Efficient mRNA Delivery with mRNA Lipoplexes Prepared Using a Modified Ethanol Injection Method

Abstract

Messenger RNA (mRNA)-based therapies are a novel class of therapeutics used in vaccination and protein replacement therapies for monogenic diseases. Previously, we developed a modified ethanol injection (MEI) method for small interfering RNA (siRNA) transfection, in which cationic liposome/siRNA complexes (siRNA lipoplexes) were prepared by mixing a lipid-ethanol solution with a siRNA solution. In this study, we applied the MEI method to prepare mRNA lipoplexes and evaluated the in vitro and in vivo protein expression efficiencies. We selected six cationic lipids and three neutral helper lipids to generate 18 mRNA lipoplexes. These were composed of cationic lipids, neutral helper lipids, and polyethylene glycol-cholesteryl ether (PEG-Chol). Among them, mRNA lipoplexes containing N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16) or 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl) amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12) with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and PEG-Chol exhibited high protein expression in cells. Furthermore, mRNA lipoplexes composed of DC-1-16, DOPE, and PEG-Chol exhibited high protein expression in the lungs and spleen of mice after systemic injection and induced high antigen-specific IgG1 levels upon immunization. These results suggest that the MEI method can potentially increase the efficiency of mRNA transfection, both in vitro and in vivo.

Effect of vorinostat on protein expression in vitro and in vivo following mRNA lipoplex administration

“…The thin film was hydrated by adding sterilized water at 60˚C, followed by vortexing and subsequent sonication for 10 min at 42 kHz, 100 W and at room temperature in a bath-type sonicator (Bransonic 2510J-MTH; Branson Ultrasonics Corporation). To prepare the mRNA lipoplexes, the cationic liposome suspension was combined with the mRNA solution at a charge ratio (+:-) of 4:1, as described in a previous study ( 8 ), thorough vortexing for 10 sec and subsequently left undisturbed at room temperature for 15 min. Particle size distribution and ζ-potential of the mRNA lipoplexes were assessed using a light-scattering photometer (ELS-Z2; Otsuka Electronics Co., Ltd.), as previously described ( 15 ).…”

“…Therefore, the development of efficient and safe mRNA delivery systems is imperative ( 5-7 ). Previously, we demonstrated that cationic liposomes, comprising N -hexadecyl- N , N -dimethylhexadecan-1-aminium bromide (DC-1-16), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and poly(ethylene glycol) cholesteryl ether (PEG-Chol), induce high protein expression in the lungs and spleen following intravenous administration of mRNA/cationic liposome complexes (mRNA lipoplexes) ( 8 ).…”

Non-cellular immunotherapies in pediatric central nervous system tumors

“…Additionally, DNA-based vaccines advance personalized medicine by being specifically manufactured from a patient’s own tumor DNA on a case-by-case basis ( 80 , 102 , 105 , 106 ). There is an abundance of pre-clinical data showing the efficacy of NA vaccines in generating an immune response, however, the use of NA vaccines in pediatric patients is still in the infancy stages, and an exciting prospect in cancer vaccine technology ( 102 – 107 ).…”

Editorial for Special Issue: “Liposomal and Lipid-Based Drug Delivery Systems and Vaccines”

“…The original research papers published in this Special Issue present new perspectives of the development of liposomal small-molecule drugs designed for different indications and modes of administration [ 1 , 2 , 3 ], anti-cancer immunogenic liposomes [ 4 ], lipid-based formulations with siRNA [ 5 ], and mRNA for effective transfection in vitro [ 6 , 7 ] and in vivo [ 8 ]. One review [ 9 ] considers the rational design of liposomal gene delivery systems that target the folic acid receptor.…”

“…A study on mRNA-transfection presented by the researchers from Hoshi University, Tokyo, Japan, is one of the most advanced contributions to this Special Issue [ 8 ]. These researchers proposed a modified ethanol injection method to prepare mRNA lipoplexes, which are formed by simply mixing a lipid–ethanol solution with an mRNA solution.…”