Cellular and Molecular Bioengineering

, Volume 9, Issue 3, pp 305–314 | Cite as

Lipidoid Tail Structure Strongly Influences siRNA Delivery Activity

  • Christopher M. Knapp
  • Penghong Guo
  • Kathryn A. Whitehead
Article

Abstract

RNA interference therapeutics have been limited, in large part, by the lack of efficient, non-toxic, non-immunogenic delivery systems. Among previously established methods, lipidoid nanoparticles (LNPs) show particular promise in delivering siRNA to diverse cell and organ targets in vitro and in vivo. However, a better understanding of structure–function relationships is needed to facilitate broad translation to clinical applications. Here, we demonstrate the critical role of tail chemistry in conferring delivery efficacy to lipidoid molecules with three or four aliphatic tails. Tail length and structure significantly affected siRNA transfection in HeLa cells, with methacrylate (vs. acrylate) tails and tails containing ethers causing reductions in efficacy. Notably, we report a novel tail precursor, isodecyl acrylate, that conveyed marked siRNA delivery ability in vitro and in vivo. LNPs with isodecyl acrylate lipidoids uniformly induced greater than 90% gene silencing, both in vitro and in mice (hepatocytes), at 40 nM and 0.1 mg/kg, respectively. Furthermore, we found that tail chemistry significantly influenced the surface pKa values of formulated LNPs, with tails that conferred higher pKa facilitating higher levels of gene knockdown. Together, these data underscore the importance of lipidoid tail structure and provide guidance for the development of next generation lipid nanoparticle siRNA delivery systems.

Keywords

Lipid nanoparticles siRNA delivery Nanomedicine Hepatocellular delivery RNA interference Structure–function 

