Journal of Molecular Medicine

, 84:774 | Cite as

Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

  • Luc Wasungu
  • Marco Scarzello
  • Gooitzen van Dam
  • Grietje Molema
  • Anno Wagenaar
  • Jan B. F. N. Engberts
  • Dick Hoekstra
Original Article

Abstract

In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH range as a consequence of an increased protonation state. In the same way, lipoplexes made with these amphiphiles exhibit a lamellar morphology at physiological pH and a non-lamellar phase at acidic pH. In this study, we confirm that the gemini surfactants are able to form complexes with plasmid DNA at physiological pH and are able to transfect efficiently CHO cells in vitro. Out of the five compounds tested here, two of these amphiphiles, GS1 and GS2, led to 70% of transfected cells with a good cell survival. These two compounds were tested further for in vivo applications. Because of their lamellar organisation, these lipoplexes exhibited a good colloidal stability in salt and in serum at physiological pH compatible with a prolonged stability in vivo. Indeed, when injected intravenously to mice, these stable lipoplexes apparently did not substantially accumulate, as inferred from the observation that transfection of the lungs was not detectable, as examined by in vivo bioluminescence. This potential of avoiding ‘preliminary capture’ in the lungs may, thus, be further exploited in developing devices for specific targeting of gemini lipoplexes.

Keywords

Transfection Gene therapy Cationic liposomes Gemini amphiphiles pH Sensitive Bioluminescence 

Abbreviations

DNA

Deoxyribonucleic acid

lipolexes

Complexes of DNA and cationic lipids

PEG

Poly(ethylene glycol)

GFP

Green fluorescent protein

GS

Gemini surfactant

DOPE

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

DOTAP

N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonim chloride

Saint-2

N-methyl-4-(dioleyl)methylpyridinium

N-Rh-PE

N-(lissamine rhodamine B sulphonyl)phosphatidylethanolamine

FACS

fluorescence-activated cell sorting

SAXS

Small angle X-ray scattering

HEPES

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

MES

2-[N-morpholino]ethanesulfonic acid

HBS solution

HEPES buffered saline solution

CHO cells

Chinese hamster ovarian cells

AU

Arbitrary unit

Notes

Acknowledgements

This work was supported by a grant from The Netherlands Organization for Scientific Research (NWO)/NDRF Innovative Drug Research (940-70-001). The authors would like to thank Dr. Marc Stuart and Dr. Jaap Klijn for helpful discussions.

