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Novel Liposomal Formulation for Targeted Gene Delivery

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Abstract

Purpose

Development of a polyethylene glycol (PEG)-stabilized immunoliposome (PSIL) formulation with high DNA content suitable for in vivo intravenous administration and targeted gene delivery.

Materials and Methods

Plasmid DNA was condensed using 40% ethanol and packaged into neutral PSILs targeted to the mouse transferrin receptor using monoclonal antibodies (MAbs; clones RI7 and 8D3) attached to their PEG maleimide moieties. PSILs size was measured by quasi-elastic light scattering. The targeting capacity of the formulation was determined by transfection of mouse neuroblastoma Neuro 2A (N2A) cells with PSIL-DNA complexes conjugated with either RI7 or 8D3 MAbs.

Results

DNA encapsulation and MAb conjugation efficiencies averaged 71 ± 14% and 69 ± 5% (mean ± SD), respectively. No alteration in mean particle size (< 100 nm) or DNA leakage were found after 48 h storage in a physiological buffer, and the in vivo terminal half-life reached 23.9 h, indicating that the PSIL-DNA formulation was stable. Addition of free RI7 MAbs prevented transfection of N2A cells with PSIL-DNA complexes conjugated with either RI7 or 8D3 MAbs, confirming that the transfection was transferrin receptor-dependent.

Conclusions

The present data suggest that our new PSIL formulation combines molecular features required for targeted gene therapy including high DNA encapsulation efficiencies and vector-specific transient transfection capacity.

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Abbreviations

2I:

2-iminothiolane

AAV:

adeno-associated virus

ATP:

adenosine 5′-triphosphate

AV:

adenovirus

BSA:

bovine serum albumine

CMV:

cytomegalovirus

[33P]-dCTP:

deoxycytidine 5′-[α-33P]triphosphate

DDAB:

didodecyldimethylammonium bromide

Dpm:

disintegrations per minute

DSPE:

distearoylphosphatidylethanolamine

EtOH:

ethanol

HEPES:

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

HSV-1:

herpes simplex virus-1

LSC:

liquid scintillation counting

MAbs:

monoclonal antibodies

MWCO:

molecular weight cut-off

N2A:

neuroblastoma neuro 2A

NC:

non-conjugated

3H-NSP:

N-succinimidyl-[2,3-3H] propionate

PBS:

phosphate buffered saline

PCR:

polymerase chain reaction

pGLuc:

pCMV-GLuc

PEG:

polyethylene glycol

PEG2000 :

2,000 da polyethylene glycol

POPC:

1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine

PSLs:

PEG-stabilized liposomes

PSILs:

PEG-stabilized immunoliposomes

RLU:

relative light units

RT:

room temperature

QELS:

quasi-elastic light scattering

SEM:

standard error mean

SCID:

severe combined immunodeficiency

SD:

standard deviation

SV40:

simian virus 40

VP-SFM:

virus production serum-free medium

References

  1. M. G. Kaplitt and D. W. Pfaff. Viral vectors for gene delivery and expression in the CNS. Methods 10:343–350 (1996).

    Article  PubMed  CAS  Google Scholar 

  2. J. H. Kordower, M. E. Emborg, J. Bloch, S. Y. Ma, Y. Chu, L. Leventhal, J. McBride, E. Y. Chen, S. Palfi, B. Z. Roitberg, W. D. Brown, J. E. Holden, R. Pyzalski, M. D. Taylor, P. Carvey, Z. Ling, D. Trono, P. Hantraye, N. Deglon, and P. Aebischer. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 290:767–773 (2000).

    Article  PubMed  CAS  Google Scholar 

  3. B. L. Davidson and X. O. Breakefield. Viral vectors for gene delivery to the nervous system. Nat. Rev., Neurosci. 4:353–364 (2003).

    Article  CAS  Google Scholar 

  4. P. R. Lowenstein and M. G. Castro. Recent advances in the pharmacology of neurological gene therapy. Curr. Opin. Pharmacol. 4:91–97 (2004).

