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A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

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Purpose.

A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.

Methods.

Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.

Results.

Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.

Conclusions.

This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.

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Abbreviations

Chol:

cholesterol

DODAC:

dioleyldimethylammonium chloride

DODAP:

1,2-dioleoyl-N,N-dimethyl-3-aminopropane

DODMA:

1,2-dioleyloxy-N,N-dimethylaminopropane

DOPE:

dioleoylphosphatidylethanolamine

DOPG:

dioleoylphosphatidylglycerol

DSPC:

distearoylphosphatidylcholine

EPC:

egg phosphatidylcholine

HBS:

Hepes buffered saline

3H-CHE:

tritium-labeled cholesteryl hexadecyl ether

OGP:

octylglucopyranoside

PBS:

phosphate buffered saline

PEG-CerC20:

1-O-(2′-(ω-methoxypolyethyleneglycol)2000)-2-N-arachidoylsphingosine

PEG-S-DSG:

3-O-(2′(ω-methoxypolyethyleneglycol)2000)-1,2-distearoyl-sn-glycerol

QELS:

quasi-elastic light scattering

SPLP:

stabilized plasmid lipid particles

SVF:

spontaneous vesicle formation

TE:

Tris EDTA buffer

TEM:

transmission electron microscopy

TNS:

potassium 2-(p-toluidino)-6-naphthalenesulfonic acid

References

  1. 1.

    1. MacLachlan, P. Cullis, and R. W. Graham. Progress towards a synthetic virus for systemic gene therapy. Curr. Opin. Mol. Ther. 1:252–259 (1999).

    Google Scholar 

  2. 2.

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

    Google Scholar 

  3. 3.

    3. A. H. Sarris, F. Hagemeister, J. Romaguera, M. A. Rodriguez, P. McLaughlin, A. M. Tsimberidou, L. J. Medeiros, B. Samuels, O. Pate, M. Oholendt, H. Kantarjian, C. Burge, and F. Cabanillas. Liposomal vincristine in relapsed non-Hodgkin’s lymphomas: early results of an ongoing phase II trial. Ann. Oncol. 11:69–72 (2000).

    Google Scholar 

  4. 4.

    4. L. D. Mayer, R. Nayar, R. L. Thies, N. L. Boman, P. R. Cullis, and M. B. Bally. Identification of vesicle properties that enhance the antitumour activity of liposomal vincristine against murine L1210 leukemia. Cancer Chemother. Pharmacol. 33:17–24 (1993).

    Google Scholar 

  5. 5.

    5. N. L. Boman, M. B. Bally, P. R. Cullis, L. D. Mayer, and M. S. Webb. Encapsulation of vincristine in liposomes reduces its toxicity and improves its anti-tumor efficacy. J. Liposome Res. 5:523–541 (1995).

    Google Scholar 

  6. 6.

    6. M. S. Webb, T. O. Harasym, D. Masin, M. B. Bally, and L. D. Mayer. Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models. Br. J. Cancer 72:896–904 (1995).

    Google Scholar 

  7. 7.

    7. 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).

    Google Scholar 

  8. 8.

    8. P. Tam, M. Monck, D. Lee, O. Ludkovski, E. Leng, K. Clow, H. Stark, P. Scherrer, R. W. Graham, and P. R. Cullis. Stabilized plasmid lipid particles for systemic gene therapy. Gene Ther. 7:1867–1874 (2000).

    Google Scholar 

  9. 9.

    9. 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).

    Google Scholar 

  10. 10.

    10. D. B. Fenske, I. MacLachlan, and P. R. Cullis. Stabilized plasmid-lipid particles: a systemic gene therapy vector. Methods Enzymol. 346:36–71 (2002).

    Google Scholar 

  11. 11.

    11. I. M. Hafez, S. Ansell, and P. R. Cullis. Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids. Biophys. J. 79:1438–1446 (2000).

    Google Scholar 

  12. 12.