References

  1. 1.
    Felgner PL (1996) Improvements in cationic liposomes for in vivo gene transfer. Hum Gene Ther 7(15):1791–1793PubMedCrossRefGoogle Scholar
  2. 2.
    Romano G, Claudio PP, Kaiser HE, Giordano A (1998) Recent advances, prospects and problems in designing new strategies for oligonucleotide and gene delivery in therapy. In Vivo 12(1):59–67PubMedGoogle Scholar
  3. 3.
    Felgner PL (1997) Nonviral strategies for gene therapy. Sci Am 276(6):102–106PubMedCrossRefGoogle Scholar
  4. 4.
    van der Woude I, Wagenaar A, Meekel AA, ter Beest MB, Ruiters MH, Engberts JB, Hoekstra D (1997) Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery. Proc Natl Acad Sci U S A 94(4):1160–1165PubMedCrossRefGoogle Scholar
  5. 5.
    Shi F, Nomden A, Oberle V, Engberts JB, Hoekstra D (2001) Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor. Nucleic Acids Res 29(10):2079–2087PubMedCrossRefGoogle Scholar
  6. 6.
    Garcia-Chaumont C, Seksek O, Grzybowska J, Borowski E, Bolard J (2000) Delivery systems for antisense oligonucleotides. Pharmacol Ther 87(2-3):255–277PubMedCrossRefGoogle Scholar
  7. 7.
    Chien PY, Wang J, Carbonaro D, Lei S, Miller B, Sheikh S, Ali SM, Ahmad MU, Ahmad I (2005) Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther 12(3):321–328PubMedCrossRefGoogle Scholar
  8. 8.
    Zelphati O, Wang Y, Kitada S, Reed JC, Felgner PL, Corbeil J (2001) Intracellular delivery of proteins with a new lipid-mediated delivery system. J Biol Chem 276(37):35103–35110PubMedCrossRefGoogle Scholar
  9. 9.
    Simberg D, Weisman S, Talmon Y, Barenholz Y (2004) DOTAP (and other cationic lipids): chemistry, biophysics, and transfection. Crit Rev Ther Drug Carr Syst 21(4):257–317CrossRefGoogle Scholar
  10. 10.
    Simeoni F, Morris MC, Heitz F, Divita G (2003) Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 31(11):2717–2724PubMedCrossRefGoogle Scholar
  11. 11.
    Mountain A (2000) Gene therapy: the first decade. Trends Biotechnol 18(3):119–128PubMedCrossRefGoogle Scholar
  12. 12.
    de Lima MC, Simoes S, Pires P, Faneca H, Duzgunes N (2001) Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications. Adv Drug Deliv Rev 47(2–3):277–294CrossRefGoogle Scholar
  13. 13.
    Simberg D, Weisman S, Talmon Y, Faerman A, Shoshani T, Barenholz Y (2003) The role of organ vascularization and lipoplex-serum initial contact in intravenous murine lipofection. J Biol Chem 278(41):39858–39865PubMedCrossRefGoogle Scholar
  14. 14.
    Mahato RI, Anwer K, Tagliaferri F, Meaney C, Leonard P, Wadhwa MS, Logan M, French M, Rolland A (1998) Biodistribution and gene expression of lipid/plasmid complexes after systemic administration. Hum Gene Ther 9(14):2083–2099PubMedCrossRefGoogle Scholar
  15. 15.
    Niven R, Pearlman R, Wedeking T, Mackeigan J, Noker P, Simpson-Herren L, Smith JG (1998) Biodistribution of radiolabeled lipid-DNA complexes and DNA in mice. J Pharm Sci 87(11):1292–1299PubMedCrossRefGoogle Scholar
  16. 16.
    Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L (1999) Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther 6(4):585–594PubMedCrossRefGoogle Scholar
  17. 17.
    Li S, Rizzo MA, Bhattacharya S, Huang L (1998) Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery. Gene Ther 5(7):930–937PubMedCrossRefGoogle Scholar
  18. 18.
    Tam P, Monck M, Lee D, Ludkovski O, Leng EC, Clow K, Stark H, Scherrer P, Graham RW, Cullis PR (2000) Stabilized plasmid-lipid particles for systemic gene therapy. Gene Ther 7(21):1867–1874PubMedCrossRefGoogle Scholar
  19. 19.
