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Pharmaceutical Research

, Volume 26, Issue 12, pp 2619–2629 | Cite as

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

  • Abdulgader Baoum
  • Sheng-Xue Xie
  • Amir Fakhari
  • Cory BerklandEmail author
Research Paper

Abstract

Purpose

Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks.

Methods

Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure.

Results

TAT/pDNA complexes were condensed into small, 50–100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these “soft” crosslinks could be competitively disrupted to facilitate DNA release.

Conclusion

Small and stable TAT-Ca/pDNA complexes were obtained via “soft” calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.

KEY WORDS

A549 cells gene delivery plasmid DNA polyethylenimine TAT 

Notes

ACKNOWLEDGMENTS

We would like to acknowledge support for this work from the Coulter Foundation, the Higuchi Biosciences Center, and the Cystic Fibrosis Foundation as well as additional lab funding from the American Heart Association, the NIH (R03 AR054035, P20 RR016443 and T32 GM08359-11) and the Department of Defense. In addition, we acknowledge the support of the NSF (CHE 0719464). We also thank Prof. C. Russ Middaugh for the use of laboratory equipment and the Microscopy Lab for assistance with electron microscopy.

REFERENCES

  1. 1.
    Check E. A tragic setback. Nature. 2002;420:116–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Felgner PL. Nonviral strategies for gene therapy. Sci Am. 1997;276:102–6.PubMedCrossRefGoogle Scholar
  3. 3.
    Hope MJ, Mui B, Ansell S, Ahkong QF. Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (Review). Mol Membr Biol. 1998;15:1–14.CrossRefPubMedGoogle Scholar
  4. 4.
    Marshall E. Clinical trials: gene therapy death prompts review of adenovirus vector. Science. 1999;286:2244.CrossRefPubMedGoogle Scholar
  5. 5.
    Peeters M, Patijn GA, Lieber A, Meuse L, Kay MA. Adenovirus-mediated hepatic gene transfer in mice: comparison of intravascular and biliary administration. Hum Gene Ther. 1996;7:1693–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Thomasand M, Klibanov AM. Non-viral gene therapy: polycation-mediated DNA delivery. Appl Microbiol Biotechnol. 2003;62:27–34.CrossRefGoogle Scholar
  7. 7.
    Yei S, Mittereder N, Tang K, O’Sullivan C, Trapnell BC. Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung. Gene Ther. 1994;1:192–200.PubMedGoogle Scholar
  8. 8.
    Huang L, Hung M. and E. Wagner. Nonviral Vectors for Gene Therapy: Academic; 1999.Google Scholar
  9. 9.
    Davis ME. Non-viral gene delivery systems. Curr Opin Biotechnol. 2002;13:128–31.CrossRefPubMedGoogle Scholar
  10. 10.
    Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci. 1987;84:7413–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Hofland HEJ, Nagy D, Liu JJ, Spratt K, Lee YL, Danos O, et al. In vivo gene transfer by intravenous administration of stable cationic Lipid/DNA complex. Pharm Res. 1997;14:742–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Hortobagyi GN, Ueno NT, Xia W, Zhang S, Wolf JK, Putnam JB, et al. Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects: a Phase I clinical trial. J Clin Oncol. 2001;19:3422.PubMedGoogle Scholar
  13. 13.
    Med JG. Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med. 2005;7:198–207.CrossRefGoogle Scholar
  14. 14.
    Ogrisand M, Wagner E. Targeting tumors with non-viral gene delivery systems. Drug Discov Today. 2002;7:479–85.CrossRefGoogle Scholar
  15. 15.
    Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol. 1997;15:647–52.CrossRefPubMedGoogle Scholar
  16. 16.
    Sambrook J, Russell DW. Introducing cloned gene into cultured mammalian cells. Molecular cloning: A laboratory manual, vol. 16. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Lab; 2001. p. 16.14–9.Google Scholar
  17. 17.
    Tang MX, Redemann CT, Szoka FC. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug Chem. 1996;7:703–14.CrossRefPubMedGoogle Scholar
  18. 18.
    Godbey WT, Wu KK, Mikos AG. Poly (ethylenimine) and its role in gene delivery. J Control Release. 1999;60:149–60.CrossRefPubMedGoogle Scholar
  19. 19.
    Lundberg M, Wikström S, Johansson M. Cell surface adherence and endocytosis of protein transduction domains. Molec Ther. 2003;8:143–50.CrossRefGoogle Scholar
  20. 20.
    Tungand CH, Weissleder R. Arginine containing peptides as delivery vectors. Adv Drug Deliv Rev. 2003;55:281–94.CrossRefGoogle Scholar