    12. M. A. Monck, A. Mori, D. Lee, P. Tam, J. J. Wheeler, P. R. Cullis, and P. Scherrer. Stabilized plasmid-lipid particles: pharmacokinetics and plasmid delivery to distal tumors following intravenous injection. J. Drug Target. 7:439–452 (2000).

    Google Scholar 

  13. 13.

    13. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. Lipofection—a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U. S. A. 84:7413–7417 (1987).

    Google Scholar 

  14. 14.

    14. H. C. Birnboim and J. Doly. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523 (1979).

    Google Scholar 

  15. 15.

    15. J. Sambrook, E. F. Fritsch, and T. Maniatis. In N. Ford, C. Nolan, and M. Ferguson (eds.), Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, 1989, pp. 1.38–31.39.

    Google Scholar 

  16. 16.

    16. S. Hirota, C. T. de Ilarduya, L. G. Barron, and F. C. J. Szoka. Simple mixing device to reproducibly prepare cationic lipid-DNA complexes (lipoplexes). Biotechniques 27:286–290 (1999).

    Google Scholar 

  17. 17.

    17. C. H. Fiske and Y. Subbarow. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375–400 (1925).

    Google Scholar 

  18. 18.

    18. E. G. Bligh and W. J. Dyer. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911–917 (1959).

    Google Scholar 

  19. 19.

    19. A. L. Bailey and P. R. Cullis. Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids. Biochemistry 33:12573–12580 (1994).

    Google Scholar 

  20. 20.

    20. R. Thomas. The denaturation of DNA. Gene. 135:77–79 (1993).

    Google Scholar 

  21. 21.

    21. T. Schlick, B. Li, and W. K. Olson. The influence of salt on the structure and energetics of supercoiled DNA. Biophys. J. 67:2146–2166 (1994).

    Google Scholar 

  22. 22.

    22. L. D. Mayer, M. J. Hope, and P. R. Cullis. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858:161–168 (1986).

    Google Scholar 

  23. 23.

    23. B. L. Mui, P. R. Cullis, E. A. Evans, and T. D. Madden. Osmotic properties of large unilamellar vesicles prepared by extrusion. Biophys. J. 64:443–453 (1993).

    Google Scholar 

  24. 24.

    24. M. Almgren, K. Edwards, and G. Karlsson. Cryo transmission electron microscopy of liposomes and related structures. Colloids and Surfaces A: Physiochemical and Engineering Aspects 174:3–21 (2000).

    Google Scholar 

  25. 25.

    25. B. L. Mui, H. G. Dobereiner, T. D. Madden, and P. R. Cullis. Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles. Biophys. J. 69:930–941 (1995).

    Google Scholar 

  26. 26.

    26. E. Maurer-Spurej, K. F. Wong, N. Maurer, D. B. Fenske, and P. R. Cullis. Factors influencing uptake and retention of amino-containing drugs in large unilamellar vesicles exhibiting transmembrane pH gradients. Biochim. Biophys. Acta 1416:1–10 (1999).

    Google Scholar 

  27. 27.

    27. M. B. Bally, M. J. Hope, C. J. A. Van Echteld, and P. R. Cullis. Uptake of safranine and other lipophilic cations into model membrane systems in response to a membrane potential. Biochim. Biophys. Acta 812:66–76 (1985).

    Google Scholar 

  28. 28.

    28. 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).

    Google Scholar 

  29. 29.

    29. Y. Perrie and G. Gregoriadis. Liposome-entrapped plasmid DNA: characterisation studies. Biochim. Biophys. Acta 1475:125–132 (2000).

    Google Scholar 

  30. 30.

    30. R. Fraley, S. Subramani, P. Berg, and D. Papahadjopoulos. Introduction of liposome-encapsulated SV-40 DNA into cells. J. Biol. Chem. 255:10431–10435 (1980).

    Google Scholar 

  31. 31.

