Pharmaceutical Research

, 26:512 | Cite as

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

  • Sherry Y. Wu
  • Lisa N. Putral
  • Mingtao Liang
  • Hsin-I. Chang
  • Nigel M. Davies
  • Nigel A. J. McMillan
Research Paper

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.

KEY WORDS

cancer liposomes PEGylation siRNA systemic gene delivery 

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

Notes

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.

References

  1. 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.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedCrossRefGoogle Scholar
  18. 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.PubMedCrossRefGoogle Scholar
  19. 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).PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 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.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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).PubMedGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 34.
    I. MacLachlan, and L. Jeffs. Compositions for the delivery of therapeutic agents and uses thereof, Protiva Biotherapeutics, US, 2006, pp. 5, 26.Google Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 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.PubMedCrossRefGoogle Scholar
  38. 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.PubMedCrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Sherry Y. Wu
    • 1
    • 2
  • Lisa N. Putral
    • 1
  • Mingtao Liang
    • 2
  • Hsin-I. Chang
    • 2
  • Nigel M. Davies
    • 2
  • Nigel A. J. McMillan
    • 1
  1. 1.Diamantina Institute for Cancer, Immunology and Metabolic MedicineUniversity of Queensland, Princess Alexandra HospitalBurandaAustralia
  2. 2.School of PharmacyUniversity of QueenslandBrisbaneAustralia

Personalised recommendations