  21. 21.
    Cao G, Pei W, Ge H, Liang Q, Luo Y, Sharp FR, et al. In vivo delivery of a Bcl-xL fusion protein containing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis. J Neurosci. 2002;22:5423.PubMedGoogle Scholar
  22. 22.
    Dietzand GPH, Bdhr M. Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol Cell Neurosci. 2004;27:85–131.CrossRefGoogle Scholar
  23. 23.
    Guptaand B, Torchilin VP. Transactivating transcriptional activator-mediated drug delivery. Expert Opin Drug Deliv. 2006;3:177–90.CrossRefGoogle Scholar
  24. 24.
    Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999;285:1569.CrossRefPubMedGoogle Scholar
  25. 25.
    Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000;10:290–5.CrossRefPubMedGoogle Scholar
  26. 26.
    Koch AM, Reynolds F, Merkle HP, Weissleder R, Josephson L. Transport of surface-modified nanoparticles through cell monolayers. ChemBioChem. 2005;6:337–45.CrossRefPubMedGoogle Scholar
  27. 27.
    Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G, Prochiantz A. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem. 1996;271:18188.CrossRefPubMedGoogle Scholar
  28. 28.
    Deshayes S, Heitz A, Morris MC, Charnet P, Divita G, Heitz F. Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis. Biochemistry. 2004;43:1449–57.CrossRefPubMedGoogle Scholar
  29. 29.
    Henriques ST, Costa J, Castanho M. Translocation of β-galactosidase mediated by the cell-penetrating peptide Pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential. Biochemistry. 2005;5:9.Google Scholar
  30. 30.
    Mano M, Teodosio C, Paiva A, Simoes S, de Lima MCP. On the mechanisms of the internalization of S413-PV cell-penetrating peptide. Biochem J. 2005;390:603.CrossRefPubMedGoogle Scholar
  31. 31.
    Patel LN, Zaro JL, Shen WC. Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm Res. 2007;24:1977–92.CrossRefPubMedGoogle Scholar
  32. 32.
    Vives E, Richard JP, Rispal C, Lebleu B. TAT peptide internalization: seeking the mechanism of entry. Current Protein and Peptide Science. 2003;4:125–32.CrossRefPubMedGoogle Scholar
  33. 33.
    Console S, Marty C, Garcia-Echeverria C, Schwendener R, Ballmer-Hofer K. Antennapedia and HIV transactivator of transcription (TAT) “Protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem. 2003;278:35109–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Foerg C, Ziegler U, Fernandez-Carneado J, Giralt E, Rennert R, Beck-Sickinger AG, et al. Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. Biochemistry. 2005;44:72–81.CrossRefPubMedGoogle Scholar
  35. 35.
    Gerbal-Chaloin S, Gondeau C, Aldrian-Herrada G, Heitz F, Gauthier-Rouviere C, Divita G. First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling. Biol Cell. 2007;99:223–38.CrossRefPubMedGoogle Scholar
  36. 36.
    Jones AT. Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides. J Cell Mol Med. 2007;11:670–84.CrossRefPubMedGoogle Scholar
  37. 37.
    Nakase I, Tadokoro A, Kawabata N, Takeuchi T, Katoh H, Hiramoto K, et al. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry. 2007;46:492–501.CrossRefPubMedGoogle Scholar
  38. 38.
    Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B, Chernomordik LV. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem. 2005;280:15300.CrossRefPubMedGoogle Scholar
  39. 39.
    Silhol M, Tyagi M, Giacca M, Lebleu B, Vives E. Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat. Eur J Biochem. 2002;269:494–501.CrossRefPubMedGoogle Scholar
  40. 40.
    Thorén PEG, Persson D, Isakson P, Goksor M, Onfelt A, Nordén B. Uptake of analogs of penetratin, Tat (48–60) and oligoarginine in live cells. Biochem Biophys Res Commun. 2003;307:100–7.CrossRefPubMedGoogle Scholar
  41. 41.
    Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med. 2004;10:310–5.CrossRefPubMedGoogle Scholar
  42. 42.
    Jarver P, Langel Ü. Cell-penetrating peptides—a brief introduction. BBA-Biomembranes. 2006;1758:260–3.CrossRefPubMedGoogle Scholar
  43. 43.
    Mae M, Myrberg H, Jiang Y, Paves H, Valkna A, Langel Ü. Internalisation of cell-penetrating peptides into tobacco protoplasts. BBA-Biomembranes. 2005;1669:101–7.CrossRefPubMedGoogle Scholar
  44. 44.
    Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, et al. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci. 1994;91:664–8.CrossRefPubMedGoogle Scholar
  45. 45.
