Cellular and Molecular Bioengineering

, Volume 11, Issue 5, pp 383–396 | Cite as

Layer-by-Layer Assembled Gold Nanoshells for the Intracellular Delivery of miR-34a

  • Ritu Goyal
  • Chintan H. Kapadia
  • Jilian R. Melamed
  • Rachel S. Riley
  • Emily S. Day



MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation.


LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-l-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles’ ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively.


Each successive coating reversed the nanoparticles’ charge and increased their hydrodynamic diameter, resulting in a final diameter of 208 ± 4 nm and a zeta potential of 53 ± 5 mV. The LbL-NS released ~ 30% of their miR-34a cargo over 5 days in 1× PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46 ± 3 and 35 ± 3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects.


LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.


MicroRNA Nanoparticles RNA interference Gene regulation Poly-l-lysine Trafficking Release SIRT1 Bcl-2 Triple-negative breast cancer 



Diethyl pyrocarbonate


Dynamic light scattering


Dulbecco’s Modified Eagle Medium




Deoxyribonucleic acid




Fetal bovine serum


Lysosomal-associated membrane protein 1




Layer-by-layer assembled nanoshells


Mander’s colocalization coefficient


Messenger RNA




11-Mercaptoundecanoic acid


Sodium chloride


Sodium hydroxide




Optical density




Radioimmunoprecipitation assay


Ribonucleic acid


RNA interference


Rotations per minute


Scanning electron microscopy


Nucleophilic aromatic substitution reaction


Triple-negative breast cancer


2,4,6-Trinitrobenzenesulfonic acid




Tetramethylrhodamine isothiocyanate


Ultra violet–visible spectrophotometry



This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number R35GM119659 (PI:Day). JRM received support from the Department of Defense through a National Defense Science and Engineering Graduate Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the views of the funding agencies. The LSM880 confocal microscope was acquired with a shared instrumentation Grant (S10 OD016361) and access was supported by the NIH-NIGMS (P20 GM103446), the NSF (IIA-1301765), and the State of Delaware. The Hitachi S4700 used in this work was acquired with the Delaware INBRE Grant P20 GM103446.

Author contributions

All authors conceptualized the experiments. RG, CK, JM, and RR performed the experiments and analyzed the data. ED secured funding for the experiments. All authors wrote and revised the manuscript.

Conflict of interest

Ritu Goyal, Chintan Kapadia, Jilian Melamed, Rachel Riley, and Emily Day declare no conflicts of interest.

Ethical standards

No animal or human studies were performed in this work.

Supplementary material

12195_2018_535_MOESM1_ESM.pdf (2.6 mb)
Supplementary material 1 (PDF 2676 kb)


