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Article

Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method

  • Authors:
    • Kumi Kawano
    • Yoshiyuki Hattori
  • View Affiliations / Copyright

    Affiliations: Department of Molecular Pharmaceutics, Hoshi University, Shinagawa, Tokyo 142‑8501, Japan
  • Article Number: 33
    |
    Published online on: January 14, 2026
       https://doi.org/10.3892/br.2026.2106
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Abstract

Cationic liposomes are useful carriers for delivering small interfering RNA (siRNA). In our previous study, a simple and efficient modified ethanol injection (MEI) method was developed for the preparation of cationic siRNA lipoplexes. However, non‑specific interactions between cationic lipoplexes and biological components, including erythrocytes, induce aggregation, and thus, siRNA is not delivered to the target site after intravenous injection. In the present study, an anionic polymer coating was applied to cationic lipoplexes prepared using the MEI method, and their gene knockdown effect and biodistribution in mice were evaluated. The gene knockdown effect of cationic lipoplexes was preserved after coating with anionic polymers [hyaluronan (HA), chondroitin sulfate (CS) and polyglutamic acid (PGA)], although anionic polymer‑coated lipoplexes showed lower cellular association than cationic lipoplexes. Coating of cationic lipoplexes with high‑molecular‑weight HA, CS and PGA reduced agglutination with erythrocytes. Following intravenous injection, CS‑ or PGA‑coated lipoplexes exhibited lower pulmonary accumulation than cationic lipoplexes, whereas hepatic accumulation of CS‑ or PGA‑coated lipoplexes increased. Collectively, cationic lipoplexes prepared using the MEI method were successfully coated with anionic polymers. Notably, CS or PGA coating of lipoplexes reduced non‑specific interactions with erythrocytes, and CS‑ or PGA‑coated lipoplexes could be potential vectors for in vivo siRNA delivery to the liver.

View Figures

Figure 1

Effect of anionic polymer coating of
siRNA lipoplexes on suppression of luciferase expression in
MCF-7-Luc cells. MCF-7-Luc cells were treated for 48 h with anionic
polymer-coated (A) LP-DOTAP or (B) LP-DDAB at a final Cont or Luc
siRNA concentration of 50 nM. Each value represents the mean ±
standard deviation (n=3). *P<0.05,
**P<0.01 compared with Cont siRNA. Cont, control; CS,
chondroitin sulfate; DDAB, dimethyldioctadecylammonium bromide;
DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate
salt; HA, hyaluronan; HA-H, high-molecular-weight HA; HA-L,
low-molecular-weight HA; HA-M, medium-molecular-weight HA; HA-UL,
ultra-low-molecular-weight HA; Luc, luciferase; LP, lipoplex; PGA,
polyglutamic acid; siRNA, small interfering RNA.

Figure 2

Cellular association of anionic
polymer-coated siRNA lipoplexes. MCF-7-Luc cells were treated for 3
h with anionic polymer-coated (A) LP-DOTAP or (B) LP-DDAB at a
final Cy5-siRNA concentration of 50 nM. The association between the
siRNA lipoplexes and cells was determined based on Cy5 fluorescence
using flow cytometry. Each value represents the mean ± standard
deviation (n=3). *P<0.05, **P<0.01
compared with LP-DOTAP or LP-DDAB. CS, chondroitin sulfate; DDAB,
dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; HA,
hyaluronan; HA-H, high-molecular-weight HA; HA-L,
low-molecular-weight HA; HA-M, medium-molecular-weight HA; HA-UL,
ultra-low-molecular-weight HA; LP, lipoplex; PGA, polyglutamic
acid; siRNA, small interfering RNA.

Figure 3

Effect of anionic polymer-coated
siRNA lipoplexes on cell viability. MCF-7-Luc cells were treated
for 24 h with anionic polymer-coated (A) LP-DOTAP or (B) LP-DDAB at
a final control siRNA concentration of 50 nM. Each value represents
the mean ± standard deviation (n=5-6). CS, chondroitin sulfate;
DDAB, dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; HA,
hyaluronan; HA-H, high-molecular-weight HA; HA-L,
low-molecular-weight HA; HA-M, medium-molecular-weight HA; HA-UL,
ultra-low-molecular-weight HA; LP, lipoplex; NS, not significant;
PGA, polyglutamic acid; siRNA, small interfering RNA.

Figure 4

Erythrocyte agglutination with
anionic polymer-coated siRNA lipoplexes. Anionic polymer-coated
LP-DOTAP or LP-DDAB with 2 µg control siRNA were added to
erythrocyte suspensions, and agglutination was observed by
microscopy. Scale bar, 200 µm. CS, chondroitin sulfate; DDAB,
dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; HA,
hyaluronan; HA-H, high-molecular-weight HA; HA-L,
low-molecular-weight HA; HA-M, medium-molecular-weight HA; HA-UL,
ultra-low-molecular-weight HA; LP, lipoplex; PGA, polyglutamic
acid; siRNA, small interfering RNA.

Figure 5

Biodistribution of siRNA in mice 1 h
after intravenous injection of anionic polymer-coated siRNA
lipoplexes. Fluorescence imaging of the tissues was performed 1 h
after injection of mice with anionic polymer-coated (A) LP-DOTAP or
(B) LP-DDAB with 10 µg Cy5-siRNA. Images were obtained from one
mouse for each siRNA lipoplex. The fluorescence intensity was
illustrated using a color-coded scale (red is the maximum, purple
is the minimum). cps, count per sec; CS, chondroitin sulfate; DDAB,
dimethyldioctadecylammonium bromide; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; HA,
hyaluronan; HA-H, high-molecular-weight HA; HA-L,
low-molecular-weight HA; HA-M, medium-molecular-weight HA; HA-UL,
ultra-low-molecular-weight HA; LP, lipoplex; PGA, polyglutamic
acid; siRNA, small interfering RNA.
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Copy and paste a formatted citation
Spandidos Publications style
Kawano K and Hattori Y: <p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>. Biomed Rep 24: 33, 2026.
APA
Kawano, K., & Hattori, Y. (2026). <p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>. Biomedical Reports, 24, 33. https://doi.org/10.3892/br.2026.2106
MLA
Kawano, K., Hattori, Y."<p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>". Biomedical Reports 24.3 (2026): 33.
Chicago
Kawano, K., Hattori, Y."<p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>". Biomedical Reports 24, no. 3 (2026): 33. https://doi.org/10.3892/br.2026.2106
Copy and paste a formatted citation
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Spandidos Publications style
Kawano K and Hattori Y: <p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>. Biomed Rep 24: 33, 2026.
APA
Kawano, K., & Hattori, Y. (2026). <p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>. Biomedical Reports, 24, 33. https://doi.org/10.3892/br.2026.2106
MLA
Kawano, K., Hattori, Y."<p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>". Biomedical Reports 24.3 (2026): 33.
Chicago
Kawano, K., Hattori, Y."<p>Evaluation of anionic polymer‑coated siRNA lipoplexes prepared using a modified ethanol injection method</p>". Biomedical Reports 24, no. 3 (2026): 33. https://doi.org/10.3892/br.2026.2106
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