The Effect of Size and Shape of RNA Nanoparticles on Biodistribution

Abstract

Drugs with ideal pharmacokinetic profile require long half-life but little organ accumulation. Generally, PK and organ accumulation are contradictory factors: smaller size leads to faster excretion and shorter half-lives and thus a lower tendency to reach targets; larger size leads to longer circulation but stronger organ accumulation that leads to toxicity. Organ accumulation has been reported to be size dependent due in large part to engulfing by macrophages. However, publications on the size effect are inconsistent because of complication by the effect of shape that varies from nanoparticle to nanoparticle. Unique to RNA nanotechnology, size could be tuned without a change in shape, resulting in a true size comparison. Here we investigated size effects using RNA squares of identical shape but varying size and shape effects using RNA triangles, squares, and pentagons of identical size but varying shape. We found that circulation time increased with increasing RNA nanoparticle size from 5-25 nm, which is the common size range of therapeutic RNA nanoparticles. Most particles were cleared from the body within 2 hr after systemic injection. Undetectable organ accumulation was found at any time for 5 nm particles. For 20 nm particles, weak signal was found after 24 hr, while accumulation in tumor was strongest during the entire study.

Keywords: RNA Nanotechnology; RNA nanoparticles; RNA nanostructure; bacteriophage phi29; motor pRNA; nanobiotechnology; pRNA 3WJ motif; phi29 DNA packaging motor; phi29 pRNA; viral DNA packaging.

Figure 1

RNA Nanoparticle Design (A) Structures of the 5, 10, and 20 nm 2′F nanosquares. (B) Structures of 10 nm RNA triangle, square, and pentagon.

Figure 2

Physical Properties of RNA Nanoparticles (A) Assembly of 2′F RNA nanoparticles. (B) DLS size summary of RNA nanoparticles. (C) DLS graphs of same-shape, different-size RNA nanosquares. (D) DLS graphs of same-size, different-shape RNA polygons. PENT, pentagon; SQR, square; TRI, triangle; ULR.

Figure 3

In Vivo Biodistribution of Different-Size RNA Nanoparticles Time-course fluorescence images of 5, 10, and 20 nm 2′F nanosquares. SQR, square.

Figure 4

Organ Images of 5, 10, and 20 nm 2′F Nanosquares Fluorescent organ images of diverse-size RNA nanosquares after 12 and 24 hr of circulation. H, heart; K, kidneys; Li, liver; Lu, lung; S, spleen; T, tumors.

Figure 5

In Vivo Biodistribution of Different-Shape RNA Nanoparticles Time-course fluorescence images of RNA triangle, square, and pentagon. H, heart; K, kidneys; Li, liver; Lu, lung; S, spleen; T, tumors.

Figure 6

Serum Assays (A) Gel images of serum degradation of 5, 10, and 20 nm 2′F nanosquares. Different sizes of squares result in different intensities seen by gel band visualization. (B) Plots of gel band intensity from serum degradation gels of 5, 10, and 20 nm 2′F nanosquares. (C) Representative gel of serum binding experiments. (D) Equilibrium serum binding concentrations for 2′F RNA nanoparticles. Error bars indicate SD from three independent experiments. PENT, pentagon; SQR, square; TRI, triangle.