Skip to main content

Advertisement

SpringerLink
Log in

Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use.

Methods

PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the size and homogeneity of resulting particles was studied. Particles prepared using this method were compared to those prepared using an established post-insertion procedure for the entrapment efficiency, stability, in vitro biological activity as well as in vivo biodistribution.

Results

Using this hydration method, a particle size of less than 200 nm can be obtained with high siRNA entrapment efficiency (>90%) and high gene-silencing efficiency. Following intravenous administration into mice, these particles achieved a similar degree of accumulation in subcutaneous tumours but displayed less liver uptake compared to the post-insertion formulations. Importantly, in contrast to post-insertion preparations, particles made by hydration method retained 100% of their gene-silencing efficiency after storage at room temperature for 1 month.

Conclusions

This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

bp:

Base pair

CMV:

Cytomegalovirus

DEPC:

Diethylpyrocarbonate

dH2O:

Distilled water

DiR:

1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide

DMEM:

Dulbecco’s modified Eagle medium

DNA:

Deoxyribonucleic acid

DODAP:

1,2-Dioleoyloxy-3-(dimethylamino)propane

DOTAP:

Dioleoyl trimethylammonium propane

dsDNA:

Double-stranded DNA

dsRNA:

Double-stranded RNA

EDTA:

Ethylenediaminetetraacetic acid

FACS:

Fluorescence activated cell sorting

FBS:

Fetal bovine serum

GFP:

Green fluorescence protein

HFDM:

Hydration of freeze-dried matrix

i.v.:

Intravenous

LP:

Lipid particle

mRNA:

Messenger RNA

N/P:

Nitrogen/phosphate

ODN:

Oligodexoynucleotides

PBS:

Phosphate buffered saline

PEG:

Polyethylene glycol

PI:

Post-insertion

RES:

Reticuloendothelial system

RNA:

Ribonucleic acid

RNAi:

RNA interference

RT:

Room temperature

SD:

Standard deviation

siRNA:

Small interfering RNA

UV:

Ultraviolet

References

  1. A. Fire, S. Q. 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). doi:10.1038/35888.

    Article  PubMed  CAS  Google Scholar 

  2. C. N. Landen, A. Chavez-Reyes, C. Bucana, R. Schmandt, M. T. Deavers, G. Lopez-Berestein, and A. K. Sood. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res. 65:6910–6918 (2005). doi:10.1158/0008-5472.CAN-05-0530.

    Article  PubMed  CAS  Google Scholar 

  3. M. Diaz-Hernandez, J. Torres-Peraza, A. Salvatori-Abarca, M. A. Moran, P. Gomez-Ramos, J. Alberch, and J. J. Lucas. Full motor recovery despite striatal neuron loss and formation of irreversible amyloid-like inclusions in a conditional mouse model of Huntington’s disease. J. Neurosci. 25:9773–9781 (2005). doi:10.1523/JNEUROSCI.3183-05.2005.

    Article  PubMed  CAS  Google Scholar 

  4. H. Giladi, M. Ketzinel-Gilad, L. Rivkin, Y. Felig, O. Nussbaum, and E. Galun. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol. Ther. 8:769–776 (2003). doi:10.1016/S1525-0016(03)00244-2.

    Article  PubMed  CAS  Google Scholar 

  5. T. S. Zimmermann, A. C. H. Lee, A. Akinc, B. Bramlage, D. Bumcrot, M. N. Fedoruk, J. Harborth, J. A. Heyes, L. B. Jeffs, M. John, A. D. Judge, K. Lam, K. McClintock, L. V. Nechev, L. R. Palmer, T. Racie, I. Rohl, S. Seiffert, S. Shanmugam, V. Sood, J. Soutschek, I. Toudjarska, A. J. Wheat, E. Yaworski, W. Zedalis, V. Koteliansky, M. Manoharan, H. P. Vornlocher, and I. MacLachlan. RNAi-mediated gene silencing in non-human primates. Nature. 441:111–114 (2006). doi:10.1038/nature04688.

