Ultrasound-Mediated Delivery of RNA to Colonic Mucosa of Live Mice

Abstract

Background & aims: It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice.

Methods: We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs ex vivo and mice in vivo. Colon tissues were collected and fluorescence was measured by confocal microscopy. We then evaluated whether ultrasound is effective in delivering small interfering (si)RNA to C57BL/6 mice with dextran sodium sulfate-induced colitis. Some mice were given siRNA against tumor necrosis factor (Tnf) mRNA for 6 days; colon tissues were collected and analyzed histologically and TNF protein levels measured by enzyme-linked immunosorbent assay. Feces were collected and assessed for consistency and occult bleeding. We delivered mRNA encoding firefly luciferase to colons of healthy C57BL/6 mice.

Results: Exposure of ex vivo pig colon tissues to 20 kHz ultrasound for 1 minute increased the level of delivery of 3 kDa dextran 7-fold compared with passive diffusion (P = .037); 40 kHz ultrasound application for 0.5 seconds increased the delivery 3.3-fold in living mice (P = .041). Confocal microscopy analyses of colon tissues from pigs revealed regions of punctuated fluorescent dextran signal, indicating intracellular delivery of macromolecules. In mice with colitis, ultrasound delivery of unencapsulated siRNA against Tnf mRNA reduced protein levels of TNF in colon tissues, compared with mice with colitis given siRNA against Tnf mRNA without ultrasound (P ≤ .014), and reduced features of inflammation (P ≤ 4.1 × 10^-5). Separately, colons of mice administered an mRNA encoding firefly luciferase with ultrasound and the D-luciferin substrate had levels of bioluminescence 11-fold greater than colons of mice given the mRNA alone (P = .0025). Ultrasound exposures of 40 kHz ultrasound for 0.5 seconds were well tolerated, even in mice with acute colitis.

Conclusions: Ultrasound can be used to deliver mRNAs and siRNAs to the colonic mucosa of mice and knock down expression of target mRNAs.

Keywords: Antisense Therapy; Inflammatory Bowel Disease; Ulcerative Colitis; Ultrasound-Mediated Gastrointestinal Drug Delivery.

Figure 1. Ultrasound-Mediated Delivery of 3 kDa Dextran Ex Vivo

(A) Ex vivo experimental setup, the Franz diffusion cell. A section of fresh GI tissue is shown sandwiched between a donor and receiver chamber. (B) Enhancement in the delivery of ¹⁴C-labeled inulin into GI tissue using 20 kHz, 60 kHz, or 1 MHz ultrasound compared to passive diffusion without ultrasound. The P-values were determined by one-way ANOVA with multiple comparisons. (C) Total fluorescent intensity of porcine colon segments immediately after administration of 3 kDa dextran tagged with Texas red with ultrasound or without ultrasound (Control). The P-value was determined by a two-tailed Student’s t-test. Representative multiphoton microscopy images at a fixed z-position within porcine colonic tissue after delivery of dextran (red) with ultrasound (D) and without ultrasound (E) followed by nuclear staining (blue). The scale bars in (Di, Ei) represent 100 µm and the scale bars in (Dii, Eii) represent 30 µm. Data (in B and C) are averages + 1SD.

Figure 2. Ultrasound-Mediated GI Delivery of 3 kDa Dextran In Vivo

(A) A custom-made 40 kHz ultrasound probe allowing for administration in the colon of mice. The protrusions enhance radial ultrasound activity. (B) Experimental setup depicting placement of an enema in the colon followed by ultrasound exposure with the custom probe shown in (A). Total fluorescent intensity (averages + 1SD) of the colon immediately after administration (C) of 3 kDa dextran tagged with Texas red with ultrasound or without ultrasound (Control) and two hours after administration (D). P-values determined by two-tailed Student’s t-tests. Representative multiphoton microscopy images of fixed murine colonic tissue after delivery of 3 kDa dextran tagged with Texas red with ultrasound (E) and without ultrasound (F) immediately and two hours after administration. The red channel and second harmonic are shown. The scale bars represent 500 µm.

Figure 3. Ultrasound-Mediated Delivery of siRNA in DSS Colitis

(A) DSS colitis induction and enema administration schedule. DSS was given for 7 consecutive days to induce acute colitis in mice. Concurrently, animals were administered enemas from day 1 through 6. Experimental groups consisted of either siRNA targeting Tnf mRNA in combination with ultrasound or siRNA targeting Tnf mRNA alone. (B) Total fecal score for animals with acute colitis receiving various enemas (n = 5 animals per group). * represents P < 0.021 for siRNA + Ultrasound compared to all other groups (determined by one-way ANOVA with multiple comparisons). (C) Histology scores of colonic tissue sections on Day 8. (D) TNF protein levels from colonic tissue biopsies normalized by total protein content on Day 8. ** represents P < 0.014 for both siRNA + Ultrasound groups compared to any other group (determined by one-way ANOVA with multiple comparisons). P-values (in C, D) determined by one-way ANOVA with multiple comparisons. Data (in B, C, D) are averages + 1SD.

Figure 4. Ultrasound-Mediated Delivery of RNA in the Colon

Radiance of dissected mouse colons (n = 5) 24 hours after rectal administration of mRNA alone or in combination with ultrasound in vivo. The median, 25^th, and 75^th percentiles are shown. The whiskers indicate the most extreme data points. The P-value was determined by a two-tailed Student’s t-tests.