    Article  PubMed  CAS  Google Scholar 

  5. P. L. Sinn, S. L. Sauter, and P. B. McCray, Jr. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors—design, biosafety, and production. Gene Ther. 12:1089–1098 (2005).

    Article  PubMed  CAS  Google Scholar 

  6. C. E. Thomas, A. Ehrhardt, and M. A. Kay. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev., Genet. 4:346–358 (2003).

    Article  CAS  Google Scholar 

  7. K. Jooss and N. Chirmule. Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Ther. 10:955–963 (2003).

    Article  PubMed  CAS  Google Scholar 

  8. M. A. Schnell, Y. Zhang, J. Tazelaar, G. P. Gao, Q. C. Yu, R. Qian, S. J. Chen, A. N. Varnavski, C. LeClair, S. E. Raper, and J. M. Wilson. Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors. Molec. Ther. 3:708–722 (2001).

    Article  CAS  Google Scholar 

  9. S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, M. P. McCormack, N. Wulffraat, P. Leboulch, A. Lim, C. S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P. Fraser, J. I. Cohen, G. de Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A. Aurias, D. Stoppa-Lyonnet, S. Romana, I. Radford-Weiss, F. Gross, F. Valensi, E. Delabesse, E. Macintyre, F. Sigaux, J. Soulier, L. E. Leiva, M. Wissler, C. Prinz, T. H. Rabbitts, F. Le Deist, A. Fischer, and M. Cavazzana-Calvo. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415–419 (2003).

    Article  PubMed  CAS  Google Scholar 

  10. D. J. Glover, H. J. Lipps, and D. A. Jans. Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev., Genet. 6:299–310 (2005).

    Article  CAS  Google Scholar 

  11. N. Smyth Templeton. Liposomal delivery of nucleic acids in vivo. DNA Cell Biol. 21:857–867 (2002).

    Article  PubMed  Google Scholar 

  12. T. M. Allen and P. R. Cullis. Drug delivery systems: entering the mainstream. Science 303:1818–1822 (2004).

    Article  PubMed  CAS  Google Scholar 

  13. D. J. Bharali, I. Klejbor, E. K. Stachowiak, P. Dutta, I. Roy, N. Kaur, E. J. Bergey, P. N. Prasad, and M. K. Stachowiak. Organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain. Proc. Natl. Acad. Sci. U. S. A. 102:11539–11544 (2005).

    Article  PubMed  CAS  Google Scholar 

  14. P. H. Tan, M. Manunta, N. Ardjomand, S. A. Xue, D. F. Larkin, D. O. Haskard, K. M. Taylor, and A. J. George. Antibody targeted gene transfer to endothelium. J. Gene Med. 5:311–323 (2003).

    Article  PubMed  CAS  Google Scholar 

  15. Y. Zhang, H. Jeong Lee, R. J. Boado, and W. M. Pardridge. Receptor-mediated delivery of an antisense gene to human brain cancer cells. J. Gene Med. 4:183–194 (2002).

    Article  PubMed  Google Scholar 

  16. P. Machy, F. Lewis, L. McMillan, and Z. L. Jonak. Gene transfer from targeted liposomes to specific lymphoid cells by electroporation. Proc. Natl. Acad. Sci. U. S. A. 85:8027–8031 (1988).

    Article  PubMed  CAS  Google Scholar 

  17. Y. Zhang, F. Calon, C. Zhu, R. J. Boado, and W. M. Pardridge. Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism. Hum. Gene Ther. 14:1–12 (2003).

    Article  PubMed  Google Scholar 

  18. N. Shi, Y. Zhang, C. Zhu, R. J. Boado, and W. M. Pardridge. Brain-specific expression of an exogenous gene after i.v. administration. Proc. Natl. Acad. Sci. U. S. A. 98:12754–12759 (2001).

    Article  PubMed  CAS  Google Scholar 

  19. C. Y. Wang and L. Huang. pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse. Proc. Natl. Acad. Sci. U. S. A. 84:7851–7855 (1987).

