Favorable biodistribution, specific targeting and conditional endosomal escape of RNA nanoparticles in cancer therapy

Abstract

The past decades have witnessed the successful transition of several nanotechnology platforms into the clinical trials. However, specific delivery of therapeutics to tumors is hindered by several barriers including cancer recognition and tissue penetration, particle heterogeneity and aggregation, and unfavorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNA nanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many of the aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles. Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can be tuned with relative ease for investigating the in vivo behavior of nanoparticles upon systemic injection. The size, shape, and surface chemistry, especially hydrophobic modifications, exert significant impacts on the in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometry and high homogeneity have been demonstrated to specifically target tumor cells while avoiding accumulation in healthy vital organs after systemic injection. RNA nanoparticles were proven to deliver therapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Although the release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regression in multiple animal models, the efficiency of endosomal escape for siRNA delivery needs further improvement. This review focuses on the advances and perspectives of this promising RNA nanotechnology platform for cancer targeting and therapy.

Keywords: Biodistribution; Cancer therapy; Nanobiotechnology; RNA nanotechnology; pRNA-3WJ motif; phi29 motor pRNA.

Figure 1

Sequence of (A) pRNA and (B) pRNA-3WJ motif. Three domains of pRNA including right-/left-hand loop, foot and central 3WJ provide three different strategies for nanoparticle construction. 3WJ is composed of three RNA oligomers in black (a_3WJ), red (b_3WJ) and blue (c_3WJ). Helical segments are represented as H1, H2 and H3. (C) AFM image of the extended pRNA-3WJ nanoparticles. (D) Sequence of re-engineered pRNA-X motif. The nucleotides in red indicate new sequences that were added to the original pRNA sequences in black. The core is composed of four RNA oligos (denoted a, b, c and d). Helical segments are represented as H1, H2, H3 and H4. Figure adapted and reproduced with permission from: (A) Ref. [14] © 2013 RNA Society; (B) Ref. [9] © 2011 Nature Publishing Group; (C) Ref. [18] © 2015 American Chemical Society; (D) Ref [48] © 2012 Elsevier.

Figure 2

Construction and characterization of multivalent RNA nanoparticles with tunable shape and stoichiometry. Three different strategies were developed for RNA nanoparticle design: (1–5) Loop-loop interactions; (6–10) Foot-to-foot interactions using palindrome sequences; (11–26) Branch grafting based on pRNA-3WJ motif. Figure adapted and reproduced with permission from ref. [14] © 2013 RNA Society, ref. [52] © 2014 Oxford University Press and ref. [71] © 2015 Elsevier.

Figure 3

Design and characterization of RNA nanoparticles of different sizes. (A) Crystal structure of pRNA-3WJ. (B) AFM images of small (5 nm), medium (10 nm), and large (20 nm) RNA squares derived from pRNA-3WJ. (C) Cryo-EM reconstruction of small (8 nm) and large (17 nm) RNA tetrahedrons derived from pRNA-3WJ. (D) Cryo-EM reconstruction of small (5 nm) and large (10 nm) RNA nanoprisms derived from pRNA-3WJ. Figure adapted and reproduced with permission from: (A) Ref. [70] © 2013 Oxford University Press; (B) Ref. [53] © 2014 American Chemical Society; (C) Ref. [67] © 2016 John Wiley & Sons, Inc. (D) Ref. [68] © 2016 John Wiley & Sons, Inc.

Figure 4

RNA nanoparticles with tunable (A) thermal stability, (B) chemical stability and (C) mechanical stability. Figure adapted and reproduced with permission from: (A) Ref. [53] © 2014 American Chemical Society; (B) Ref. [53] © 2014 American Chemical Society; (C) Ref. [82] © AAAS.

Figure 5

Specific targeting of RNA nanoparticles to various xenografts, metastasis, and ocular tissues. Whole-body and organ imaging of (A) Brain cancer; (B) Breast cancer; (C) Gastric cancer; (D) Prostate cancer; (E) Colorectal cancer; (F) Head & neck cancer; (G) CRC metastasis in the lung, liver, lymph node and bones. Green: GFP-cancer cells; blue: DAPI; red: RNA nanoparticles. (H) Ocular fluorescence imaging of the eye after subconjunctival injection of pRNA-X. Figure adapted and reproduced with permission from: (A) Ref. [21] © 2015 Impact Journals; (B) Ref. [18] © 2015 American Chemical Society; (C) Ref. [19] © 2015 Macmillan Publishers Limited, part of Springer Nature; (D) Ref. [20] © 2016 Elsevier; (E) Ref. [49] © 2015 American Chemical Society; (F) Ref. [14] © 2013 RNA Society; (G) Ref. [49] © 2015 American Chemical Society; (H) Ref. [110] © 2013 Springer US.

Figure 6

Specific targeting and accumulation of pRNA-X nanoparticles to KB cells xenograft. (A) Whole-body imaging at different time points. (B) Internal organ imaging. Figure adapted and reproduced with permission from ref [48] © 2012 Elsevier.

Figure 7

Evaluation of the therapeutic effects of 3WJ-EGFRapt/anti-miRNA21. (A) Fluorescence images showing specific targeting and retention in TNBC tumors 8 h post-injection. (B) Histological assay of breast tumor frozen cross sections showing binding and internalization. Blue: nuclei; red: RNA nanoparticle. (C) Tumor growth curve over the course of 5 injections. (*P<0.05, **P<0.01, error bars indicate SEM). (D) Tumor inhibition over the course of 5 injections. (E) Western blot and (F) Real-time PCR showing the down-regulation of miRNA21 after treatment, resulting in up-regulation of two target genes PTEN and PDCD4. RQ: relative quantification. Figures adapted with permission from ref [18] © 2015 American Chemical Society.