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. 2015 Oct 27;9(10):9731-40.
doi: 10.1021/acsnano.5b02471. Epub 2015 Sep 21.

Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology

Dan Shu, Hui Li, Yi Shu, Gaofeng Xiong, William E Carson 3rd  1 , Farzin Haque, Ren Xu, Peixuan Guo
Affiliations

Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology

Dan Shu et al. ACS Nano. .

Abstract

MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; "active" targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.

Keywords: EGFR RNA aptamer; RNA nanotechnology; miRNA; three-way junction; triple negative breast cancer.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Peixuan Guo is the cofounder of Biomotor and RNA Nanotechnology Development Corp. Ltd..

Figures

Figure 1
Figure 1
Characterization and introduction of the system for pRNA-3WJ nanoparticle construction. (A) Sequence of phi29 pRNA-3WJ core. (B) 3D model of arm-extended RNA nanoparticles using 3WJ as scaffold. (C) Atomic force microscopy (AFM) image of the nanoparticle in Figure 1B. (D) Size of the 3WJ core determined by dynamic light scattering (DLS). (E) Zeta potential of the 3WJ core.
Figure 2
Figure 2
Design and physicochemical characterization of 3WJ-EGFRapt/anti-miR-21 nanoparticles. (A) 2D sequence of the nanoparticle harboring three functional modules: EGFR RNA aptamer for targeted delivery, anti-miR-21 LNA for therapy, and Alexa-647 dye for imaging. (B) Native PAGE showing stepwise highly efficient assembly of the RNA nanoparticle. (C) DLS measurements showing the hydrodynamic size. (D) Zeta potential. (E) Serum stability assay. (F) Apparent Tm extracted from temperature gradient gel electrophoresis (TGGE, insert).
Figure 3
Figure 3
Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles in vitro. (A) Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cytoplasm; blue: nuclei; and red: RNA nanoparticles. (B) Flow cytometry assay showing the binding to MDA-MB-231 cells. (C) Dual-luciferase assay demonstrating in vitro delivery of anti-miR-21 LNA into MDA-MB-231 cells. (D) qRT-PCR assay depicting the effect of miR-21 knockdown on target gene expression level of PTEN and PDCD4 after treatment. RQ: relative quantification. (E) Caspase-3 assay showing the cellular apoptotic effects of MDA-MB-231 cells after treatment.
Figure 4
Figure 4
Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles using orthotopic TNBC mouse model. (A) Tumor inhibition over the course of 5 injections. The end point luminescence indicates the tumor volume. (B) Tumor growth curve over the course of 5 injections. (*P < 0.05, **P < 0.01, error bars indicate SEM). (C) Fluorescence images showing specific targeting and retention in TNBC tumors 8 h postinjection. (D) Histological assay of breast tumor frozen cross sections (10 μm thick) by fluorescence confocal microscopy showing binding and internalization. Blue: nuclei; red: RNA nanoparticle. (E) Real-time PCR at the mRNA level and (F) Western blot at the protein level showing the down-regulation of miR-21 after treatment, resulting in up-regulation of two target genes PTEN and PDCD4. Lamin A/C was internal control. RQ: relative quantification. (G) Immunohistochemistry assay showing inhibition of tumor cell growth after treatment, using Ki67 as indicator of tumor cell proliferation, and active caspase-3 as indicator of tumor cell apoptosis.
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