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Pharmaceutical Research

, 36:37 | Cite as

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery

  • Kaushik Thanki
  • Simon Papai
  • Abhijeet Lokras
  • Fabrice Rose
  • Emily Falkenberg
  • Henrik Franzyk
  • Camilla FogedEmail author
Research Paper

Abstract

Background

Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties.

Methods

LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS).

Results

A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid).

Conclusions

The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

Key Words

antisense oligonucleotides in vitro splice correction HeLa pLuc/705 cells lipidoids lipid-polymer hybrid nanoparticles (LPNs) quality-by-design statistical optimisation 

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

We gratefully acknowledge the support from the Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115363 resources, which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 600207. We are also grateful to the Lundbeck Foundation – Denmark (R219–2016-908), the Novo Nordisk Foundation – Denmark (grant no. NNF17OC0026526) and Hørslev-Fonden – Denmark for financial support. We also acknowledge Stat-Ease Inc. for generously providing the evaluation license of Design Expert version 11 and ChemAxon for kindly providing the academic license of Marvin Suite including calculator plugins. The authors report no potential conflicts of interest.

Supplementary material

11095_2018_2566_MOESM1_ESM.docx (342 kb)
ESM 1 (DOCX 342 kb)

References

  1. 1.
    Hood L, Rowen L. The human genome project: big science transforms biology and medicine. Genome Med. 2013;5(9):79.PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Auffray C, Chen Z, Hood L. Systems medicine: the future of medical genomics and healthcare. Genome Med. 2009;1(1):2.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Kaczmarek JC, Kowalski PS, Anderson DG. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. 2017;9(1):60.PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Russ AP, Lampel S. The druggable genome: an update. Drug Discov Today. 2005;10(23–24):1607–10.PubMedCrossRefGoogle Scholar
  5. 5.
    Jorgensen R. Altered gene-expression in plants due to trans interactions between homologous genes. Trends Biotechnol. 1990;8(12):340–4.PubMedCrossRefGoogle Scholar
  6. 6.
    Wong WS, Melendez AJ. Frontiers in research reviews: cutting-edge molecular approach to therapeutics - introduction. Clin Exp Pharmacol Physiol. 2006;33(5–6):480–1.CrossRefGoogle Scholar
  7. 7.
    Dias N, Stein CA. Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther. 2002;1(5):347–55.PubMedGoogle Scholar
  8. 8.
    Havens MA, Hastings ML. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016;44(14):6549–63.PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Kurreck J. Antisense technologies - improvement through novel chemical modifications. Eur J Biochem. 2003;270(8):1628–44.PubMedCrossRefGoogle Scholar
  10. 10.
    Chery J. RNA therapeutics: RNAi and antisense mechanisms and clinical applications. Postdoc J. 2016;4(7):35–50.PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Sharma VK, Sharma RK, Singh SK. Antisense oligonucleotides: modifications and clinical trials. Medchemcomm. 2014;5(10):1454–71.CrossRefGoogle Scholar
  12. 12.
    Stein CA, Castanotto D. FDA-approved oligonucleotide therapies in 2017. Mol Ther. 2017;25(5):1069–75.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Askari FK, WM MD. Molecular medicine - Antisense oligonucleotide therapy. N Engl J Med. 1996;334(5):316–8.PubMedCrossRefGoogle Scholar
  14. 14.
    Paterson BM, Roberts BE, Kuff EL. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977;74(10):4370–4.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Moccia M, Adamo MF, Saviano M. Insights on chiral, backbone modified peptide nucleic acids: properties and biological activity. Artif DNA PNA XNA. 2014;5(3):e1107176.PubMedCrossRefGoogle Scholar
  16. 16.
    Nielsen PE, Egholm M. An introduction to peptide nucleic acid. Curr Issues Mol Biol. 1999;1(1–2):89–104.PubMedGoogle Scholar
  17. 17.
    Kole R, Krainer AR, Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov. 2012;11(2):125–40.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Hadinoto K, Sundaresan A, Cheow WS. Lipid-polymer hybrid nanoparticles as a new generation therapeutic delivery platform: a review. Eur J Pharm Biopharm. 2013;85(3):427–43.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhang LF, Chan JM, Gu FX, Rhee JW, Wang AZ, Radovic-Moreno AF, et al. Self-assembled lipid-polymer hybrid nanoparticles: a robust drug delivery platform. ACS Nano. 2008;2(8):1696–702.PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Maurer N, Fenske DB, Cullis PR. Developments in liposomal drug delivery systems. Expert Opin Biol Ther. 2001;1(6):923–47.PubMedCrossRefGoogle Scholar