  1. 1.
    Adams, B. D., C. Parsons, and F. J. Slack. The tumor-suppressive and potential therapeutic functions of miR-34a in epithelial carcinomas. Expert Opin. Ther. Targets 20(6):737–753, 2016.CrossRefGoogle Scholar
  2. 2.
    Adams, B., V. Wali, C. Cheng, S. Inukai, C. Booth, S. Agarwal, D. Rimm, B. Győrffy, L. Santarpia, L. Pusztai, W. Saltzman, and F. Slack. mir-34a silences c-SRC to attenuate tumor growth in triple-negative breast cancer. Cancer Res. 76(4):927–939, 2016.CrossRefGoogle Scholar
  3. 3.
    Avery-Kiejda, K. A., S. G. Braye, A. Mathe, J. F. Forbes, and R. J. Scott. Decreased expression of key tumor suppressor microRNAs is associated with lymph node metastasis in triple negative breast cancer. BMC Cancer 14:51, 2014.CrossRefGoogle Scholar
  4. 4.
    Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233, 2009.CrossRefGoogle Scholar
  5. 5.
    Beg, M. S., A. J. Brenner, J. Sachdev, M. Borad, Y. K. Kang, J. Stoudemire, S. Smith, A. G. Bader, S. Kim, and D. S. Hong. Phase I study of MEX34, a liposomal mir-34a mimic, administered twice weekly in patients with advanced solid tumors. Invest New Drugs 35(2):180–188, 2017.CrossRefGoogle Scholar
  6. 6.
    Ben-Shushan, D., E. Markovsky, H. Gibori, G. Tiram, A. Scomparin, and R. Satchi-Fainaro. Overcoming obstacles in microRNA delivery towards improved cancer therapy. Drug Deliv. Transl. Res. 4(1):38–49, 2014.CrossRefGoogle Scholar
  7. 7.
    Calin, G. A., and C. M. Croce. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6(11):857–866, 2006.CrossRefGoogle Scholar
  8. 8.
    Deng, X., M. Cao, J. Zhang, K. Hu, Z. Yin, Z. Zhou, X. Xiao, Y. Yang, W. Sheng, Y. Wu, and Y. Zeng. Hyaluronic acid-chitosan nanoparticles for co-delivery of miR-34a and doxorubicin in therapy against triple negative breast cancer. Biomaterials 35(14):4333–4344, 2014.CrossRefGoogle Scholar
  9. 9.
    Deng, Z. J., S. W. Morton, E. Ben-Akiva, E. C. Dreaden, K. E. Shopsowitz, and P. T. Hammond. Layer-by-layer nanoparticles for systemic codelivery of an anticancer drug and siRNA for potential triple-negative breast cancer treatment. ACS Nano 7(11):9571–9584, 2013.CrossRefGoogle Scholar
  10. 10.
    Duff, D. G., A. Baiker, and P. P. Edwards. A new hydrosol of gold clusters. 1. Formation and particle-size variation. Langmuir 9(9):2301–2309, 1993.CrossRefGoogle Scholar
  11. 11.
    Elbakry, A., A. Zaky, R. Liebl, R. Rachel, A. Goepferich, and M. Breunig. Layer-by-layer assembled gold nanoparticles for siRNA delivery. Nano Lett. 9(5):2059–2064, 2009.CrossRefGoogle Scholar
  12. 12.
    Gilleron, J., W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S. Seifert, C. Andree, M. Stoter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M. Bickle, Y. Kalaidzidis, A. Akinc, M. Maier, and M. Zerial. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat Biotechnol. 31(7):638–646, 2013.CrossRefGoogle Scholar
  13. 13.
    Goyal, R., S. K. Tripathi, S. Tyagi, A. Sharma, K. R. Ram, D. K. Chowdhuri, Y. Shukla, P. Kumar, and K. C. Gupta. Linear PEI nanoparticles: efficient pDNA/siRNA carriers in vitro and in vivo. Nanomedicine 8(2):167–175, 2012.CrossRefGoogle Scholar
  14. 14.
    Goyal, R., S. K. Tripathi, E. Vazquez, P. Kumar, and K. C. Gupta. Biodegradable poly(vinyl alcohol)-polyethylenimine nanocomposites for enhanced gene expression in vitro and in vivo. Biomacromolecules 13(1):73–83, 2012.CrossRefGoogle Scholar
  15. 15.
    Grotzky, A., Y. Manaka, S. Fornera, M. Willeke, and P. Walde. Quantification of alpha-polylysine: a comparison of four UV/Vis spectrophotometric methods. Anal. Methods 2(10):1448–1455, 2010.CrossRefGoogle Scholar
  16. 16.
    Gu, L., Z. J. Deng, S. Roy, and P. T. Hammond. A combination RNAi-chemotherapy layer-by-layer nanoparticle for systemic targeting of KRAS/p53 with cisplatin to treat non-small cell lung cancer. Clin. Cancer Res. 23(23):7312–7323, 2017.CrossRefGoogle Scholar
  17. 17.
    Hao, L., P. C. Patel, A. H. Alhasan, D. A. Giljohann, and C. A. Mirkin. Nucleic acid-gold nanoparticle conjugates as mimics of microRNA. Small 7(22):3158–3162, 2011.CrossRefGoogle Scholar
  18. 18.
    Kim, V. N. MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6:376–385, 2005.CrossRefGoogle Scholar
  19. 19.
    Kouri, F. M., L. A. Hurley, W. L. Daniel, E. S. Day, Y. Hua, L. Hao, C.-Y. Peng, T. J. Merkel, M. A. Queisser, C. Ritner, H. Zhang, C. D. James, J. I. Sznajder, L. Chin, D. A. Giljohann, J. A. Kessler, M. E. Peter, C. A. Mirkin, and A. H. Stegh. miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma. Genes Dev. 29(7):732–745, 2015.CrossRefGoogle Scholar
  20. 20.
    Kreuzberger, N. L., J. R. Melamed, and E. S. Day. Nanoparticle-mediated gene regulation as a novel strategy for cancer therapy. Del. J. Public Health 3(3):20–24, 2017.Google Scholar
  21. 21.
