In vitro and in vivo transfections using siRNA lipoplexes prepared by mixing siRNAs with a lipid-ethanol solution

Hattori, Yoshiyuki; Saito, Hiromu; Nakamura, Kayo; Yamanaka, Akihiro; Tang, Min; Ozaki, Kei-ichi

doi:10.1016/j.jddst.2022.103635

Cited by 3 publications

(3 citation statements)

References 27 publications

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“…An advantage of the MEI method for preparing mRNA lipoplexes is the production of homogeneous liposomes without sonication or dialysis [26]. In the present study, we prepared monodisperse mRNA lipoplexes (<0.20 PDI) for LP-DC-1-16/DOPE or LP-TC-1-12/DOPE by simply mixing lipid-ethanol solution and PBS containing mRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…A lipid-ethanol solution was prepared by dissolving cationic lipid (DOTAP, DDAB, DC-1-16, DC-1-14, DC-6-14, or TC-1-12), neutral helper lipid (DOPE, DOPC, or Chol), and PEG-Chol at a molar ratio of 49.5:49.5:1 in ethanol (Table 1) (2 mg/mL for cationic lipids; for example, 2 mg TC-1-12, 1.53 mg DOPE, and 0.08 mg PEG-Chol were dissolved in 1 mL ethanol) as previously reported [26]. For in vitro transfection, 0.5 µL of 1 mg/mL mRNA solution was transferred into a 1.5 mL tube containing 100 µL of PBS (pH 7.4), and the obtained solution was rapidly added to the lipid-ethanol solution (2.4 µL, 1.6 µL, 1.8 µL, 2.0 µL, 2.1 µL, and 3 µL for DOTAP, DC-1-14, DC-1-16, DDAB, DC-6-14, and TC-1-12 formulation) in another 1.5 mL tube at a charge ratio (+:-) of 4:1, based on previous reports [27,28]; subsequently, the mixture was vortex-mixed for 10 s.…”

Section: Preparation Of Mrna Lipoplexes For In Vitro Transfectionmentioning

confidence: 99%

“…Previously, we developed a simple technique, known as the modified ethanol injection (MEI) method, for preparing small interfering RNA (siRNA) lipoplexes by rapidly mixing a lipid-ethanol solution with phosphate-buffered saline (PBS) containing siRNAs [26]. We demonstrated that the siRNA lipoplexes prepared using this method exhibited high gene knockdown activity both in vitro and in vivo.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Mrna Lipoplexes For In Vitro Transfectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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Evaluation of mRNA lipoplexes prepared using modified ethanol injection method as a tumour vaccine

Hattori,

Tang,

Sato

et al. 2024

Journal of Drug Targeting

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Nano-Encapsulation and Conjugation Applied in the Development of Lipid Nanoparticles Delivering Nucleic Acid Materials to Enable Gene Therapies

Dinh,

Mahon,

Yan

2024

Applied Nano

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Nano-encapsulation and conjugation are the main strategies employed for drug delivery. Nanoparticles help improve encapsulation and targeting efficiency, thus optimizing therapeutic efficacy. Through nanoparticle technology, replacement of a defective gene or delivery of a new gene into a patient’s genome has become possible. Lipid nanoparticles (LNPs) loaded with genetic materials are designed to be delivered to specific target sites to enable gene therapy. The lipid shells protect the fragile genetic materials from degradation, then successfully release the payload inside of the cells, where it can integrate into the patient’s genome and subsequently express the protein of interest. This review focuses on the development of LNPs and nano-pharmaceutical techniques for improving the potency of gene therapies, reducing toxicities, targeting specific cells, and releasing genetic materials to achieve therapeutic effects. In addition, we discuss preparation techniques, encapsulation efficiency, and the effects of conjugation on the efficacy of LNPs in delivering nucleic acid materials.

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In vitro and in vivo transfections using siRNA lipoplexes prepared by mixing siRNAs with a lipid-ethanol solution

Cited by 3 publications

References 27 publications

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

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

Evaluation of mRNA lipoplexes prepared using modified ethanol injection method as a tumour vaccine

Nano-Encapsulation and Conjugation Applied in the Development of Lipid Nanoparticles Delivering Nucleic Acid Materials to Enable Gene Therapies

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