    Article  PubMed  CAS  Google Scholar 

  6. S. D. Li, S. Chono, and L. Huang. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Mol. Ther. 16:942–946 (2008). doi:10.1038/mt.2008.51.

    Article  PubMed  CAS  Google Scholar 

  7. D. Sorensen, M. Leirdal, and M. Sioud. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327:761–766 (2003). doi:10.1016/S0022-2836(03)00181-5.

    Article  PubMed  CAS  Google Scholar 

  8. M. Stevenson, V. Ramos-Perez, S. Singh, M. Soliman, J. A. Preece, S. S. Briggs, M. L. Read, and L. W. Seymour. Delivery of siRNA mediated by histidine-containing reducible polycations. J. Control Release. 130:46–56 (2008). doi:10.1016/j.jconrel.2008.05.014.

    Article  PubMed  CAS  Google Scholar 

  9. J. Pille, H. Li, E. Blot, J. Bertrand, L. Pritchard, P. Opolon, A. Maksimenko, H. Lu, J. Vannier, J. Soria, C. Malvy, and C. Soria. Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer. Hum. Gene Ther. 17:1019–1026 (2006). doi:10.1089/hum.2006.17.1019.

    Article  PubMed  CAS  Google Scholar 

  10. W. Zamboni. Liposomal, nanoparticle, and conjugated formulations of anticancer agents. Clin. Cancer Res. 11:8230–8234 (2005). doi:10.1158/1078-0432.CCR-05-1895.

    Article  PubMed  CAS  Google Scholar 

  11. J. Kim, S. Choi, C. Kim, J. Park, W. Ahn, and C. Kim. Enhancement of polyethylene glycol (PEG)-modified cationic liposome-mediated gene deliveries: effects on serum stability and transfection efficiency. J. Pharm. Pharmacol. 55:453–460 (2003). doi:10.1211/002235702928.

    Article  PubMed  CAS  Google Scholar 

  12. P. Opanasopit, M. Nishikawa, and M. Hashida. Factors affecting drug and gene delivery: effects of interaction with blood components. Crit. Rev. Ther. Drug Carrier Syst. 19:191–233 (2002). doi:10.1615/CritRevTherDrugCarrierSyst.v19.i3.10.

    Article  PubMed  CAS  Google Scholar 

  13. W. Li, and F. C. Szoka Jr. Lipid-based nanoparticles for nucleic acid delivery. Pharm. Res. 24:438–449 (2007). doi:10.1007/s11095-006-9180-5.

    Article  PubMed  CAS  Google Scholar 

  14. P. Sapra, P. Tyagi, and T. M. Allen. Ligand-targeted liposomes for cancer treatment. Curr. Drug Deliv. 2:369–381 (2005). doi:10.2174/156720105774370159.

    Article  PubMed  CAS  Google Scholar 

  15. E. Wagner, R. Kircheis, and G. Walker. Targeted nucleic acid delivery into tumors: new avenues for cancer therapy. Biomed. Pharmacother. 58:152–161 (2004). doi:10.1016/j.biopha.2004.01.003.

    Article  PubMed  CAS  Google Scholar 

  16. S. D. Li, and L. Huang. Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Mol. Pharm. 3:579–588 (2006). doi:10.1021/mp060039w.

    Article  PubMed  CAS  Google Scholar 

  17. D. Stuart, and T. Allen. A new liposomal formulation for antisense oligodeoxynucleotides with small size, high incorporation efficiency and good stability. Biochimica et Biophysica Acta. 1463:219–229 (2000). doi:10.1016/S0005-2736(99)00209-6.

    Article  PubMed  CAS  Google Scholar 

  18. J. J. Wheeler, L. Palmer, M. Ossanlou, I. MacLachlan, R. W. Graham, Y. P. Zhang, M. J. Hope, P. Scherrer, and P. R. Cullis. Stabilized plasmid-lipid particles: construction and characterization. Gene Ther. 6:271–281 (1999). doi:10.1038/sj.gt.3300821.

    Article  PubMed  CAS  Google Scholar 

  19. N. Maurer, K. F. Wong, H. Stark, L. Louie, D. McIntosh, T. Wong, P. Scherrer, S. C. Semple, and P. R. Cullis. Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes. Biophys. J. 80:2310–2326 (2001).