References

  1. 1.
    Akinc, A., W. Querbes, S. De, J. Qin, M. Frank-Kamenetsky, K. N. Jayaprakash, M. Jayaraman, K. G. Rajeev, W. L. Cantley, J. R. Dorkin, J. S. Butler, L. Qin, T. Racie, A. Sprague, E. Fava, A. Zeigerer, M. J. Hope, M. Zerial, D. W. Sah, K. Fitzgerald, M. A. Tracy, M. Manoharan, V. Koteliansky, A. de Fougerolles, and M. A. Maier. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18:1357–1364, 2010.CrossRefGoogle Scholar
  2. 2.
    Akinc, A., A. Zumbuehl, M. Goldberg, E. S. Leshchiner, V. Busini, N. Hossain, S. A. Bacallado, D. N. Nguyen, J. Fuller, R. Alvarez, A. Borodovsky, T. Borland, R. Constien, A. De Fougerolles, J. R. Dorkin, K. Narayanannair Jayaprakash, M. Jayaraman, M. John, V. Koteliansky, M. Manoharan, L. Nechev, J. Qin, T. Racie, D. Raitcheva, K. G. Rajeev, D. W. Y. Sah, J. Soutschek, I. Toudjarska, H. Vornlocher, T. S. Zimmermann, R. Langer, and D. G. Anderson. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26:561–569, 2008.CrossRefGoogle Scholar
  3. 3.
    Alabi, C. A., K. T. Love, G. Sahay, H. Yin, K. M. Luly, R. Langer, and D. G. Anderson. Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery. Proc. Natl. Acad. Sci. 110:12881–12886, 2013.CrossRefGoogle Scholar
  4. 4.
    Ameres, S. L., J. Martinez, and R. Schroeder. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130:101–112, 2007.CrossRefGoogle Scholar
  5. 5.
    Chen, C.-K., W.-C. Law, R. Aalinkeel, B. Nair, A. Kopwitthaya, S. D. Mahajan, J. L. Reynolds, J. Zou, S. A. Schwartz, P. N. Prasad, and C. Cheng. Well-defined degradable cationic polylactide as nanocarrier for the delivery of siRNA to silence angiogenesis in prostate cancer. Adv. Healthc. Mater. 1:751–761, 2012.CrossRefGoogle Scholar
  6. 6.
    Dong, Y., K. T. Love, J. R. Dorkin, S. Sirirungruang, Y. Zhang, D. Chen, R. L. Bogorad, H. Yin, Y. Chen, A. J. Vegas, C. A. Alabi, G. Sahay, K. T. Olejnik, W. Wang, A. Schroeder, A. K. R. Lytton-Jean, D. J. Siegwart, A. Akinc, C. Barnes, S. A. Barros, M. Carioto, K. Fitzgerald, J. Hettinger, V. Kumar, T. I. Novobrantseva, J. Qin, W. Querbes, V. Koteliansky, R. Langer, and D. G. Anderson. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. Proc. Natl. Acad. Sci. 111:3955–3960, 2014.CrossRefGoogle Scholar
  7. 7.
    Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811, 1998.CrossRefGoogle Scholar
  8. 8.
    Fitzgerald, K., M. Frank-Kamenetsky, S. Shulga-Morskaya, A. Liebow, B. R. Bettencourt, J. E. Sutherland, R. M. Hutabarat, V. A. Clausen, V. Karsten, J. Cehelsky, S. V. Nochur, V. Kotelianski, J. Horton, T. Mant, J. Chiesa, J. Ritter, M. Munisamy, A. K. Vaishnaw, J. A. Gollob, and A. Simon. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial. Lancet 383:60–68, 2014.CrossRefGoogle Scholar
  9. 9.
    Frank-Kamenetsky, M., A. Grefhorst, N. N. Anderson, T. S. Racie, B. Bramlage, A. Akinc, D. Butler, K. Charisse, R. Dorkin, Y. Fan, C. Gamba-Vitalo, P. Hadwiger, M. Jayaraman, M. John, K. N. Jayaprakash, M. Maier, L. Nechev, K. G. Rajeev, T. Read, I. Röhl, J. Soutschek, P. Tan, J. Wong, G. Wang, T. Zimmermann, A. de Fougerolles, H.-P. Vornlocher, R. Langer, D. G. Anderson, M. Manoharan, V. Koteliansky, J. D. Horton, and K. Fitzgerald. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. 105:11915–11920, 2008.CrossRefGoogle Scholar
  10. 10.
    Gilleron, J., W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S. Seifert, C. Andree, M. Stöter, 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. 31:638–646, 2013.CrossRefGoogle Scholar
  11. 11.
    Heyes, J., L. Palmer, K. Bremner, and I. MacLachlan. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. J. Control. Release 107:276–287, 2005.CrossRefGoogle Scholar
  12. 12.
    Jayaraman, M., S. M. Ansell, B. L. Mui, Y. K. Tam, J. Chen, X. Du, D. Butler, L. Eltepu, S. Matsuda, J. K. Narayanannair, K. G. Rajeev, I. M. Hafez, A. Akinc, M. A. Maier, M. A. Tracy, P. R. Cullis, T. D. Madden, M. Manoharan, and M. J. Hope. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. 51:8529–8533, 2012.CrossRefGoogle Scholar
  13. 13.
    Kumar, V., J. Qin, Y. Jiang, R. G. Duncan, B. Brigham, S. Fishman, J. K. Nair, A. Akinc, S. A. Barros, and P. V. Kasperkovitz. Shielding of lipid nanoparticles for siRNA delivery: impact on physicochemical properties, cytokine induction, and efficacy. Mol. Ther. Nucl. Acids 3:e210, 2014.CrossRefGoogle Scholar
  14. 14.
    Love, K. T., K. P. Mahon, C. G. Levins, K. A. Whitehead, W. Querbes, J. R. Dorkin, J. Qin, W. Cantley, L. L. Qin, T. Racie, M. Frank-Kamenetsky, K. N. Yip, R. Alvarez, D. W. Y. Sah, A. de Fougerolles, K. Fitzgerald, V. Koteliansky, A. Akinc, R. Langer, and D. G. Anderson. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. 107:1864–1869, 2010.CrossRefGoogle Scholar