    Article  PubMed  CAS  Google Scholar 

  20. N. Zhu, D. Liggitt, Y. Liu, and R. Debs. Systemic gene expression after intravenous DNA delivery into adult mice. Science 261:209–211 (1993).

    Article  PubMed  CAS  Google Scholar 

  21. A. Gabizon, H. Shmeeda, and Y. Barenholz. Pharmacokinetics of pegylated liposomal Doxorubicin: review of animal and human studies. Clin. Pharmacokinet. 42:419–436 (2003).

    Article  PubMed  CAS  Google Scholar 

  22. P. R. Cullis, A. Chonn, and S. C. Semple. Interactions of liposomes and lipid-based carrier systems with blood proteins: relation to clearance behaviour in vivo. Adv. Drug Deliv. Rev. 32:3–17 (1998).

    Article  PubMed  Google Scholar 

  23. K. Maruyama, O. Ishida, T. Takizawa, and K. Moribe. Possibility of active targeting to tumor tissues with liposomes. Adv. Drug Deliv. Rev. 40:89–102 (1999).

    Article  PubMed  CAS  Google Scholar 

  24. A. L. Klibanov, K. Maruyama, V. P. Torchilin, and L. Huang. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 268:235–237 (1990).

    Article  PubMed  CAS  Google Scholar 

  25. A. Schnyder and J. Huwyler. Drug transport to brain with targeted liposomes. NeuroRx 2:99–107 (2005).

    Article  PubMed  Google Scholar 

  26. P. Goyal, K. Goyal, S. G. Kumar, A. Singh, O. P. Katare, and D. N. Mishra. Liposomal drug delivery systems—clinical applications. Acta Pharm. 55:1–25 (2005).

    PubMed  CAS  Google Scholar 

  27. N. Shi and W. M. Pardridge. Noninvasive gene targeting to the brain. Proc. Natl. Acad. Sci. U. S. A. 97:7567–7572 (2000).

    Article  PubMed  CAS  Google Scholar 

  28. L. Cattel, M. Ceruti, and F. Dosio. From conventional to stealth liposomes: a new frontier in cancer chemotherapy. Tumori 89:237–249 (2003).

    PubMed  CAS  Google Scholar 

  29. R. I. Mahato, K. Kawabata, Y. Takakura, and M. Hashida. in vivo disposition characteristics of plasmid DNA complexed with cationic liposomes. J. Drug Target. 3:149–157 (1995).

    PubMed  CAS  Google Scholar 

  30. G. Osaka, K. Carey, A. Cuthbertson, P. Godowski, T. Patapoff, A. Ryan, T. Gadek, and J. Mordenti. Pharmacokinetics, tissue distribution, and expression efficiency of plasmid [33P]DNA following intravenous administration of DNA/cationic lipid complexes in mice: use of a novel radionuclide approach. J. Pharm. Sci. 85:612–618 (1996).

    Article  PubMed  CAS  Google Scholar 

  31. L. D. Leserman, P. Machy, and J. Barbet. Cell-specific drug transfer from liposomes bearing monoclonal antibodies. Nature 293:226–228 (1981).

    Article  PubMed  CAS  Google Scholar 

  32. P. A. Monnard, T. Oberholzer, and P. Luisi. Entrapment of nucleic acids in liposomes. Biochim. Biophys. Acta 1329:39–50 (1997).

    Article  PubMed  CAS  Google Scholar 

  33. N. H. Kim, H. M. Park, S. Y. Chung, E. J. Go, and H. J. Lee. Immunoliposomes carrying plasmid DNA: preparation and characterization. Arch. Pharm. Res. 27:1263–1269 (2004).

    Article  PubMed  CAS  Google Scholar 

  34. D. D. Lasic, B. Ceh, M. C. Stuart, L. Guo, P. M. Frederik, and Y. Barenholz. Transmembrane gradient driven phase transitions within vesicles: lessons for drug delivery. Biochim. Biophys. Acta 1239:145–156 (1995).

