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Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Abstract

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

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Fig. 1: RNA nanotechnology for decorating native EVs.
Fig. 2: Comparison of the role of arrowhead and arrowtail 3WJ.
Fig. 3: Specific binding and siRNA delivery to cells in vitro using PSMA aptamer-displaying EVs.
Fig. 4: Animal trials using ligand-displaying EVs for tumour inhibition.
Fig. 5: EGFR-aptamer-displaying EVs can deliver survivin siRNA to breast cancer orthotopic xenograft mouse model.
Fig. 6: Folate-displaying EVs can deliver survivin siRNA to patient-derived colorectal cancer xenograft (PDX-CRC) mouse model.

References

  1. 1.

    Shu, D., Shu, Y., Haque, F., Abdelmawla, S. & Guo, P. Thermodynamically stable RNA three-way junctions for constructing multifuntional nanoparticles for delivery of therapeutics. Nat. Nanotech. 6, 658–667 (2011).

    Article  Google Scholar 

  2. 2.

    Zhang, H. et al. Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA. RNA 19, 1226–1237 (2013).

    Article  Google Scholar 

  3. 3.

    Guo, P., Erickson, S. & Anderson, D. A small viral RNA is required for in vitro packaging of bacteriophage phi29 DNA. Science 236, 690–694 (1987).

    Article  Google Scholar 

  4. 4.

    Guo, P., Zhang, C., Chen, C., Trottier, M. & Garver, K. Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol. Cell. 2, 149–155 (1998).

    Article  Google Scholar 

  5. 5.

    Lamichhane, T. N., Raiker, R. S. & Jay, S. M. Exogenous DNA loading into extracellular vesicles via electroporation is size-dependent and enables limited gene delivery. Mol. Pharm. 12, 3650–3657 (2015).

    Article  Google Scholar 

  6. 6.

    Rak, J. Organ-seeking vesicles. Nature 527, 312–314 (2015).

    Article  Google Scholar 

  7. 7.

    Melo, S. A. et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177–182 (2015).

    Article  Google Scholar 

  8. 8.

    Witwer, K. W. et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles. 2, (2013).

  9. 9.

    Shelke, G. V., Lasser, C., Gho, Y. S. & Lotvall, J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J. Extracell. Vesicles. 3, (2014).

  10. 10.

    Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22 (2006).

  11. 11.

    Kumar, D., Gupta, D., Shankar, S. & Srivastava, R. K. Biomolecular characterization of exosomes released from cancer stem cells: possible implications for biomarker and treatment of cancer. Oncotarget 6, 3280–3291 (2015).

    Google Scholar 

  12. 12.

    Bunge, A. et al. Lipid membranes carrying lipophilic cholesterol-based oligonucleotides: characterization and application on layer-by-layer coated particles. J. Phys. Chem. B 113, 16425–16434 (2009).

    Article  Google Scholar 

  13. 13.

    Pfeiffer, I. & Hook, F. Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies. J. Am. Chem. Soc. 126, 10224–10225 (2004).

    Article  Google Scholar 

  14. 14.

    Marcus, M. & Leonard, J. N. FedExosomes: engineering therapeutic biological nanoparticles that truly deliver. Pharmaceuticals (Basel) 6, 659–680 (2013).

    Article  Google Scholar 

  15. 15.

    van Dongen, H. M., Masoumi, N., Witwer, K. W. & Pegtel, D. M. Extracellular vesicles exploit viral entry routes for cargo delivery. Microbiol. Mol. Biol. Rev. 80, 369–386 (2016).

    Article  Google Scholar 

  16. 16.

    Parker, N. et al. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal. Biochem. 338, 284–293 (2005).

    Article  Google Scholar 

  17. 17.

    Dassie, J. P. et al. Targeted inhibition of prostate cancer metastases with an RNA aptamer to prostate-specific membrane antigen. Mol. Ther. 22, 1910–1922 (2014).

    Article  Google Scholar 

  18. 18.

    Rockey, W. M. et al. Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling. Nucleic Acid Ther. 21, 299–314 (2011).

    Article  Google Scholar 

  19. 19.

    Binzel, D. et al. Specific delivery of MiRNA for high efficient inhibition of prostate cancer by RNA nanotechnology. Mol. Ther. 24, 1267–1277 (2016).

    Article  Google Scholar 

  20. 20.

    Hynes, N. E. & Lane, H. A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354 (2005).

    Article  Google Scholar 

  21. 21.

    Esposito, C. L. et al. A neutralizing RNA aptamer against EGFR causes selective apoptotic cell death. PLoS One 6, e24071 (2011).

    Article  Google Scholar 

  22. 22.

    Shu, D. et al. Systemic delivery of anti-miRNA for suppression of triple negative breast cancer utilizing RNA nanotechnology. ACS Nano 9, 9731–9740 (2015).

    Article  Google Scholar 

  23. 23.

    Paduano, F. et al. Silencing of survivin gene by small interfering RNAs produces supra-additive growth suppression in combination with 17-allylamino-17-demethoxygeldanamycin in human prostate cancer cells. Mol. Cancer Ther. 5, 179–186 (2006).

    Article  Google Scholar 

  24. 24.

    Khaled, A., Guo, S., Li, F. & Guo, P. Controllable self-assembly of nanoparticles for specific delivery of multiple therapeutic molecules to cancer cells using RNA nanotechnology. Nano Lett. 5, 1797–1808 (2005).

    Article  Google Scholar 

  25. 25.

    Cui, D. et al. Regression of gastric cancer by systemic injection of RNA nanoparticles carrying both ligand and siRNA. Sci. Rep. 5, 10726 (2015).

    Article  Google Scholar 

  26. 26.