  15. 15.
    Mahon, K. P., K. T. Love, K. A. Whitehead, J. Qin, A. Akinc, E. Leshchiner, I. Leshchiner, R. Langer, and D. G. Anderson. Combinatorial approach to determine functional group effects on lipidoid-mediated siRNA delivery. Bioconjugate Chem. 21:1448–1454, 2010.CrossRefGoogle Scholar
  16. 16.
    Rand, T. A., S. Petersen, F. Du, and X. Wang. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123:621–629, 2005.CrossRefGoogle Scholar
  17. 17.
    Sahay, G., W. Querbes, C. Alabi, A. Eltoukhy, S. Sarkar, C. Zurenko, E. Karagiannis, K. Love, D. Chen, R. Zoncu, Y. Buganim, A. Schroeder, R. Langer, and D. G. Anderson. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31:653–658, 2013.CrossRefGoogle Scholar
  18. 18.
    Sehgal, A., S. Barros, L. Ivanciu, B. Cooley, J. Qin, T. Racie, J. Hettinger, M. Carioto, Y. Jiang, J. Brodsky, H. Prabhala, X. Zhang, H. Attarwala, R. Hutabarat, D. Foster, S. Milstein, K. Charisse, S. Kuchimanchi, M. A. Maier, L. Nechev, P. Kandasamy, A. V. Kel’in, J. K. Nair, K. G. Rajeev, M. Manoharan, R. Meyers, B. Sorensen, A. R. Simon, Y. Dargaud, C. Negrier, R. M. Camire, and A. Akinc. An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia. Nat. Med. 21:492–497, 2015.CrossRefGoogle Scholar
  19. 19.
    Semple, S. C., A. Akinc, J. Chen, A. P. Sandhu, B. L. Mui, C. K. Cho, D. W. Y. Sah, D. Stebbing, E. J. Crosley, E. Yaworski, I. M. Hafez, J. R. Dorkin, J. Qin, K. Lam, K. G. Rajeev, K. F. Wong, L. B. Jeffs, L. Nechev, M. L. Eisenhardt, M. Jayaraman, M. Kazem, M. A. Maier, M. Srinivasulu, M. J. Weinstein, Q. Chen, R. Alvarez, S. A. Barros, S. De, S. K. Klimuk, T. Borland, V. Kosovrasti, W. L. Cantley, Y. K. Tam, M. Manoharan, M. A. Ciufolini, M. A. Tracy, A. de Fougerolles, I. MacLachlan, P. R. Cullis, T. D. Madden, and M. J. Hope. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28:172–176, 2010.CrossRefGoogle Scholar
  20. 20.
    Wang, M., S. Sun, K. A. Alberti, and Q. Xu. A combinatorial library of unsaturated lipidoids for efficient intracellular gene delivery. ACS Synth. Biol. 1:403–407, 2012.CrossRefGoogle Scholar
  21. 21.
    Whitehead, K.A., J.R. Dorkin, A.J. Vegas, P.H. Chang, O. Veiseh, J. Matthews, O.S. Fenton, Y. Zhang, K.T. Olejnik, V. Yesilyurt, D. Chen, S. Barros, B. Klebanov, T. Novobrantseva, R. Langer, and D.G. Anderson. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 5, 2014 [cited 2014 Oct 7]. Available from: http://www.nature.com/ncomms/2014/140627/ncomms5277/full/ncomms5277.html.
  22. 22.
    Whitehead, K. A., R. Langer, and D. G. Anderson. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8:129–138, 2009.CrossRefGoogle Scholar
  23. 23.
    Whitehead, K. A., J. Matthews, P. H. Chang, F. Niroui, J. R. Dorkin, M. Severgnini, and D. G. Anderson. In vitro–in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery. ACS Nano 6:6922–6929, 2012.CrossRefGoogle Scholar
  24. 24.
    Wisse, E., F. Jacobs, B. Topal, P. Frederik, and B. De Geest. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 15:1193–1199, 2008.CrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Wooddell, C. I., D. B. Rozema, M. Hossbach, M. John, H. L. Hamilton, Q. Chu, J. O. Hegge, J. J. Klein, D. H. Wakefield, C. E. Oropeza, J. Deckert, I. Roehl, K. Jahn-Hofmann, P. Hadwiger, H.-P. Vornlocher, A. McLachlan, and D. L. Lewis. Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection. Mol. Ther. 21:973–985, 2013.CrossRefGoogle Scholar
  27. 27.
    Yameen, B., W. I. Choi, C. Vilos, A. Swami, J. Shi, and O. C. Farokhzad. Insight into nanoparticle cellular uptake and intracellular targeting. J. Control. Release 190:485–499, 2014.CrossRefGoogle Scholar
  28. 28.
    Yu, B., S.-H. Hsu, C. Zhou, X. Wang, M. C. Terp, Y. Wu, L. Teng, Y. Mao, F. Wang, W. Xue, S. T. Jacob, K. Ghoshal, R. J. Lee, and L. J. Lee. Lipid nanoparticles for hepatic delivery of small interfering RNA. Biomaterials 33:5924–5934, 2012.CrossRefGoogle Scholar
  29. 29.
    Zhou, K., L. H. Nguyen, J. B. Miller, Y. Yan, P. Kos, H. Xiong, L. Li, J. Hao, J. T. Minnig, H. Zhu, and D. J. Siegwart. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. 113:520–525, 2016.CrossRefGoogle Scholar
  30. 30.
    Zuckerman, J. E., and M. E. Davis. Clinical experiences with systemically administered siRNA-based therapeutics in cancer. Nat. Rev. Drug Discov. 14:843–856, 2015.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2016

Authors and Affiliations

  • Christopher M. Knapp
    • 1
  • Penghong Guo
    • 1
  • Kathryn A. Whitehead
    • 1
    • 2
  1. 1.Department of Chemical EngineeringCarnegie Mellon UniversityPittsburghUSA
  2. 2.Department of Biomedical EngineeringCarnegie Mellon UniversityPittsburghUSA

Personalised recommendations