    Article  PubMed  CAS  Google Scholar 

  20. L. B. Jeffs, L. R. Palmer, E. G. Ambegia, C. Giesbrecht, S. Ewanick, and I. MacLachlan. A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm. Res. 22:362–372 (2005). doi:10.1007/s11095-004-1873-z.

    Article  PubMed  CAS  Google Scholar 

  21. D. Morrissey, J. Lockridge, L. Shaw, K. Blanchard, K. Jensen, W. Breen, K. Hartsough, L. Machemer, S. Radka, V. Jadhav, N. Vaish, S. Zinnen, C. Vargeese, K. Bowman, C. Shaffer, L. Jeffs, A. Judge, I. MacLachlan, and B. Polisky. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23:1002–1007 (2005). doi:10.1038/nbt1122.

    Article  PubMed  CAS  Google Scholar 

  22. C. Li, and Y. Deng. A novel method for the preparation of liposomes: freeze drying of monophase solutions. J. Pharm. Sci. 93:1403–1414 (2004). doi:10.1002/jps.20055.

    Article  PubMed  CAS  Google Scholar 

  23. T. J. Anchordoquy, J. F. Carpenter, and D. J. Kroll. Maintenance of transfection rates and physical characterization of lipid/DNA complexes after freeze-drying and rehydration. Arch. Biochem. Biophys. 348:199–206 (1997). doi:10.1006/abbi.1997.0385.

    Article  PubMed  CAS  Google Scholar 

  24. J. Clement, K. Kiefer, A. Kimpfler, P. Garidel, and R. Peschka-Suss. Large-scale production of lipoplexes with long shelf-life. Eur. J. Pharm. Biopharm. 59:35–43 (2005). doi:10.1016/j.ejpb.2004.06.001.

    Article  PubMed  CAS  Google Scholar 

  25. M. T. Liang, N. M. Davies, and I. Toth. Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation. Int. J. Pharm. 301:247–254 (2005). doi:10.1016/j.ijpharm.2005.06.010.

    Article  PubMed  CAS  Google Scholar 

  26. W. Gu, L. Putral, K. Hengst, K. Minto, N. A. Saunders, G. Leggatt, and N. A. McMillan. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes. Cancer Gene Ther. 13:1023–1032 (2006). doi:10.1038/sj.cgt.7700971.

    Article  PubMed  CAS  Google Scholar 

  27. C. H. Lee, Y. H. Ni, C. C. Chen, C. K. Chou, and F. H. Chang. Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human cancer cells. Biochim. Biophys. Acta. 1611:55–62 (2003). doi:10.1016/S0005-2736(03)00027-0.

    Article  PubMed  CAS  Google Scholar 

  28. A. Toubaji, S. Hill, M. Terabe, J. Qian, T. Floyd, R. M. Simpson, J. A. Berzofsky, and S. N. Khleif. The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection. Vaccine. 25:5882–5891 (2007). doi:10.1016/j.vaccine.2007.05.040.

    Article  PubMed  CAS  Google Scholar 

  29. F. Brau, J. C. Bernengo, K. L. Min, and J. P. Steghens. Firefly luciferase generates two low-molecular-weight light-emitting species. Biochem. Biophys. Res. Commun. 270:247–253 (2000). doi:10.1006/bbrc.2000.2394.

    Article  PubMed  CAS  Google Scholar 

  30. J. Heyes, K. Hall, V. Tailor, R. Lenz, and I. MacLachlan. Synthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug delivery. J. Control Release. 112:280–290 (2006). doi:10.1016/j.jconrel.2006.02.012.

    Article  PubMed  CAS  Google Scholar 

  31. W. L. Monsky, D. Fukumura, T. Gohongi, M. Ancukiewcz, H. A. Weich, V. P. Torchilin, F. Yuan, and R. K. Jain. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res. 59:4129–4135 (1999).

