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. 2020 Feb 20;11(1):972.
doi: 10.1038/s41467-020-14780-5.

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

Sijin Guo #  1   2   3   4 , Mario Vieweger #  1   2 , Kaiming Zhang #  5 , Hongran Yin  1   2   3   4 , Hongzhi Wang  1   2   3   4 , Xin Li  1   2   3   4 , Shanshan Li  5 , Shuiying Hu  2   3 , Alex Sparreboom  2   3 , B Mark Evers  6 , Yizhou Dong  1   2   3   4 , Wah Chiu  5   7 , Peixuan Guo  8   9   10   11
Affiliations

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy

Sijin Guo et al. Nat Commun. .

Abstract

Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.

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Conflict of interest statement

P.G. is the consultant of Oxford Nanopore Technologies, the co-founder of Shenzhen P&Z Bio-medical Co. Ltd, and the co-founder of the ExonanoRNA, LLC and its subsidiary ExonanoRNA (Foshan) Biomedicine Co., Ltd.

Figures

Fig. 1
Fig. 1. Design and construction of thermostable RNA 4WJ-X nanostructure.
a Hypothetical 3D model of RNA 4WJ-X nanostructure (left), redesigned from pRNA-3WJ motif (right),. b Step-wise self-assembly of 4WJ-X, evaluated by native polyacrylamide gel electrophoresis (PAGE) (size marker: ultra-low range DNA ladder; M, D, and T indicate RNA monomer, dimer, and trimer, respectively). c Size comparison of 4WJ-X (purple) and pRNA-3WJ (pink) by dynamic light scattering (DLS) (mean ± SD of one size distribution). d Ta comparison of 4WJ-X (purple) and pRNA-3WJ (pink) in representative annealing curves, measured by real-time-PCR (RT-PCR) (n = 3 independent samples over three independent measurements). Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Conjugation of RNA-PTX and construction of 4WJ-X-24 PTXs nanoparticles.
a Schematic of RNA-6 PTXs chemical conjugation and 4WJ-X-24 PTXs self-assembly. b Conjugating six PTXs to an RNA, evaluated by denaturing PAGE. c HPLC chromatogram (absorbance 260 nm) with an inserted gel image of RNA-6 alkynes (blue) and RNA-6 PTXs (red). d Turbidity changes of PTX and RNA-6 PTXs in aqueous solution at two-fold serial dilution. e Step-wise self-assembly of 4WJ-X-24 PTXs and 4WJ-X-24 PTXs-EGFRapt, evaluated by native PAGE (M, D, and T indicate RNA monomer, dimer, and trimer conjugated with PTX, respectively). f Size comparison of 4WJ-X, 4WJ-X-24 PTXs, and PTX in aqueous solution by DLS (n = 3 independent samples, mean ± SD). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Comparison of 4WJ-X and 4WJ-X-24 PTXs nanoparticles by single-particle cryo-EM.
a Representative motion-corrected cryo-EM micrographs of 4WJ-X and b 4WJ-X-24 PTXs (scale bar: 500 Å). c Reference-free 2D class averages of 4WJ-X and d 4WJ-X-24 PTXs computed in Relion. e Gold standard Fourier shell correlation (FSC) plots to measure resolution at FSC = 0.143 for the 3D reconstruction of 4WJ-X and f 4WJ-X-24 PTXs. g 3D reconstructed cryo-EM maps of 4WJ-X and 4WJ-24 PTXs in four views, with a Supplementary Movie 1. A 3D design model is shown. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Thermodynamic and chemical stability of 4WJ-X-24 PTXs nanoparticles.
a Representative TGGE showing the Tm of 3WJ-10 PTXs (M = 20 nucleotides monomer) and 4WJ-X-24 PTXs (M = 40 nucleotides monomer) nanoparticles. b Quantitative calculation of Tm from the representative TGGE for 3WJ-10 PTXs and 4WJ-X-24 PTXs nanoparticles. c Ta comparison of 4WJ-X-24 PTXs (orange) and 3WJ-10 PTXs (green) nanoparticles in a representative annealing curve, measured by RT-PCR (n = 3 independent samples over three independent measurements). d Enzymatic stability curve of 4WJ-X nanoparticles after incubation in 50% FBS at 37 °C over time points. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. In vitro cell binding, cytotoxicity and apoptotic effects of 4WJ-X-24 PTXs nanoparticles.
a In vitro cell binding of 4WJ-X-24 PTXs-EGFRapt nanoparticles, shown by confocal microscopy (blue: nucleus; green: cytoskeleton; red: RNA nanoparticles. Scale bar: 100 μm for original images, and 20 μm for magnified image). b In vitro cytotoxicity study of 4WJ-X-24 PTXs-EGFRapt nanoparticles by MTT assay (n = 3 independent samples, mean ± SD). c In vitro apoptosis effect of 4WJ-X-24 PTXs-EGFRapt nanoparticles by propidium iodide (PI)/Annexin V-FITC dual staining and fluorescence-activated cell sorting (FACS) analysis (Q2 = Annexin V-FITC and PI positive, indicating cells in late apoptosis or already dead; Q3 = PI negative & Annexin V-FITC positive, indicating early apoptotic cells). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. In vivo biodistribution and tumor inhibition of 4WJ-X-24 PTXs nanoparticles.
a Representative organ images showing specific tumor targeting of Alexa Fluor 647 labeled 4WJ-X-EGFRapt nanoparticles 8 h post-injection into mice bearing MDA-MB-231 xenograft (T: tumor, H: heart, S: spleen, L: lung, K: kidney, and Li: liver; Color scale: radiant efficiency, [p s−1 cm−2 sr−1] [μW cm−2]−1). Very low radiant mark was revealed in the liver of 4WJ-X-EGFRapt treated sample. b Quantitative analysis of biodistribution in tumors and normal organs, quantified from the organ images. c Intravenous treatment of nude mice bearing orthotopic MDA-MB-231 xenografts with 4WJ-X-24 PTXs-EGFRapt nanoparticles (purple) and control groups (turquoise: 4WJ-X-24 PTXs, red: PTX, blue: PBS) every other day for a total of five injections (8 mg kg−1, PTX per body weight, indicated by arrows). Mice body weight was monitored during the time course of treatments (n = 5 biologically independent animals, statistics was calculated by two-tailed unpaired t-test presented as mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001; p = 0.038, 9.99 × 10−4, and 6 × 10−6 comparing 4WJ-X-24 PTXs-EGFRapt to PTX, 4WJ-X-24 PTXs, and PBS, respectively). d Representative images of breast cancer tumors harvested from mice after treatments (n = 5 biologically independent animals, statistics was calculated by two-tailed unpaired t-test presented as mean ± SD, *p < 0.05, ****p < 0.0001; p = 0.033 and 2.2 × 10−5 comparing 4WJ-X-24 PTXs-EGFRapt to 4WJ-X-24 PTXs, and PBS, respectively). Source data are provided as a Source Data file.
Fig. 7
Fig. 7. In vivo immunostimulation study of 4WJ-X-24 PTXs nanoparticles.
Evaluation of a IFN-α, b IFN-γ, c TNF-α, and d IL-6 secretion in mice after systemic injection of 4WJ-X-24 PTXs nanoparticles, evaluated by enzyme-linked immunosorbent assay (ELISA) (n = 3 biologically independent animals, statistics was calculated by two-tailed unpaired t-test presented as mean ± SD, p = 5.4 × 10−4, 0.013, and 5.9 × 10−3 comparing 4WJ-X-24 PTXs to PTX for IFN-γ, TNF-α, and IL-6, respectively). Source data are provided as a Source Data file.
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