    Krzeszinski, J. Y., W. Wei, H. Huynh, Z. Jin, X. Wang, T.-C. Chang, X.-J. Xie, L. He, L. S. Mangala, G. Lopez-Berestein, A. K. Sood, J. T. Mendell, and Y. Wan. miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2. Nature 512(7515):431–435, 2014.CrossRefGoogle Scholar
  22. 22.
    Kuo, W.-H., Y.-Y. Chang, L.-C. Lai, M.-H. Tsai, C. K. Hsiao, K.-J. Chang, and E. Y. Chuang. Molecular characteristics and metastasis predictor genes of triple-negative breast cancer: a clinical study of triple-negative breast carcinomas. PLOS ONE 7(9):e45831, 2012.CrossRefGoogle Scholar
  23. 23.
    Li, L., X. Xie, J. Luo, M. Liu, S. Xi, J. Guo, Y. Kong, M. Wu, J. Gao, Z. Xie, J. Tang, X. Wang, W. Wei, M. Yang, M.-C. Hung, and X. Xie. Targeted expression of mir-34a using the t-visa system suppresses breast cancer growth and invasion. Mol. Ther. 20(12):2326–2334, 2012.CrossRefGoogle Scholar
  24. 24.
    Li, L., L. Yuan, J. Luo, J. Gao, J. Guo, and X. Xie. miR-34a inhibits proliferation and migration of breast cancer through down-regulation of Bcl-2 and SIRT1. Clin. Exp. Med. 13:109–117, 2013.CrossRefGoogle Scholar
  25. 25.
    Liedtke, C., C. Mazouni, K. R. Hess, F. Andre, A. Tordai, J. A. Mejia, W. F. Symmans, A. M. Gonzalez-Angulo, B. Hennessy, M. Green, M. Cristofanilli, G. N. Hortobagyi, and L. Pusztai. Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J. Clin. Oncol. 26:1275–1281, 2008.CrossRefGoogle Scholar
  26. 26.
    MacFarlane, L.-A., and P. R. Murphy. MicroRNA: biogenesis, function and role in cancer. Curr. Genom. 11(7):537–561, 2010.CrossRefGoogle Scholar
  27. 27.
    Manders, E. M. M., F. J. Verbeek, and J. A. Aten. Measurement of co-localization of objects in dual-colour confocal images. J. Microsc. 169(3):375–382, 1993.CrossRefGoogle Scholar
  28. 28.
    Melamed, J., R. Riley, D. Valcourt, M. Billingsley, N. Kreuzberger, and E. Day. Quantification of siRNA duplexes bound to gold nanoparticle surfaces. In: Biomedical Nanotechnology: Methods and Protocols2nd, edited by S. H. Petrosko, and E. S. Day. New York: Humana Press, 2017, pp. 1–15.Google Scholar
  29. 29.
    Misso, G., M. T. D. Martino, G. D. Rosa, A. A. Farooqi, A. Lombardi, V. Campani, M. R. Zarone, A. Gullà, P. Tagliaferri, P. Tassone, and M. Caraglia. miR-34: a new weapon against cancer? Molec. Ther. Nucleic Acids 3:e194, 2014.CrossRefGoogle Scholar
  30. 30.
    Oldenburg, S. J., R. D. Averitt, S. L. Westcott, and N. J. Halas. Nanoengineering of optical resonances. Chem. Phys. Lett. 288(2–4):243–247, 1998.CrossRefGoogle Scholar
  31. 31.
    Poon, Z., D. Chang, X. Zhao, and P. T. Hammond. Layer-by-layer nanoparticles with a pH-sheddable layer for in vivo targeting of tumor hypoxia. ACS Nano 5(6):4284–4292, 2011.CrossRefGoogle Scholar
  32. 32.
    Riley, R. S., and E. S. Day. Frizzled7 antibody-functionalized nanoshells enable multivalent binding for Wnt signaling inhibition in triple negative breast cancer cells. Small 13(26):1700544, 2017.CrossRefGoogle Scholar
  33. 33.
    Rokavec, M., H. Li, L. Jiang, and H. Hermeking. The p53/miR-34 axis in development and disease. J. Mol. Cell Biol. 6(3):214–230, 2014.CrossRefGoogle Scholar
  34. 34.
    Saito, Y., T. Nakaoka, and H. Saito. MicroRNA-34a as a therapeutic agent against human cancer. J. Clin. Med. 4(11):1951–1959, 2015.CrossRefGoogle Scholar
  35. 35.
    Sparrow, J. T., V. V. Edwards, C. Tung, M. J. Logan, M. S. Wadhwa, J. Duguid, and L. C. Smith. Synthetic peptide-based DNA complexes for nonviral gene delivery. Adv. Drug Deliv. Rev. 30(1–3):115–131, 1998.Google Scholar
  36. 36.
    Stern, J. M., V. V. Kibanov Solomonov, E. Sazykina, J. A. Schwartz, S. C. Gad, and G. P. Goodrich. Initial evaluation of the safety of nanoshell-directed photothermal therapy in the treatment of prostate disease. Int. J. Toxicol. 35(1):38–46, 2016.CrossRefGoogle Scholar
  37. 37.
    Swami, A., R. Goyal, S. K. Tripathi, N. Singh, N. Katiyar, A. K. Mishra, and K. C. Gupta. Effect of homobifunctional crosslinkers on nucleic acids delivery ability of PEI nanoparticles. Int. J. Pharm. 374(1–2):125–138, 2009.CrossRefGoogle Scholar
  38. 38.
    Wittrup, A., A. Ai, X. Liu, P. Hamar, R. Trifonova, K. Charisse, M. Manoharan, T. Kirchhausen, and J. Lieberman. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33:870–876, 2015.CrossRefGoogle Scholar
  39. 39.
    Wu, Z. W., C. T. Chien, C. Y. Liu, J. Y. Yan, and S. Y. Lin. Recent progress in copolymer-mediated siRNA delivery. J. Drug Target. 20(7):551–560, 2012.CrossRefGoogle Scholar
  40. 40.
    Yamakuchi, M., M. Ferlito, and C. J. Lowenstein. miR-34a repression of SIRT1 regulates apoptosis. Proc. Natl. Acad. Sci. USA 105(36):13421–13426, 2008.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2018

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of DelawareNewarkUSA
  2. 2.Department of Materials Science & EngineeringUniversity of DelawareNewarkUSA
  3. 3.Helen F. Graham Cancer Center & Research InstituteNewarkUSA

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