  21. 21.
    Wu XY. Strategies for optimizing polymer-lipid hybrid nanoparticle-mediated drug delivery. Expert Opin Drug Deliv. 2016;13(5):609–12.PubMedCrossRefGoogle Scholar
  22. 22.
    Thanki K, Zeng X, Justesen S, Tejlmann S, Falkenberg E, Van Driessche E, et al. Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: a quality by design-based approach. Eur J Pharm Biopharm. 2017;120:22–33.PubMedCrossRefGoogle Scholar
  23. 23.
    Cheow WS, Hadinoto K. Factors affecting drug encapsulation and stability of lipid-polymer hybrid nanoparticles. Colloids Surf B: Biointerfaces. 2011;85(2):214–20.PubMedCrossRefGoogle Scholar
  24. 24.
    Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm. 2008;5(4):505–15.PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Bauman J, Jearawiriyapaisarn N, Kole R. Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides. 2009;19(1):1–13.PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Disterer P, Kryczka A, Liu Y, Badi YE, Wong JJ, Owen JS, et al. Development of therapeutic splice-switching oligonucleotides. Hum Gene Ther. 2014;25(7):587–98.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol. 2008;26(5):561–9.PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Kang SH, Cho MJ, Kole R. Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay developments. Biochemistry. 1998;37(18):6235–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Overbeek JTG. Monodisperse colloidal systems, fascinating and useful. Adv Colloid Interf Sci. 1982;15(3–4):251–77.CrossRefGoogle Scholar
  30. 30.
    Frohlich E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine. 2012;7:5577–91.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Zelphati O, Szoka FC. Mechanism of oligonucleotide release from cationic liposomes. Proc Natl Acad Sci U S A. 1996;93(21):11493–8.PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Semple SC, Klimuk SK, Harasym TO, Dos Santos N, Ansell SM, Wong KF, et al. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochim Biophys Acta Biomembr. 2001;1510(1–2):152–66.CrossRefGoogle Scholar
  33. 33.
    Khvedelidze M, Mdzinarashvili T, Partskhaladze T, Nafee N, Schaefer UF, Lehr CM, et al. Calorimetric and spectrophotometric investigation of PLGA nanoparticles and their complex with DNA. J Therm Anal Calorim. 2010;99(1):337–48.CrossRefGoogle Scholar
  34. 34.
    Kapoor DN, Bhatia A, Kaur R, Sharma R, Kaur G, Dhawan S. PLGA: a unique polymer for drug delivery. Ther Deliv. 2015;6(1):41–58.PubMedCrossRefGoogle Scholar
  35. 35.
    Mir M, Ahmed N, Rehman AU. Recent applications of PLGA based nanostructures in drug delivery. Colloids Surf B: Biointerfaces. 2017;159:217–31.PubMedCrossRefGoogle Scholar
  36. 36.
    Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers-Basel. 2011;3(3):1377–97.PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Sharma N, Madan P, Lin SS. Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: a co-surfactant study. Asian J Pharm Sci. 2016;11(3):404–16.CrossRefGoogle Scholar
  38. 38.
    Yang Y, Xie XY, Mei XG. Preparation and in vitro evaluation of thienorphine-loaded PLGA nanoparticles. Drug Deliv. 2016;23(3):787–93.PubMedGoogle Scholar
  39. 39.
    Kim SH, Jeong JH, Chun KW, Park TG. Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate. Langmuir. 2005;21(19):8852–7.PubMedCrossRefGoogle Scholar
  40. 40.
    Colombo SF, Cun DM, Remaut K, Bunker M, Zhang JX, Martin-Bertelsen B, et al. Mechanistic profiling of the siRNA delivery dynamics of lipid-polymer hybrid nanoparticles. J Control Release. 2015;201:22–31.PubMedCrossRefGoogle Scholar
  41. 41.
    Kedmi R, Ben-Arie N, Peer D. The systemic toxicity of positively charged lipid nanoparticles and the role of toll-like receptor 4 in immune activation. Biomaterials. 2010;31(26):6867–75.PubMedCrossRefGoogle Scholar
  42. 42.
    Lv HT, Zhang SB, Wang B, Cui SH, Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release. 2006;114(1):100–9.PubMedCrossRefGoogle Scholar
  43. 43.
    Xia Y, Chen E, Liang D. Recognition of single- and double-stranded oligonucleotides by bovine serum albumin via nonspecific interactions. Biomacromolecules. 2010;11(11):3158–66.PubMedCrossRefGoogle Scholar
  44. 44.
    Cheng X, Lee RJ. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. Adv Drug Deliv Rev. 2016;99(Pt A):129–37.PubMedCrossRefGoogle Scholar
  45. 45.
    Li W, Szoka FC Jr. Lipid-based nanoparticles for nucleic acid delivery. Pharm Res. 2007;24(3):438–49.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pharmacy, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagen ØDenmark
  2. 2.Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,University of CopenhagenCopenhagen ØDenmark

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