LNP delivers mRNA, transfection So easy!

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August 19, 2025

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Background Introduction

Bare RNA is a negatively charged hydrophilic macromolecule. Because of the electrostatic repulsion of the cell membrane, it is difficult to enter the cell, and it is easily degraded quickly by the ubiquitous RNase, so it needs a protective shell to enter the cell. The composition of the cell membrane is mainly lipids, and lipid vesicles are used to encapsulate RNA across the cell membrane and release RNA into the cytoplasm. The vesicle should be a positively charged lipid to bind to negatively charged RNA.

However, vesicles composed of permanent cationic lipids can cause cytotoxicity due to electrostatic interaction with negatively charged cell membranes, and lipid structures can be positively charged molecules in response to the acidic environment of endosomes.

Lipid nanoparticle structure

LNPs typically consist of four key components: ionizable cationic lipids, phospholipids (zwitterionic lipids) (PL), cholesterol, and PEGylated lipids.

		Ionizable Cationic Lipids (40-60%) • Under low pH conditions, they transiently acquire a positive charge, forming reverse micelle complexes with negatively charged oligonucleotides (e.g., siRNA, mRNA) via electrostatic interactions. • They circumvent the toxicity issues associated with traditional cationic lipids and are a critical component of lipid nanoparticles for in vivo delivery.
		Phospholipids (8-12%) • Maintain the structural stability of the particles and promote fusion between nanoparticles and cell membranes (commonly neutral or zwitterionic phospholipids, such as DSPC, are used).
		Sterol Lipids (30-50%) • Enhance the structural integrity of the particles and assist in the fusion of nanoparticles with target cell membranes (e.g., cholesterol).
		PEGylated Lipids (1-2%) • Serve as "stealth lipids," reducing serum protein adsorption and particle aggregation, thereby prolonging circulation time in vivo. • Can be functionalized for targeted delivery.
		Carrier • Ionizable cationic lipids can efficiently encapsulate negatively charged oligonucleotides (e.g., siRNA, mRNA, plasmids).

Lipid nanoparticle delivery mechanism

Lipid nanoparticles (LNPs) act as delivery systems for mRNA, and their mechanism of action involves multiple steps, ensuring safe and efficient delivery of mRNA to target cells and protein expression. In LNPs, ionizable cationic lipid plays an important role because it can be positively charged below its Ka to mediate loading of negatively charged RNA during self-assembly into LNPs, uncharged at neutral pH to minimize toxicity, and positively charged again after cellular uptake to facilitate endosomal escape as endosomal pH decreases. Variants of cholesterol and PEG lipids have been shown to modulate LNP physical properties, nanostructure, and ultimate efficacy. Zwitterionic phospholipids are often referred to as "helper lipids". LNPs play a key role in protecting mRNA, promoting intracellular transmission, and activating immune responses.

LNP Transfection Experimental Steps

6-well plate single-well reference conditions are used in the transfection steps described below. For other culture forms, please refer to Table 1.

1. Preparation of mRNA premix

2.5 ug mRNA (concentration of 1 ug/uL) was mixed with 60 uL Buffer.

2. mRNA-easyLNP complex

Mix 62.5 uL of mRNA premix with 62.5 uL of easyLNP nanosuspension, vortex for 2-3s, and mix well.

3. Add cells

125 uL of the mRNA-easyLNP complex was added to the cells and gently shaken to disperse uniformly.

4. Analysis time

Analysis after 24-48 hours of culture.

Table 1 mRNA transfection fingers according to cell culture vessel used per well

mRNA cell transfection step	Reagent	96-well plate	24-well plate	6-well plate
(1) Preparation of 40ng/μL mRNA premix	Buffer (taking mRNA stock solution concentration of 1 μg/μL as an example)	2.4 μL	12 μL	60 μL
(1) Preparation of 40ng/μL mRNA premix	mRNA	0.1 μg	0.5 μg	2.5 μg
(2) Preparation of mRNA-easyLNP complex	easyLNP nanosuspension	2.5 μL	12.5 μL	62.5 μL
(2) Preparation of mRNA-easyLNP complex	mRNA premix	2.5 μL	12.5 μL	62.5 μL
(3) Adding cells	mRNA-easyLNP complex	5 μL	25 μL	125 μL

Experimental schematic diagram:

Product Recommendations:

Transfection mode	Item number	Product name	Specifications
Cells	abs60339	mRNA Transfection Reagent	1mL
	abs60620	easyLNP ™ mRNA immune cell transfection kit	1.5 mL/3mL/5mL
	abs60621	easyLNP ™ Plus universal RNA cell transfection kit	1.5 mL/3mL/5mL
	abs60322	Lipofect5000 plasmid transfection reagent	0.5 mL/1. 0 mL
	abs60255	Plasmid DNA Transfection Kit (Adherent Cell)	0.5 mL/1. 0mL/1. 5mL
	abs60256	PM Primary Cell Plasmid DNA Transfection Kit	0.1 mL/0. 5mL/1. 0mL/1. 5mL
	abs60327	SP suspension cell plasmid DNA transfection kit	0.1 mL/0. 5mL/1. 0mL/1. 5mL
	abs60369	RF 293 Cell DNA Transfection Kit	0.5 mL/1. 5 mL
	abs60259	Nucleic acid transfection kit	0.5 mL/1. 0mL/1. 5mL
	abs60324	PM Primary Cell Nucleic Acid Transfection Kit	0.5 mL/1. 0mL/1. 5mL
	abs60325	SP suspension cell nucleic acid transfection kit	0.5 mL/1. 0mL/1. 5mL
	abs60252	Small nucleic acid transfection reagent	0.5 mL/1. 0mL/1. 5mL
	abs60253	PM primary cell small nucleic acid transfection reagent	0.1 mL/0. 5mL/1. 0mL/1. 5mL
	abs60254	SP suspension cell small nucleic acid transfection reagent	0.1 mL/0. 5mL/1. 0mL/1. 5mL
	abs60317	Nucleic acid co-transfection reagent	0.5 mL/1. 0mL/1. 5mL
	abs7993	50nm magnetic transfection reagent	100uL
Transfection Enhancer	abs42025397	Polybrene solution	1mL
	abs42085824	Hemelonium bromide	5g
	abs42085198	Protamine sulfate	5g/25g
In animal body	abs60622	easyLNP ™ mRNA in vivo transfection kit (systemic action)	0.3 mL/0. 5mL/1mL
In animal body	abs60623	easyLNP ™ mRNA in vivo transfection kit (local action)	0.3 mL/0. 5mL/1mL
Nucleic acid transfection (in animals)	abs60258	Elite Fect-1 small nucleic acid biopsy transfection reagent (mouse transfection)	3/6

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