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Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery 

  • Authors:
    • Yoshiyuki Hattori
    • Aya Kurihara
    • Mizuki Shinkawa
  • View Affiliations / Copyright

    Affiliations: Department of Molecular Pharmaceutics, Hoshi University, Tokyo 142‑8501, Japan
    Copyright: © Hattori et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 12
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    Published online on: November 12, 2025
       https://doi.org/10.3892/br.2025.2085
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Abstract

Our previous study introduced a modified ethanol injection (MEI) method for preparing positively charged small interfering RNA (siRNA) lipoplexes by mixing a lipid‑ethanol solution of cationic lipid, 1,2‑dioleoyl‑sn‑glycero‑3‑­phosphoethanolamine (DOPE), and poly(ethylene glycol) cholesteryl ether (PEG‑Chol) with siRNA in phosphate‑­buffered saline (PBS). This method was adapted in the current study to develop a two‑step MEI process for creating anionic siRNA lipoplexes. First, a lipid‑ethanol solution comprising one of four cationic lipids [1,2‑dioleoyl‑­3‑trimethylammonium‑propane methyl sulfate salt (DOTAP), dimethyldioctadecylammonium bromide (DDAB), 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)], DOPE and PEG‑Chol was combined with siRNA in PBS. Next, a lipid‑ethanol solution of cholesteryl hemisuccinate (CHS) and DOPE was added. The gene‑silencing activity of anionic siRNA lipoplexes was evaluated in human breast cancer (MCF‑7) and human cervical carcinoma (HeLa) cells. Additionally, their interactions with erythrocytes were investigated. Regardless of the cationic lipid used, adding the CHS and DOPE‑ethanol solution inverted the ζ‑potentials of all siRNA lipoplexes from positive to negative. Notably, TC‑1‑12‑based lipoplexes achieved strong gene silencing while minimizing interactions with erythrocytes. This study demonstrates the effectiveness of the two‑step MEI method for preparing anionic siRNA lipoplexes.
View Figures

Figure 1

Structures of cationic lipids, anionic
lipid, neutral lipid and PEG-lipid. DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; 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; CHS,
cholesteryl hemisuccinate; PEG-Chol, poly(ethylene glycol)
cholesteryl ether.

Figure 2

Schematic diagram of anionic siRNA
lipoplex preparation. To prepare cationic siRNA lipoplexes, a 100
µl PBS solution (pH 7.4) containing 50 pmol siRNA was rapidly added
to a lipid-ethanol solution of cationic lipid, DOPE and PEG-Chol at
a molar ratio of 49.5:49.5:1. To prepare anionic lipoplexes, the
cationic lipoplex suspension was added to a lipid-ethanol solution
of CHS and DOPE at a molar ratio of 1:1, achieving charge ratios
(+:-) of 1:1 or 2:1 for cationic lipid:CHS. siRNA, small
interfering RNA; DOPE,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PEG-Chol,
poly(ethylene glycol) cholesteryl ether; CHS, cholesteryl
hemisuccinate.

Figure 3

Luc activity suppression by cationic
and anionic siRNA lipoplexes. Cationic and anionic lipoplexes
containing Cont and Luc siRNAs were prepared using the modified
ethanol injection method. These lipoplexes were then added to (A)
MCF-7-Luc and (B) HeLa-Luc cells at a final siRNA concentration of
50 nM. Luc activity was measured 48 h post-transfection. Data are
represented as the mean + standard deviation (n=3).
*P<0.05, **P<0.01 and
***P<0.001 vs. Cont siRNA. Luc, luciferase; siRNA,
small interfering RNA; Cont, control; LP, liposome; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; CHS, cholesteryl hemisuccinate.

Figure 4

Cell viability after transfection
with cationic and anionic siRNA lipoplexes. Cationic and anionic
lipoplexes containing Cont siRNA were prepared by the modified
ethanol injection method and subsequently added to (A) MCF-7-Luc
and (B) HeLa-Luc cells at a final siRNA concentration of 50 nM.
Cell viability was assessed 48 h after transfection. Data are
represented as the mean + standard deviation (n=5).
*P<0.05, **P<0.01 and
***P<0.001 vs. untreated cells. Luc, luciferase;
siRNA, small interfering RNA; Cont, control; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; CHS, cholesteryl hemisuccinate.

