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. 2021 Jan 7;19(1):13.
doi: 10.1186/s12951-020-00758-4.

Development of targeted therapy therapeutics to sensitize triple-negative breast cancer chemosensitivity utilizing bacteriophage phi29 derived packaging RNA

Long Zhang  1   2 , Chaofeng Mu  3 , Tinghong Zhang  4   5 , Dejun Yang  1   2 , Chenou Wang  1 , Qiong Chen  1 , Lin Tang  1 , Luhui Fan  3 , Cong Liu  3 , Jianliang Shen  6   7 , Huaqiong Li  8   9
Affiliations

Development of targeted therapy therapeutics to sensitize triple-negative breast cancer chemosensitivity utilizing bacteriophage phi29 derived packaging RNA

Long Zhang et al. J Nanobiotechnology. .

Abstract

Background: To date, triple-negative breast cancer (TNBC) treatment options are limited because of the loss of target receptors and, as a result, are only managed with chemotherapy. What is worse is that TNBC is frequently developing resistance to chemotherapy. By using small interfering RNA (siRNA)-based therapeutics, our recent work demonstrated X-box-binding protein 1 (XBP1) was linked to human epidermal growth factor receptor 2 positive (HER2+) breast cancer development and chemoresistance. Given the instability, off-target effects, net negative charge, and hydrophobicity of siRNA in vivo utilization and clinical transformation, its use in treatment is hampered. Thus, the development of a siRNA-based drug delivery system (DDS) with ultra-stability and specificity is necessary to address the predicament of siRNA delivery.

Results: Here, we assembled RNase resistant RNA nanoparticles (NPs) based on the 3WJ structure from Phi29 DNA packaging motor. To improved targeted therapy and sensitize TNBC to chemotherapy, the RNA NPs were equipped with an epidermal growth factor receptor (EGFR) targeting aptamer and XBP1 siRNA. We found our RNA NPs could deplete XBP1 expression and suppress tumor growth after intravenous administration. Meanwhile, RNA NPs treatment could promote sensitization to chemotherapy and impede angiogenesis in vivo.

Conclusions: The results further demonstrate that our RNA NPs could serve as an effective and promising platform not only for siRNA delivery but also for chemotherapy-resistant TNBC therapy.

Keywords: Chemoresistance; RNA nanoparticles; TNBC; XBP1; siRNA.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Characterizations of pRNA-EGFRapt-siXBP1 NPs. a Scheme of pRNA-EGFRapt-siXBP1NPs. b Native PAGE showing stepwise highly efficient assembly of the NPs. c Atomic force microscopy (AFM) images of pRNA nanoparticles. d Stability analyze by 8% native PAGE gel electrophoresis after RNase A and10% FBS-supplemented DMEM medium treatments for the indicated time at 37 °C. e Tm value of pRNA-EGFRapt-siXBP1 NPs determined by SYBR Green assay. f DLS measurements showing the hydrodynamic size. g Zeta potential
Fig. 2
Fig. 2
pRNA-EGFRapt-siXBP1 NPs cell uptake, specific binding to tumor cells in vitro. a, b Cell binding is assessed by flow cytometry. c Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cell membrane; blue: nuclei; and red: RNA nanoparticles. Scale bars, 10 µm. d Representative gating strategies in mixture of MDA-MB-231 and MCF10A cells. e Cell specific binding is assessed by flow cytometry. f pRNA-EGFRapt-siXBP1 NPs binding efficiency were evaluated. g MFI of Cy5 positive cells
Fig. 3
Fig. 3
pRNA-EGFRapt-siXBP1 NPs silenced XBP1 expression, sensitizes MDA-MB-231 cells to doxorubicin treatment in vitro, reduces cell viability, impairs mammospheres forming ability, but not alters cell apoptosis and cell cycle. a RT-PCR result of XBP1 gene expression in MDA-MB-231 cells after treated with 50 nM NPs. (mean ± s.d., n = 3). ***, p < 0.001. b Western blot analysis of XBP1s and XBP1u expression in MDA-MB-231cells after pRNA-EGFRapt-siXBP1 NPs treatment for 72 h. c Flow cytometry analysis of MDA-MB-231 cells cell cycle alteration after treated by pRNA-EGFRapt-siXBP1 or pRNA-EGFRapt-siScr control, respectively. (mean ± s.d., n = 3). d Cell apoptosis determined by annexin V and 7AAD staining. e Cell viability analysis of MDA-MB-231 cells after treated by NPs or NPs plus dox for 72 h. (mean ± s.d., n = 5–7). ***, p < 0.001. f Quantification of soft agar colony formation in different groups. (mean ± s.d., n = 3). **, p < 0.01. **
Fig. 4
Fig. 4
pRNA-EGFRapt-siXBP1 NPs specifically bind to tumor cells and efficiently silence XBP1 expression in vivo. a Pharmacokinetic study of pRNA-EGFRapt-siXBP1. b Quantitative RT-PCR analysis of XBP1 expression in MDA-MB-231 xenograft tumor. Data are presented relative to β-actin. n = 3. c Western blot analysis of XBP1s and XBP1u expression in MDA-MB-231 tumor mouse. d Biodistribution of Cy5-labeled pRNA-EGFRapt-siXBP1 NPs 16 h post intravenously injection. e Confocal microscopic images of tumor sections from mice injected intravenously with pRNA-EGFRapt-siXBP1 or control NPs without EGFR aptamer. Red, Cy5-labelled pRNA; green, Alexa488-wheat germ agglutinin-labelled cell membrane; blue, nuclear staining with DAPI. Scale bars, 20 µm and 7 µm, respectively
Fig. 5
Fig. 5
pRNA-EGFRapt-siXBP1 NPs treated inhibits TNBC in vivo. a Tumor growth in different group treated TNBC mice. (mean ± s.d., n = 5). *, p < 0.05. b, c CD31immunostaining of TNBC tumor. Scale bars, 50 µm. d Quantitative RT-PCR analysis of VEGFA, PDK1, GLUT1, and DDIT4 expression in TNBC tumor after pRNA-EGFRapt-siScr plus dox and pRNA-EGFRapt-siXBP1 plus dox treatment. Results are presented relative to β-actin expression. (mean ± s.d., n = 5). ***, p < 0.001
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