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. 2018 Jun 6;26(6):1520-1528.
doi: 10.1016/j.ymthe.2018.03.019. Epub 2018 Apr 4.

Hydrophobicity of Lipid-Conjugated siRNAs Predicts Productive Loading to Small Extracellular Vesicles

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Free PMC article

Hydrophobicity of Lipid-Conjugated siRNAs Predicts Productive Loading to Small Extracellular Vesicles

Annabelle Biscans et al. Mol Ther. .
Free PMC article

Abstract

Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic RNA, but efficient loading of therapeutic RNA remains a challenge. We have recently shown that the attachment of cholesterol to small interfering RNAs (siRNAs) enables efficient and productive loading into sEVs. Here, we systematically explore the ability of lipid conjugates-fatty acids, sterols, and vitamins-to load siRNAs into sEVs and support gene silencing in primary neurons. Hydrophobicity of the conjugated siRNAs defined loading efficiency and the silencing activity of siRNA-sEVs complexes. Vitamin-E-conjugated siRNA supported the best loading into sEVs and productive RNA delivery to neurons.

Keywords: hydrophobicity; lipid-conjugated siRNA; small extracellular vesicles.

Figures

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Figure 1
Figure 1
Cholesterol-Conjugated hsiRNAs Load into sEVs (A) Representation of sEV membrane loaded with cholesterol-conjugated hsiRNAs. Cholesterol is the driving force for efficient loading of siRNA into sEVs. (B) Schematic of hydrophobically modified siRNAs (hsiRNAs) is shown.
Figure 2
Figure 2
The Chemical Compositions of Lipid Conjugates Significantly Affect hsiRNA Hydrophobicity (A) Library of lipophilic moieties attached to hsiRNAsHtt. (B) HPLC retention time of lipid-conjugated Cy3-hsiRNAHtt sense strands is shown. C18, buffer A, 0.1 M triethylammonium acetate in water; buffer B, acetonitrile; gradient, 0%–100% in B in 15 min; temperature, 60°C; flow, 1 mL/min.
Figure 3
Figure 3
The Number of hsiRNAs Loaded into sEVs Depends on the Hydrophobicity of the Conjugate (A) Ultracentrifugation of conjugated Cy3-hsiRNAs incubated without sEV (left) showing absence of pellet and after co-incubation of Cy3-hsiRNAs and sEVs (right) showing formation of pellet. Representative pictures are shown. (B) Cy3-hsiRNA accumulation in pellet following co-incubation of hsiRNAs and sEVs with varying hsiRNA:sEV ratio is shown (n = 3; mean ± SEM for the last point). (C) Exponential relationship between loading efficiency and hydrophobicity of conjugated hsiRNA is shown (n = 3; mean ± SEM). (D) Linear correlation between the surface charge of hsiRNA-loaded sEVs and hsiRNA loading efficiency (n = 2; mean ± SEM).
Figure 4
Figure 4
Silencing Activity of hsiRNA-Loaded sEVs Correlates with the Loading Efficiency of hsiRNAs (A) Htt mRNA levels in primary mouse neurons incubated with increasing concentrations of hsiRNAHtt-loaded sEVs (sEV:hsiRNA ratio, 1:25,000) for one week. Htt mRNA levels were normalized to Hprt (hypoxanthine-guanine phosphoribosyl transferase) and presented as percent of untreated control (n = 3; mean ± SEM). UNT, untreated. (B) Correlation between IC50 of hsiRNA-loaded sEVs and loading efficiency of hsiRNAs is shown (n = 2).

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