    PubMed  CAS  Google Scholar 

  32. H. K. de Wolf, C. J. Snel, F. J. Verbaan, R. M. Schiffelers, W. E. Hennink, and G. Storm. Effect of cationic carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous administration. Int. J. Pharm. 331:167–175 (2007). doi:10.1016/j.ijpharm.2006.10.029.

    Article  PubMed  CAS  Google Scholar 

  33. Y. Zhang, E. L. Bradshaw-Pierce, A. Delille, D. L. Gustafson, and T. J. Anchordoquy. In vivo comparative study of lipid/DNA complexes with different in vitro serum stability: effects on biodistribution and tumor accumulation. J. Pharm. Sci. 97:237–250 (2008). doi:10.1002/jps.21076.

    Article  PubMed  CAS  Google Scholar 

  34. I. MacLachlan, and L. Jeffs. Compositions for the delivery of therapeutic agents and uses thereof, Protiva Biotherapeutics, US, 2006, pp. 5, 26.

  35. S. D. Li, Y. C. Chen, M. J. Hackett, and L. Huang. Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol. Ther. 16:163–169 (2008). doi:10.1038/sj.mt.6300323.

    Article  PubMed  CAS  Google Scholar 

  36. S. Semple, S. Klimuk, T. Harasym, N. Santos, S. Ansell, K. Wong, N. Maurer, H. Stark, P. Cullis, M. Hope, and P. Scherrer. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochimica et Biophysica Acta. 1510:152–166 (2001). doi:10.1016/S0005-2736(00)00343-6.

    Article  PubMed  CAS  Google Scholar 

  37. L. Putral, W. Gu, and N. McMillan. RNA interference for the treatment of cancer. Drug News Perspect. 19:317–324 (2006). doi:10.1358/dnp.2006.19.6.985937.

    Article  PubMed  CAS  Google Scholar 

  38. T. J. Anchordoquy, L. G. Girouard, J. F. Carpenter, and D. J. Kroll. Stability of lipid/DNA complexes during agitation and freeze–thawing. J. Pharm. Sci. 87:1046–1051 (1998). doi:10.1021/js9801891.

    Article  PubMed  CAS  Google Scholar 

  39. B. Li, S. Li, Y. Tan, D. B. Stolz, S. C. Watkins, L. H. Block, and L. Huang. Lyophilization of cationic lipid–protamine–DNA (LPD) complexes. J. Pharm. Sci. 89:355–364 (2000). doi:10.1002/(SICI)1520-6017(200003)89:3<355::AID-JPS7>3.0.CO;2-H.

    Article  PubMed  CAS  Google Scholar 

  40. C. Zhang, N. Tang, X. Liu, W. Liang, W. Xu, and V. P. Torchilin. siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene. J. Control Release. 112:229–239 (2006). doi:10.1016/j.jconrel.2006.01.022.

    Article  PubMed  CAS  Google Scholar 

  41. P. Yadava, M. Gibbs, C. Castro, and J. A. Hughes. Effect of lyophilization and freeze–thawing on the stability of siRNA–liposome complexes. AAPS PharmSciTech. 9:335–341 (2008). doi:10.1208/s12249-007-9000-1.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by National Health and Medical Research Council (NHMRC) and we thank Australian Institute for Bioengineering & Nanotechnology for providing Malvern Nano Zetasizer for this study. The authors also gratefully acknowledge Dr Montree Jaturanpinyo for technical assistance and Dr Wenyi Gu for providing cell lines.

Author information

Authors and Affiliations

  1. Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Ipswich Rd, Buranda, QLD 4102, Australia

    Sherry Y. Wu, Lisa N. Putral & Nigel A. J. McMillan

  2. School of Pharmacy, University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia

    Sherry Y. Wu, Mingtao Liang, Hsin-I. Chang & Nigel M. Davies

Authors
  1. Sherry Y. Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisa N. Putral
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mingtao Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hsin-I. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nigel M. Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nigel A. J. McMillan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nigel A. J. McMillan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, S.Y., Putral, L.N., Liang, M. et al. Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use. Pharm Res 26, 512–522 (2009). https://doi.org/10.1007/s11095-008-9766-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-008-9766-1

KEY WORDS

Search

Navigation