    Bartsch M, Weeke-Klimp AH, Hoenselaar EP, Stuart MC, Meijer DK, Scherphof GL, Kamps JA (2004) Stabilized lipid coated lipoplexes for the delivery of antisense oligonucleotides to liver endothelial cells in vitro and in vivo. J Drug Target 12(9–10):613–621PubMedCrossRefGoogle Scholar
  20. 20.
    Holland JW, Cullis PR, Madden TD (1996) Poly(ethylene glycol)-lipid conjugates promote bilayer formation in mixtures of non-bilayer-forming lipids. Biochemistry 35(8):2610–2617PubMedCrossRefGoogle Scholar
  21. 21.
    Shi F, Wasungu L, Nomden A, Stuart MCA, Polushkin E, Engberts JBFN, Hoekstra D (2002) Interference of polyethylene glycol-lipid analogues with cationic lipid-mediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions. Biochem J 366:333–341PubMedGoogle Scholar
  22. 22.
    Martin-Herranz A, Ahmad A, Evans HM, Ewert K, Schulze U, Safinya CR (2004) Surface functionalized cationic lipid-DNA complexes for gene delivery: PEGylated lamellar complexes exhibit distinct DNA-DNA interaction regimes. Biophys J 86(2):1160–1168PubMedGoogle Scholar
  23. 23.
    Woodle MC (1998) Controlling liposome blood clearance by surface-grafted polymers. Adv Drug Deliv Rev 32(1–2):139–152PubMedCrossRefGoogle Scholar
  24. 24.
    Song LY, Ahkong QF, Rong Q, Wang Z, Ansell S, Hope MJ, Mui B (2002) Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes. Biochim Biophys Acta 1558(1):1–13PubMedCrossRefGoogle Scholar
  25. 25.
    Bergstrand N, Arfvidsson MC, Kim JM, Thompson DH, Edwards K (2003) Interactions between pH-sensitive liposomes and model membranes. Biophys Chem 104(1):361–379PubMedCrossRefGoogle Scholar
  26. 26.
    Li W, Huang Z, MacKay JA, Grube S, Szoka FC Jr (2005) Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery. J Gene Med 7(1):67–79PubMedCrossRefGoogle Scholar
  27. 27.
    Guo X, MacKay JA, Szoka FC Jr (2003) Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid. Biophys J 84(3):1784–1795PubMedGoogle Scholar
  28. 28.
    Ambegia E, Ansell S, Cullis P, Heyes J, Palmer L, MacLachlan I (2005) Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression. Biochim Biophys Acta 1669(2):155–163PubMedCrossRefGoogle Scholar
  29. 29.
    Rejman J, Wagenaar A, Engberts JB, Hoekstra D (2004) Characterization and transfection properties of lipoplexes stabilized with novel exchangeable polyethylene glycol-lipid conjugates. Biochim Biophys Acta 1660(1-2):41–52PubMedCrossRefGoogle Scholar
  30. 30.
    Johnsson M, Wagenaar A, Stuart MCA, Engberts JBFN (2003) Sugar-based gemini surfactants with pH-dependent aggregation behavior: vesicle-to-micelle transition, critical micelle concentration, and vesicle surface charge reversal. Langmuir 19(11):4609–4618CrossRefGoogle Scholar
  31. 31.
    Johnsson M, Wagenaar A, Engberts JBFN (2003) Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH. J Am Chem Soc 125(3):757–760PubMedCrossRefGoogle Scholar
  32. 32.
    Johnsson M, Engberts JBFN (2004) Novel sugar-based gemini surfactants: aggregation properties in aqueous solution. J Phys Org Chem 17(11):934–944CrossRefGoogle Scholar
  33. 33.
    Scarzello M, Klijn JE, Wagenaar A, Stuart MCA, Hulst R, Engberts JBFN (2006) pH-dependent aggregation properties of mixtures of sugar-based gemini surfactants with phospholipids and single-tailed surfactants. Langmuir 22(6):2558–2568PubMedCrossRefGoogle Scholar
  34. 34.
    Fielden ML, Perrin C, Kremer A, Bergsma M, Stuart MCA, Camilleri P, Engberts JBFN (2001) Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro. Eur J Biochem 268(5):1269–1279PubMedCrossRefGoogle Scholar
  35. 35.
    Bell PC, Bergsma M, Dolbnya IP, Bras W, Stuart MCA, Rowan AE, Feiters MC, Engberts JBFN (2003) Transfection mediated by gemini surfactants: engineered escape from the endosomal compartment. J Am Chem Soc 125(6):1551–1558PubMedCrossRefGoogle Scholar