    31. P. Soriano, J. Dijkstra, A. Legrand, H. Spanjer, D. Londos-Gagliardi, F. Roerdink, G. Scherphof, and C. Nicolau. Targeted and non-targeted liposomes for in vivo transfer to rat liver cells of plasmid containing the preproinsulin I gene. Proc. Natl. Acad. Sci. USA 80:7128–7131 (1983).

    Google Scholar 

  32. 32.

    32. M. Nakanishi, T. Uchida, H. Sugawa, M. Ishiura, and Y. Okada. Efficient introduction of contents of liposomes into cells using HVJ (sendai virus). Exp. Cell Res. 159:399–409 (1985).

    Google Scholar 

  33. 33.

    33. 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).

    Google Scholar 

  34. 34.

    34. R. T. Fraley, C. S. Fornari, and S. Kaplan. Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene therapy. Proc. Natl. Acad. Sci. USA 76:3348–3352 (1979).

    Google Scholar 

  35. 35.

    35. C. Nicolau and S. Rottem. Expression of beta-lactamase activity in Mycoplasma carpicolum transfected with the liposome-encapsulated E. coli pBR32 plasmid. Biochim. Biophys. Res. Comm. 108:982–986 (1982).

    Google Scholar 

  36. 36.

    36. J. C. Stavridis, G. Deliconstantinos, M. C. Psallidopoulos, N. A. Armenakas, D. J. Hadjiminas, and J. Hadjiminas. Construction of transferrin-coated liposomes for in vivo transport of exogenous DNA to bone marrow erythroblasts in rabbits. Exp. Cell Res. 164:568–572 (1986).

    Google Scholar 

  37. 37.

    37. 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. USA. 84:7851–7855 (1987).

    Google Scholar 

  38. 38.

    38. P. F. Lurquin. Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. Nucleic Acids Res. 6:3773–3784 (1979).

    Google Scholar 

  39. 39.

    39. S. F. Alino, M. Bobadilla, M. Garcia-Sanz, M. Lejarreta, F. Unda, and E. Hilario. In vivo delivery of human alpha 1-antitrypsin gene to mouse hepatocytes by liposomes. Biochim. Biophys. Res. Comm. 192:174–181 (1993).

    Google Scholar 

  40. 40.

    40. M. Baru, J. H. Axelrod, and I. Nur. Liposome-encapsulated DNA-mediated gene transfer and synthesis of human factor IX in mice. Gene. 161:143–150 (1995).

    Google Scholar 

  41. 41.

    41. D. G. Jay and W. Gilbert. Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells. Proc. Natl. Acad. Sci. USA 84:1978–1980 (1987).

    Google Scholar 

  42. 42.

    42. C. Puyal, P. Milhaud, A. Bienvenue, and J. R. Philippot. A new cationic liposome encapsulating genetic material. A potential delivery system for polynucleotides. Eur. J. Biochem. 228:697–703 (1995).

    Google Scholar 

  43. 43.

    43. M. Ibanez, P. Gariglio, P. Chavez, R. Santiago, C. Wong, and I. Baeza. Spermidine-condensed DNA and cone-shaped lipids improve delivery and expression of exogenous DNA transfer by liposomes. Biochem. Cell Biol. 74:633–643 (1996).

    Google Scholar 

  44. 44.

    44. J. Szelei and E. Duda. Entrapment of high molecular mass DNA molecules in liposomes for the genetic transformation of animal cells. Biochem. J. 259:549–553 (1989).

    Google Scholar 

  45. 45.

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

    Google Scholar 

  46. 46.

    46. J. S. Choi, J. A. MacKay, and F. C. Szoka. Low-pH-sensitive PEG-stabilized plasmid-lipid nanoparticles: Preparation and characterization. Bioconjugate Chem. 14:420–429 (2003).

    Google Scholar 

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Correspondence to Ian MacLachlan.

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Jeffs, L., Palmer, L., Ambegia, E. et al. A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA. Pharm Res 22, 362–372 (2005). https://doi.org/10.1007/s11095-004-1873-z

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Key Words:

  • DNA
  • liposome
  • plasmid
  • systemic gene delivery