Figure 5

Cellular uptake of siRNA by MCF-7-Luc
cells after transfection with cationic and anionic lipoplexes.
Cationic and anionic lipoplexes containing Cy5-siRNA were prepared
using the modified ethanol injection method and added to MCF-7-Luc
cells at a final siRNA concentration of 50 nM. As a control,
Cy5-siRNA solution was added to the MCF-7-Luc cells. Localization
of Cy5-siRNA (green) was observed at (A) 5 and (B) 24 h
post-incubation, respectively. Scale bar, 100 µm. siRNA, small
interfering RNA; Luc, luciferase; LP, liposome; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; CHS, cholesteryl hemisuccinate.

Figure 6

Cellular uptake of siRNA by MCF-7-Luc
cells after transfection with cationic and anionic lipoplexes. (A)
Cationic and (B) anionic lipoplexes containing AF555-siRNA were
prepared using the modified ethanol injection method and added to
MCF-7-Luc cells at a final siRNA concentration of 50 nM. As a
control, AF555-siRNA solution (free siRNA) was added to the
MCF-7-Luc cells. After 24 h, the cells were lysed, and the amount
of AF555-siRNA within the cells was measured using a fluorescence
plate reader. Data are represented as the mean + standard deviation
(n=3). ***P<0.001 vs. LP-TC112;
###P<0.001 vs. LP-TC112-2CHS. LP, liposome; siRNA,
small interfering RNA; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; CHS, cholesteryl hemisuccinate.

Figure 7

Hemolysis and erythrocyte
agglutination induced by cationic and anionic siRNA lipoplexes.
Cationic and anionic lipoplexes containing 50 pmol of Cont siRNA
were prepared using the modified ethanol injection method and
incubated with erythrocyte suspensions at 37˚C for 15 min. (A)
Erythrocyte agglutination after mixing with the siRNA lipoplexes.
Scale bar, 200 µm. (B) Hemolysis (%) calculated relative to the
absorbance of the hypotonic solution and presented as the mean +
standard deviation (n=3). Erythrocytes suspended in a hypotonic
solution (water) serve as a positive control for 100% hemolysis.
***P<0.001 vs. positive control. LP, liposome; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DDAB,
dimethyldioctadecylammonium bromide; DC-1-16,
N-hexadecyl-N, N-dimethylhexadecan-1-aminium
bromide; TC-1-12,
11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium
bromide; CHS, cholesteryl hemisuccinate.
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Copy and paste a formatted citation
Spandidos Publications style
Hattori Y, Kurihara A and Shinkawa M: Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;. Biomed Rep 24: 12, 2026.
APA
Hattori, Y., Kurihara, A., & Shinkawa, M. (2026). Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;. Biomedical Reports, 24, 12. https://doi.org/10.3892/br.2025.2085
MLA
Hattori, Y., Kurihara, A., Shinkawa, M."Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;". Biomedical Reports 24.1 (2026): 12.
Chicago
Hattori, Y., Kurihara, A., Shinkawa, M."Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;". Biomedical Reports 24, no. 1 (2026): 12. https://doi.org/10.3892/br.2025.2085
Copy and paste a formatted citation
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Spandidos Publications style
Hattori Y, Kurihara A and Shinkawa M: Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;. Biomed Rep 24: 12, 2026.
APA
Hattori, Y., Kurihara, A., & Shinkawa, M. (2026). Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;. Biomedical Reports, 24, 12. https://doi.org/10.3892/br.2025.2085
MLA
Hattori, Y., Kurihara, A., Shinkawa, M."Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;". Biomedical Reports 24.1 (2026): 12.
Chicago
Hattori, Y., Kurihara, A., Shinkawa, M."Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery&nbsp;". Biomedical Reports 24, no. 1 (2026): 12. https://doi.org/10.3892/br.2025.2085
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