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Article

Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes

  • Authors:
    • Ryohei Shimizu
    • Yoshiyuki Hattori
  • View Affiliations / Copyright

    Affiliations: Department of Molecular Pharmaceutics, Hoshi University, Shinagawa, Tokyo 142‑8501, Japan
  • Article Number: 239
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    Published online on: October 3, 2025
       https://doi.org/10.3892/etm.2025.12989
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Abstract

Recently, the development of messenger RNA (mRNA) therapeutics has received increased attention, and mRNA/cationic liposome complexes (mRNA lipoplexes) have been studied as effective mRNA delivery carriers. However, owing to the vast diversity of the mRNA and carrier components employed, establishing a comprehensive analysis of their efficacy remains a critical issue. Reverse transfection using lyophilized mRNA lipoplexes has the potential to simplify and automate the efficacy evaluation processes. The present study investigated the effects of cationic lipids in cationic liposomes and disaccharides as cryoprotectants during reverse transfection with lyophilized mRNA lipoplexes. Overall, five types of dialkyl or trialkyl cationic lipids were used to prepare cationic liposomes. The mRNA lipoplexes were lyophilized in the presence of trehalose or sucrose in multi‑well plates. An increase in the concentration of the disaccharide solution during the lyophilization of mRNA lipoplexes enhanced the transfection activity. Furthermore, mRNA lipoplexes lyophilized in 150 mM sucrose solution exhibited long‑term stability for up to 1 month. The transfection activity of mRNA lipoplexes composed of dialkyl cationic lipids was largely unaffected by lyophilization, whereas a significant reduction in transfection activity was observed for mRNA lipoplexes composed of trialkyl cationic lipids. These findings suggest that although dependent on the lipid type used for the preparation of cationic liposomes, the reverse transfection method using lyophilized mRNA lipoplexes has the potential to be applied for screening the transfection efficiency of mRNA lipoplexes and the function of proteins translated from mRNA.
View Figures

Figure 1

Structures of the lipid components of
liposomes used for mRNA lipoplexes. DDAB,
dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt;
DC-1-16,
N-hexadecyl-N,N-dimethylhexadecan-1-aminium
bromide; DC-6-14,
2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium
chloride; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl)
amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; DOPE,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
PEG1600-Chol, polyethylene glycol-cholesteryl ether.

Figure 2

Luciferase activity following reverse
transfection with FLuc mRNA lipoplexes. (A) non-lyophilized FLuc
mRNA lipoplexes containing each cationic lipid were reverse
transfected into HeLa cells and luciferase activity was measured 24
h after the transfection. (B) Lyophilized FLuc mRNA lipoplexes with
0-150 mM trehalose or sucrose were reverse transfected into HeLa
cells. Luciferase activity was measured 24 h after the
transfection. **P<0.01 and ***P<0.001
(trehalose vs. sucrose). Data are presented as mean ± standard
deviation (n=3 in each group). DDAB, dimethyldioctadecylammonium
bromide; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane methyl
sulfate salt; DC-1-16,
N-hexadecyl-N,N-dimethylhexadecan-1-aminium
bromide; DC-6-14,
2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium
chloride; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl)
amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; cps, count per sec.

Figure 3

Toxicity test of lyophilized mRNA
lipoplexes. FLuc mRNA lipoplexes containing each cationic lipid
were lyophilized with 150 mM trehalose or sucrose solution. The
lipoplexes were reverse transfected into HeLa cells and the cell
viability was measured using WST-8 assay 24 h after the
transfection. ***P<0.001 vs. UNT. Data are presented
as mean ± standard deviation (n=5 in each group). UNT, untreated
group; DDAB, dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt;
DC-1-16,
N-hexadecyl-N,N-dimethylhexadecan-1-aminium
bromide; DC-6-14,
2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium
chloride; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl)
amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide.

Figure 4

Analysis of transfection efficiency
and cellular uptake of the lyophilized mRNA lipoplexes. (A) EGFP
mRNA lipoplexes containing DC-1-16, DC-6-14 or TC-1-12 were reverse
transfected into HeLa cells. The percentage of EGFP positive cells
was measured 24 h after transfection. (B) Cy5-mRNA lipoplexes
containing DC-1-16, DC-6-14 or TC-1-12 were reverse transfected
into HeLa cells. The fluorescence intensity was measured 3 h after
transfection. Control, Group subjected to reverse transfection with
non-lyophilized mRNA lipoplexes. *P<0.05,
**P<0.01 and ***P<0.001. Data are
presented as mean ± standard deviation (n=3 in each group). EGFP,
enhanced green fluorescent protein; DC-1-16,
N-hexadecyl-N,N-dimethylhexadecan-1-aminium
bromide; DC-6-14,
2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium
chloride; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl)
amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide.

Figure 5

Luciferase activity following reverse
transfection with lyophilized FLuc mRNA lipoplexes after 1 month of
storage. FLuc mRNA lipoplexes containing each cationic lipid were
lyophilized with 150 mM trehalose or sucrose and stored for 1
month. The lipoplexes were reverse transfected into HeLa cells and
luciferase activity was measured 24 h after transfection.
*P<0.05 and **P<0.01. Data are
presented as mean ± standard deviation (n=3 in each group).
DC-1-16,
N-hexadecyl-N,N-dimethylhexadecan-1-aminium
bromide; DC-6-14,
2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium
chloride; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl)
amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; cps, count per sec.
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Copy and paste a formatted citation
Spandidos Publications style
Shimizu R and Hattori Y: Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes. Exp Ther Med 30: 239, 2025.
APA
Shimizu, R., & Hattori, Y. (2025). Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes. Experimental and Therapeutic Medicine, 30, 239. https://doi.org/10.3892/etm.2025.12989
MLA
Shimizu, R., Hattori, Y."Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes". Experimental and Therapeutic Medicine 30.6 (2025): 239.
Chicago
Shimizu, R., Hattori, Y."Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes". Experimental and Therapeutic Medicine 30, no. 6 (2025): 239. https://doi.org/10.3892/etm.2025.12989
Copy and paste a formatted citation
x
Spandidos Publications style
Shimizu R and Hattori Y: Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes. Exp Ther Med 30: 239, 2025.
APA
Shimizu, R., & Hattori, Y. (2025). Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes. Experimental and Therapeutic Medicine, 30, 239. https://doi.org/10.3892/etm.2025.12989
MLA
Shimizu, R., Hattori, Y."Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes". Experimental and Therapeutic Medicine 30.6 (2025): 239.
Chicago
Shimizu, R., Hattori, Y."Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes". Experimental and Therapeutic Medicine 30, no. 6 (2025): 239. https://doi.org/10.3892/etm.2025.12989
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