  36. 36.
    Smisterova J, Wagenaar A, Stuart MCA, Polushkin E, ten Brinke G, Hulst R, Engberts JBFN, Hoekstra D (2001) Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery. J Biol Chem 276(50):47615–47622PubMedCrossRefGoogle Scholar
  37. 37.
    Zuhorn IS, Bakowsky U, Polushkin E, Visser WH, Stuart MCA, Engberts JBFN, Hoekstra D (2005) Nonbilayer phase of lipoplex-membrane mixture determines endosomal escape of genetic cargo and transfection efficiency. Mol Ther 11(5):801–810PubMedCrossRefGoogle Scholar
  38. 38.
    Scarzello M, Chupin V, Wagenaar A, Stuart MCA, Engberts JBFN, Hulst R (2005) Polymorphism of pyridinium amphiphiles for gene delivery: influence of ionic strength, helper lipid content, and plasmid DNA complexation. Biophys J 88(3):2104–2113PubMedCrossRefGoogle Scholar
  39. 39.
    Oberle V, Bakowsky U, Zuhorn IS, Hoekstra D (2000) Lipoplex formation under equilibrium conditions reveals a three-step mechanism. Biophys J 79(3):1447–1454PubMedCrossRefGoogle Scholar
  40. 40.
    Zuhorn IS, Visser WH, Bakowsky U, Engberts JB, Hoekstra D (2002) Interference of serum with lipoplex-cell interaction: modulation of intracellular processing. Biochim Biophys Acta 1560(1–2):25–36Google Scholar
  41. 41.
    McElroy WD, DeLuca MA (1983) Firefly and bacterial luminescence: basic science and applications. J Appl Biochem 5(3):197–209PubMedGoogle Scholar
  42. 42.
    Wilson T, Hastings JW (1998) Bioluminescence. Annu Rev Cell Dev Biol 14:197–230PubMedCrossRefGoogle Scholar
  43. 43.
    Scarzello M, Smisterova J, Wagenaar A, Stuart MC, Hoekstra D, Engberts JB, Hulst R (2005) Sunfish cationic amphiphiles: toward an adaptative lipoplex morphology. J Am Chem Soc 127(29):10420–10429PubMedCrossRefGoogle Scholar
  44. 44.
    Audouy SA, de Leij LF, Hoekstra D, Molema G (2002) In vivo characteristics of cationic liposomes as delivery vectors for gene therapy. Pharm Res 19(11):1599–1605PubMedCrossRefGoogle Scholar
  45. 45.
    Iyer M, Berenji M, Templeton NS, Gambhir SS (2002) Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice. Mol Ther 6(4):555–562PubMedCrossRefGoogle Scholar
  46. 46.
    Stuart MCA, van de Pas JC, Engberts JBFN (2005) The use of Nile Red to monitor the aggregation behavior in ternary surfactant-water-organic solvent systems. J Phys Org Chem 18(9):929–934CrossRefGoogle Scholar
  47. 47.
    Li W, Ishida T, Okada Y, Oku N, Kiwada H (2005) Increased gene expression by cationic liposomes (TFL-3) in lung metastases following intravenous injection. Biol Pharm Bull 28(4):701-706PubMedCrossRefGoogle Scholar
  48. 48.
    Audouy S, Molema G, de Leij L, Hoekstra D (2000) Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. J Gene Med 2(6):465-476PubMedCrossRefGoogle Scholar
  49. 49.
    Crook K, Stevenson BJ, Dubouchet M, Porteous DJ (1998) Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther 5(1):137-143PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Luc Wasungu
    • 1
  • Marco Scarzello
    • 2
  • Gooitzen van Dam
    • 3
  • Grietje Molema
    • 4
  • Anno Wagenaar
    • 2
  • Jan B. F. N. Engberts
    • 2
  • Dick Hoekstra
    • 1
  1. 1.Department of Cell Biology/Section Membrane Cell BiologyUniversity Medical Center GroningenGroningenThe Netherlands
  2. 2.Physical Organic Chemistry Unit, Stratingh InstituteUniversity of GroningenGroningenThe Netherlands
  3. 3.BioOptical Imaging Center, Department of SurgeryUniversity Medical Center GroningenGroningenThe Netherlands
  4. 4.Department of Pathology and Laboratory Medicine, Medical Biology SectionUniversity Medical Center GroningenGroningenThe Netherlands

Personalised recommendations