Deliver mRNA with LNP, transfection is so easy!

18 Jun 2025 by AntBio

Background Introduction

Naked RNA is a negatively charged hydrophilic macromolecule, which, due to electrostatic repulsion by the cell membrane, struggles to enter cells and is easily decomposed by ubiquitous RNases. Therefore, it requires a protective shell to penetrate the cell membrane. The cell membrane is primarily composed of lipids, and lipid vesicles can be utilized to encapsulate RNA, facilitating its passage through the cell membrane and release into the cytoplasm. The vesicle should be a positively charged lipid to bind with the negatively charged RNA.

However, vesicles composed of permanent cationic lipids pose a risk of cytotoxicity due to their electrostatic interactions with the negatively charged cell membrane. A lipid structure that can respond to the acidic environment of endosomes by becoming positively charged is necessary.

Lipid Nanoparticle Structure

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

Lipid Nanoparticle Delivery Mechanism

As an mRNA delivery system, LNPs operate through several steps to ensure the safe and efficient intracellular delivery and protein expression of mRNA. Ionizable cationic lipids in LNPs are crucial. They carry a positive charge (below their pKa) to load negatively charged RNA during LNP self-assembly, are neutral at physiological pH to minimize toxicity, and regain a positive charge for endosomal escape as the endosomal pH decreases. Cholesterol and PEG-lipid variants modulate LNP physical properties, nanostructure, and efficacy. Zwitterionic phospholipids serve as "helper lipids." LNPs play a key role in mRNA protection, intracellular delivery, and immune response activation.

LNP Transfection Protocol

The following transfection protocol is based on a single well in a 6-well plate. For other culture formats, refer to Table 1.

1. Prepare mRNA Pre-mix Mix 2.5 µg of mRNA (at a concentration of 1 µg/µL) with 60 µL of Buffer.

2. Form mRNA-easyLNP Complexes Combine 62.5 µL of the mRNA pre-mix with 62.5 µL of easyLNP nano-suspension. Vortex for 2-3 seconds to mix thoroughly.

3. Add to Cells Add 125 µL of the mRNA-easyLNP complex to the cells. Gently rock the culture to ensure even distribution.

4. Analysis Timing Analyze the cells after 24-48 hours of incubation.

Table 1: mRNA Transfection Guide Based on Cell Culture Vessel Used per Well

mRNA Transfection Steps	Reagent	96-Well Plate	24-Well Plate	6-Well Plate
（1） Prepare 40ng/µL mRNA Pre-mix	Buffer (assuming mRNA stock concentration is 1µg/µL)	2.4μL	12μL	60μL
（1） Prepare 40ng/µL mRNA Pre-mix	mRNA	0.1μg	0.5μg	2.5μg
（2）Prepare mRNA-easyLNP Complex	easyLNP Nanosuspension	2.5μL	12.5μL	62.5μL
（2）Prepare mRNA-easyLNP Complex	mRNA Pre-mix	2.5μL	12.5μL	62.5μL
（3）Add to Cells	mRNA-easyLNP Complex	5μL	25μL	125μL

Experimental Diagram:

Product Recommendation:

Transfection Method	Catalog Number	Product Name	Specification
Cell	abs60339	mRNA Transfection Reagent	1mL
	abs60620	easyLNP™ mRNA Transfection Kit	1.5mL/3mL/5mL
	abs60621	easyLNP™ Plus Universal RNA Transfection Kit	1.5mL/3mL/5mL
	abs60322	Lipofect5000 Lipid Transfection Reagent	0.5mL/1.0mL
	abs60255	Particle DNA Transfection Kit (for adherent cells)	0.5mL/1.0mL/1.5mL
	abs60256	PM Transfection Reagent for Cell Nucleus DNA	0.1mL/0.5mL/1.0mL/1.5mL
	abs60327	SP Suspension Cell Nucleus DNA Transfection Kit	0.1mL/0.5mL/1.0mL/1.5mL
	abs60369	RF 293 Cell DNA Transfection Kit	0.5mL/1.5mL
	abs60259	Nucleic Acid Transfection Reagent	0.5mL/1.0mL/1.5mL
	abs60324	PM Transfection Reagent for Cell Nucleus Acid	0.5mL/1.0mL/1.5mL
	abs60325	SP Suspension Cell Nucleus Transfection Kit	0.5mL/1.0mL/1.5mL
	abs60252	Oligonucleotide Transfection Reagent	0.5mL/1.0mL/1.5mL
	abs60253	PM Transfection Reagent for Cell Small Nucleic Acid	0.1mL/0.5mL/1.0mL/1.5mL
	abs60254	SP Suspension Cell Nucleus Transfection Kit	0.1mL/0.5mL/1.0mL/1.5mL
	abs60317	Nucleic Acid Co-transfection Reagent	0.5mL/1.0mL/1.5mL
	abs7993	50nm Magnetic Bead Transfection Reagent	100uL
Transfection Enhancer	abs42025397	Polyethyleneimine Solution	1mL
	abs42085824	Polybrene	5g
	abs42085198	Protamine Sulfate	5g/25g
In Vivo (Animal Models)	abs60622	easyLNP™ mRNA In Vivo Transfection Kit	0.3mL/0.5mL/1mL
In Vivo (Animal Models)	abs60623	easyLNP™ mRNA In Vivo Transfection Kit	0.3mL/0.5mL/1mL
Nucleic Acid Transfection (In Vivo)	abs60258	Elite Fect-I Small Animal In Vivo Transfection Reagent (for mice)	3vials/6vials

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