Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest.
Here, University of Oxford researchers evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 μL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics.
Structure and Sequence of Cholesterol-Conjugated siRNAs and Characterization of EVs after Optimized Incubation with Cholesterol-Conjugated siRNA
(A) Structure of cholesterol-conjugated siRNA (cc-siRNA) showing the 5′ TEG cholesterol, 2′-deoxy-2-fluoro pyrimidines, 19 bp duplex region and phosphorothioate linkages in the 2 bp overhangs. (B) Sequence of human antigen R (HuR) siRNAs. Sequences are written in the 5′ to 3′ direction. (C) Size distribution histogram (n = 3) of EVs from Neuro2A cells (N2A EVs) alone or following co-incubation with cc-siRNA and washing (N2A EVs + cc-siRNA), as determined by Nanoparticle tracking analysis (NTA). (D) Table displaying the mean and mode particle size and total number of particles secreted from a 15 cm dish of Neuro2A cells (n = 3) alone (N2A EVs) or following co-incubation with cc-siRNA and washing (N2A EVs + cc-siRNA), as determined by NTA. (E) Western blot probed for the exosomal markers Alix and Tsg101 and the endoplasmic reticulum marker calnexin showing cell lysates (N2A CL), washed EVs (N2A EVs), and washed EVs that had been co-incubated with cc-siRNA (N2A EVs + cc-siRNA). chol, cholesterol; fC 2′-deoxy-2′-fluoro cytidine; fU, 2′-deoxy-2′-fluoro uridine; “ps,” phosphorothioate linkage.