaccination has been heralded as one of the most efficient medical interventions for reducing the morbidity and mortality caused by infectious diseases¹. The importance of vaccines has been demonstrated since the outbreak of coronavirus disease 2019 (COVID-19). As opposed to the traditional concept of injecting inactivated or live-attenuated pathogens, state-of-the-art vaccine approaches, such as nucleic acid-based vaccines, concentrate on preserving the immune-stimulatory properties of the target pathogens while avoiding the potential safety issues associated with the conventional whole-microbial virus- or in bacteria-based vaccines. Both DNA-....

Protocol

1. In vitro transcription of chemically modified mRNA

NOTE: It is required to use nuclease-free tubes, reagents, glassware, pipette tips, etc., because RNases are ubiquitous in the environment, such as laboratory solutions, instrument surfaces, hair, skin, dust, etc. Clean the bench surfaces and pipettes thoroughly before use, and wear gloves to avoid RNase contamination.

1. Perform linearization of the DNA template.
   1. Synthesize the open reading frame (ORF) of Metridia luciferase (MetLuc) flanked by a T7 polymerase promoter, 5' untranslated region (UTR), 3'UTR, and poly (A) tails and clo....

Results

The recombinant plasmid was digested to produce the linearized DNA template (Figure 2A). Using the protocol described, The T7 in vitro transcription kit can produce up to 80-120 µg of uncapped MetLuc-mRNA per 20 µL reaction and 50-60 µg of capped MetLuc-mRNA per 100 µL reaction. When analyzed with electrophoresis, intact MetLuc-mRNA with high quality should show a single and clear band, as displayed in Figure 2B. Contaminants introduced into the reactio.......

Discussion

The protocol described here not only allows the cost-effective and rapid production of IVT mRNA vaccine formulations with defined properties, but it also offers the possibility to customize the PP-sNp formulation according to specific therapeutic purposes, such as gene therapy. In order to ensure the successful generation of IVT mRNA/PP-sNp, it is suggested to pay extra attention to some critical steps. When working with mRNA, always remember that RNase-free conditions should be maintained throughout the process because .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BamHI	Takara	1010
cap 1 capping system	Jinan	M082
Dendritic cell-line	Sigma	SCC142
DNA sequence	Genescript
Human bronchial epithelial cells	Sigma	SCC150
KpnI	Takara	1068
LP	Beyotime	C0533
Lithium chloride	APEXBio	B6083
Malvern Zetasizer Nano ZS90	Malvern	NB007605
Microfluidic chip	ZHONGXINQIHENG	Standard PDMS chip
Microplate readers	ThermoFisher	Varioskan lux
NanoDrop One	ThermoFisher	ND-ONE-W (A30221)
Nuclease-free water	ThermoFisher	AM9932
OptiMEM	Gibco	31985070
Penicillin-streptomycin	Gibco	15140122
Pseudouridine	APE×Bio	B7972
QIAprep Spin Miniprep Kit	Qiagen	27106
Quanti-Luc	InvivoGen	Rep-qlc2
RiboRuler High Range RNA Ladder	ThermoFisher	SM1821
RNase-free conical tube	Biosharp	BS-100-M
RPMI Medium 1640	ThermoFisher	C11875500BT
Syringe pump	Chemyx	Fusion 101
T7 transcription Kit	Jinan	E131