Technologies for investigating the physiological barriers to efficient lipid nanoparticle-siRNA delivery

Abstract

Small interfering RNA (siRNA) therapeutics have advanced from bench to clinical trials in recent years, along with new tools developed to enable detection of siRNA delivered at the organ, cell, and subcellular levels. Preclinical models of siRNA delivery have benefitted from methodologies such as stem-loop quantitative polymerase chain reaction, histological in situ immunofluorescent staining, endosomal escape assay, and RNA-induced silencing complex loading assay. These technologies have accelerated the detection and optimization of siRNA platforms to overcome the challenges associated with delivering therapeutic oligonucleotides to the cytosol of specific target cells. This review focuses on the methodologies and their application in the biodistribution of siRNA delivered by lipid nanoparticles.

Keywords: LNP; QWBA; RISC; biodistribution; delivery; endosomal escape; immunofluorescence staining; intravital imaging; lipid nanoparticle; siRNA; stem-loop PCR.

Figure 1.

(A) The components of a lipid nanoparticle (LNP): a cationic lipid (CLinDMA, 30–50 mol%), a PEGylated lipid (DMG-PEG_2K, 2–6 mol%), cholesterol (20–50 mol%), and possibly a helper lipid. (B) Cryo–electron microscopy (Cryo-EM) image of LNP to show the shape, size, and uniformity of lipid-siRNA nanoparticles. Bar = 100 nm. (With permission from Matthew Haas and Ye Zhang, unpublished data.)

Figure 2.

Barriers to lipid nanoparticle (LNP)–mediated small interfering RNA (siRNA) delivery and assay development. (A) Delivery is the key challenge to be addressed before an siRNA therapeutic can be fully realized. Successful delivery must enable bypass of multiple biological barriers, including blocking blood nuclease digestion of siRNA by chemical modification of the siRNA duplex, improving target-specific tissue biodistribution and cellular uptake, increasing cell binding via electrostatic interaction of the LNP and cell membrane or ligand and cell surface receptor, enhancing receptor-mediated endocytosis, increasing the efficiency of unpacking siRNA from the lipid nanoparticle delivery vehicle, enhancing the endosomal escape of siRNA to the cytosol, and improving siRNA loading into the RNA-induced silencing complex (RISC) in the cytosol for target mRNA cleavage (courtesy of Matt Stanton and Steve Colletti). (B) Tissue distribution of LNP-siRNA-Cy5 in liver, spleen, lung, kidney, and KB3 (a nasopharyngeal carcinoma cell line) tumor xenograft. LNP-siRNA labeled with Cy5 was intravenously injected into a mouse at 3 mg/kg. Different tissues were collected 2 hr postdosing and cryosections were analyzed by epifluorescence microscopy. All images were taken at ×20 magnification. siRNA-Cy5 (purple) mainly distributed to the liver, the red pulp of the spleen, and the proximal tubules of the kidney but not much to the lung. Phalloidin (green) outlines cell membrane and CD68 (green) stains macrophages. In the tumor section, siRNA-Cy5 (red) was mainly located at CD31-stained vessels and adjacent KB3 tumor cells but did not penetrate very far. (C) Dextran endosomal escape assay measuring the endosomal release of dextran (green) from punctate endosomes into diffused signals in the cytosol in HepG2 cells in vitro (with permission from Eileen Walsh and Bonnie Howell, unpublished data). Bars = 50 µm (B: kidney); 20 µm (B: liver and lung); 20 µm (B: spleen and tumor); 10 µm (C).

Figure 3.

Quantitative whole-body autoradiography (QWBA) of ¹⁴C-labeled lipid nanoparticle (LNP)–small interfering RNA (siRNA) in rats. (A) The chemical structure of ¹⁴C-labeled lipid nanoparticle delivery system LNP201-siRNA. (B) The biodistribution of ¹⁴C-LNP201-siRNA was examined at various time points in rats following intravenous administration of 3 mg/kg siRNA and 100 µCi/kg; the autoradioluminograph image at 1 hr postdose is illustrated. Dark areas represent ¹⁴C-related material. For QWBA, at the terminal time point, rats were frozen rapidly in a dry ice/hexane bath after whole blood was sampled. Rats were embedded in 2% carboxymethycellulose, and 40-µm-thick sagittal cryo-sections were taken at various levels. Sections were exposed to phosphor imaging plates for 4 days and imaged with a Fuji FLA-5100 phosphor imager (Fujifilm; Tokyo, Japan). ¹⁴C-LNP-siRNA was delivered to the liver, spleen, lung, and kidney 1 hr after intravenous dosing, suggesting a quick and broad distribution of LNP201-siRNA after systemic administration. (With permission from Marissa Vavrek and Kenneth Koeplinger, unpublished data.)