    Article  PubMed  Google Scholar 

  35. M. L. Lee, W. Y. Poon, and H. S. Kingdon. A two-phase linear regression model for biologic half-life data. J. Lab. Clin. Med. 115:745–748 (1990).

    PubMed  CAS  Google Scholar 

  36. I. J. Hildebrandt, M. Iyer, E. Wagner, and S. S. Gambhir. Optical imaging of transferrin targeted PEI/DNA complexes in living subjects. Gene Ther. 10:758–764 (2003).

    Article  PubMed  CAS  Google Scholar 

  37. R. R. Nixon. Prion-associated increases in Src-family kinases. J. Biol. Chem. 280:2455–2462 (2005).

    Article  PubMed  CAS  Google Scholar 

  38. A. L. Bailey and S. M. Sullivan. Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium. Biochim. Biophys. Acta 1468:239–252 (2000).

    Article  PubMed  CAS  Google Scholar 

  39. Y. Fang, T. S. Spisz, and J. H. Hoh. Ethanol-induced structural transitions of DNA on mica. Nucleic Acids Res. 27:1943–1949 (1999).

    Article  PubMed  CAS  Google Scholar 

  40. A. Cudd and C. Nicolau. Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation. Biochim. Biophys. Acta 845:477–491 (1985).

    Article  PubMed  CAS  Google Scholar 

  41. 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).

    Article  PubMed  CAS  Google Scholar 

  42. J. Huwyler, D. Wu, and W. M. Pardridge. Brain drug delivery of small molecules using immunoliposomes. Proc. Natl. Acad. Sci. U. S. A. 93:14164–14169 (1996).

    Article  PubMed  CAS  Google Scholar 

  43. Z. M. Qian, H. Li, H. Sun, and K. Ho. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol Rev. 54:561–587 (2002).

    Article  PubMed  CAS  Google Scholar 

  44. W. A. Jefferies, M. R. Brandon, S. V. Hunt, A. F. Williams, K. C. Gatter, and D. Y. Mason. Transferrin receptor on endothelium of brain capillaries. Nature 312:162–163 (1984).

    Article  PubMed  CAS  Google Scholar 

  45. D. C. Mash, J. Pablo, D. D. Flynn, S. M. Efange, and W. J. Weiner. Characterization and distribution of transferrin receptors in the rat brain. J. Neurochem. 55:1972–1979 (1990).

    Article  PubMed  CAS  Google Scholar 

  46. R. J. Boado and W. M. Pardridge. Ten nucleotide cis element in the 3′-untranslated region of the GLUT1 glucose transporter mRNA increases gene expression via mRNA stabilization. Mol. Brain Res. 59:109–113 (1998).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

Grants from the Canadian Institutes of Health Research (CIHR) (FC-RMP72549 and M2C-63922), the Alzheimer Society Canada (FC-ASC 0516), and the Parkinson Society Canada (FC 2004) funded this research. VR was supported by FORMSAV-CIHR and Laval University “Fonds d’Enseignement et de Recherche” faculty of pharmacy studentships.

Competing interests statement

The authors declare that they have no competing financial interests.

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Authors and Affiliations

  1. Molecular Endocrinology and Oncology Research Center, Centre Hospitalier de l’Université Laval (CHUL) Research Center, 2705 Laurier Blvd, Quebec, QC, Canada, G1V 4G2

    Véronique Rivest, Alix Phivilay, Carl Julien, Sandra Bélanger, Cyntia Tremblay,  Vincent Émond & Frédéric Calon

  2. Faculty of Pharmacy, Laval University, Quebec, QC, Canada

    Véronique Rivest, Alix Phivilay, Carl Julien, Sandra Bélanger, Cyntia Tremblay,  Vincent Émond & Frédéric Calon

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  1. Véronique Rivest
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Correspondence to Frédéric Calon.

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Rivest, Phivilay, contributed equally to this work.

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Rivest, V., Phivilay, A., Julien, C. et al. Novel Liposomal Formulation for Targeted Gene Delivery. Pharm Res 24, 981–990 (2007). https://doi.org/10.1007/s11095-006-9224-x

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