    Lee, T. J. et al. RNA nanoparticles as a vector for targeted siRNA delivery into glioblastoma mouse model. Oncotarget 6, 14766–14776 (2015).

    Google Scholar 

  27. 27.

    varez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341–345 (2011).

    Article  Google Scholar 

  28. 28.

    Ohno, S. et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21, 185–191 (2013).

    Article  Google Scholar 

  29. 29.

    Tian, Y. et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 35, 2383–2390 (2014).

    Article  Google Scholar 

  30. 30.

    Hung, M. E. & Leonard, J. N. Stabilization of exosome-targeting peptides via engineered glycosylation. J. Biol. Chem. 290, 8166–8172 (2015).

    Article  Google Scholar 

  31. 31.

    Binzel, D. W., Khisamutdinov, E. F. & Guo, P. Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments. Biochemistry 53, 2221–2231 (2014).

    Article  Google Scholar 

  32. 32.

    Haque, F. et al. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today 7, 245–257 (2012).

    Article  Google Scholar 

  33. 33.

    Li, Y., Tian, Z., Rizvi, S. M., Bander, N. H. & Allen, B. J. In vitro and preclinical targeted alpha therapy of human prostate cancer with Bi-213 labeled J591 antibody against the prostate specific membrane antigen. Prostate Cancer Prostatic Dis. 5, 36–46 (2002).

    Article  Google Scholar 

  34. 34.

    Pettaway, C. A. et al. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin. Cancer Res. 2, 1627–1636 (1996).

    Google Scholar 

  35. 35.

    Rimawi, M. F. et al. Epidermal growth factor receptor expression in breast cancer association with biologic phenotype and clinical outcomes. Cancer 116, 1234–1242 (2010).

    Article  Google Scholar 

  36. 36.

    Pecot, C., Calin, G. A., Coleman, R. L., Lopez-Berestein, G. & Sood, A. K. RNA interference in the clinic: challenges and future directions. Nat. Rev. Cancer 11, 59–67 (2011).

    Article  Google Scholar 

  37. 37.

    El Andaloussi, S., Mager, I., Breakefield, X. O. & Wood, M. J. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).

    Article  Google Scholar 

  38. 38.

    Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    Article  Google Scholar 

  39. 39.

    El Andaloussi, S., Lakhal, S., Mager, I. & Wood, M. J. Exosomes for targeted siRNA delivery across biological barriers. Adv. Drug Deliv. Rev. 65, 391–397 (2013).

    Article  Google Scholar 

  40. 40.

    van Dommelen, S. M. et al. Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J. Control Release 161, 635–644 (2012).

    Article  Google Scholar 

  41. 41.

    Wiklander, O. P. et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J. Extracell. Vesicles 4, 26316 (2015).

    Article  Google Scholar 

  42. 42.

    Guo, P. The emerging field of RNA nanotechnology. Nat. Nanotech. 5, 833–842 (2010).

    Article  Google Scholar 

  43. 43.

    Shu, D., Khisamutdinov, E., Zhang, L. & Guo, P. Programmable folding of fusion RNA complex driven by the 3WJ motif of phi29 motor pRNA. Nucleic Acids Res. 42, e10 (2013).

    Article  Google Scholar 

  44. 44.

    Varkouhi, A. K., Scholte, M., Storm, G. & Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J. Control Release 151, 220–228 (2011).

    Article  Google Scholar 

  45. 45.

    Kilchrist, K. V., Evans, B. C., Brophy, C. M. & Duvall, C. L. Mechanism of enhanced cellular uptake and cytosolic retention of MK2 inhibitory peptide nano-polyplexes. Cell. Mol. Bioeng. 9, 368–381 (2016).

    Article  Google Scholar 

  46. 46.

    Jasinski, D., Schwartz, C., Haque, F. & Guo, P. Large scale purification of RNA nanoparticles by preparative ultracentrifugation. Methods Mol. Biol. 1297, 67–82 (2015).

    Article  Google Scholar 

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Acknowledgements

We thank H. G. Zhang for his communication during the investigation of the exosome project, and J. Zhang, H. Weiss, D. Wu and D. Gao for assistance with statistical analysis. The research was supported mainly by National Institutes of Health grants UH3TR000875 and U01CA207946 (P. G.), and partially by R01CA186100 (B. G.), R35CA197706 (C.M.C.), P30CA177558 and R01CA195573 (B.M.E.).

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P.G. generated the original idea of using the 3WJ structure orientation to control cell entry or cell surface anchoring, respectively, and designed the arrowhead and arrowtail technology. P.G. and F.P. conceived and designed the experiments. H.L. and S.W. developed the method for RNA insertion to EV. F.P., H.L., D.W.B. and Z.L. performed the experiments. M.S. and B. Guo performed the prostate cancer mouse studies. T.J.L. performed the breast cancer mouse studies. P.R. performed the colorectal cancer mouse studies. P.G. and F.H. supervised the project. P.G., C.M.C. and B.M.E. provided the funding and resources. P.G., F.P., F.H. and D.W.B. co-wrote the manuscript, and all authors refined the manuscript.

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Correspondence to Peixuan Guo.

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P.G.’s Sylvan G. Frank Endowed Chair position in Pharmaceutics and Drug Delivery is funded by the CM Chen Foundation, and he is a consultant of Oxford Nanopore, Nanobio Delivery Pharmaceutical Co., Ltd, and the cofounder of P&Z Biological Technology LLC. F.P. now works for Nanobio Delivery Pharmaceutical Co., Ltd. S.W. and F.H. now work for P&Z Biological Technology LLC.

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Pi, F., Binzel, D.W., Lee, T.J. et al. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nature Nanotech 13, 82–89 (2018). https://doi.org/10.1038/s41565-017-0012-z

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