    Frankeland AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988;55:1189–93.CrossRefGoogle Scholar
  46. 46.
    Truantand R, Cullen BR. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct Importin β-dependent nuclear localization signals. Mol Cell Biol. 1999;19:1210–7.Google Scholar
  47. 47.
    Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem. 1997;272:16010–7.CrossRefPubMedGoogle Scholar
  48. 48.
    Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, et al. Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J Biol Chem. 2001;276:26204–10.CrossRefPubMedGoogle Scholar
  49. 49.
    Torchilin VP, Rammohan R, Weissig V, Levchenko TS. TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl Acad Sci. 2001;98:8786.CrossRefPubMedGoogle Scholar
  50. 50.
    Tungand CH, Stein S. Preparation and applications of peptide-oligonucleotide conjugates. Bioconjug Chem. 2000;11:605–18.CrossRefGoogle Scholar
  51. 51.
    Futaki S, Ohashi W, Suzuki T, Niwa M, Tanaka S, Ueda K, et al. Stearylated arginine-rich peptides: a new class of transfection systems. Bioconjug Chem. 2001;12:1005–11.CrossRefPubMedGoogle Scholar
  52. 52.
    Ignatovich IA, Dizhe EB, Pavlotskaya AV, Akifiev BN, Burov SV, Orlov SV, et al. Complexes of plasmid DNA with basic domain 47–57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways. J Biol Chem. 2003;278:42625–36.CrossRefPubMedGoogle Scholar
  53. 53.
    Sandgren S, Cheng F, Belting M. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans. J Biol Chem. 2002;277:38877–83.CrossRefPubMedGoogle Scholar
  54. 54.
    Tung CH, Mueller S, Weissleder R. Novel branching membrane translocational peptide as gene delivery vector. Bioorg Med Chem. 2002;10:3609–14.CrossRefPubMedGoogle Scholar
  55. 55.
    Haberland A, Knaus T, Zaitsev SV, Buchberger B, Lun A, Haller H, et al. Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ Ions. Pharm Res. 2000;17:229–35.CrossRefPubMedGoogle Scholar
  56. 56.
    Nchinda G, Uberla K, Zschornig O. Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition. BMC Biotechnol. 2002;2:12.CrossRefPubMedGoogle Scholar
  57. 57.
    Zelphati O, Uyechi LS, Barron LG, Szoka FC. Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1998;1390:119–33.CrossRefGoogle Scholar
  58. 58.
    Kwon DS, Lin CH, Chen S, Coward JK, Walsh CT, Bollinger JM Jr. Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains. J Biol Chem. 1997;272:2429.CrossRefPubMedGoogle Scholar
  59. 59.
    Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther. 1996;7:1947–54.CrossRefPubMedGoogle Scholar
  60. 60.
    Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci. 1995;92:7297–301.CrossRefPubMedGoogle Scholar
  61. 61.
    Huang CY, Ma SS, Lee S, Radhakrishnan R, Braun CS, Choosakoonkriang S, et al. Enhancements in gene expression by the choice of plasmid DNA formulations containing neutral polymeric excipients. J Pharm Sci. 2002;91:1371–81.CrossRefPubMedGoogle Scholar
  62. 62.
    Lobo BA, Vetro JA, Suich DM, Zuckermann RN, Middaugh CR. Structure/function analysis of peptoid/lipitoid: DNA complexes. J Pharm Sci. 2003;92:1905–18.CrossRefPubMedGoogle Scholar
  63. 63.
    Tiyaboonchai W, Woiszwillo J, Middaugh CR. Formulation and characterization of DNA–polyethylenimine–dextran sulfate nanoparticles. Eur J Pharm Sci. 2003;19:191–202.CrossRefPubMedGoogle Scholar
  64. 64.
    Wiethoff CM, Koe JG, Koe GS, Middaugh CR. Compositional effects of cationic lipid/DNA delivery systems on transgene expression in cell culture. J Pharm Sci. 2004;93:108–23.CrossRefPubMedGoogle Scholar
  65. 65.
    Choosakoonkriang S, Lobo BA, Koe GS, Koe JG, Middaugh CR. Biophysical characterization of PEI/DNA complexes. J Pharm Sci. 2003;92:1710–22.CrossRefPubMedGoogle Scholar
  66. 66.
    Forrest ML, Koerber JT, Pack DW. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug Chem. 2003;14:934–40.CrossRefPubMedGoogle Scholar
  67. 67.
    Lindsay MA. Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol. 2002;2:587–94.CrossRefPubMedGoogle Scholar
  68. 68.
    Snyderand EL, Dowdy SF. Cell penetrating peptides in drug delivery. Pharm Res. 2004;21:389–93.CrossRefGoogle Scholar
  69. 69.
    Pouton CW, Lucas P, Thomas BJ, Uduehi AN, Milroy DA, Moss SH. Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. J Control Release. 1998;53:289–99.CrossRefPubMedGoogle Scholar
  70. 70.
    Simberg D, Danino D, Talmon Y, Minsky A, Ferrari ME, Wheeler CJ, et al. Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity. J Biol Chem. 2001;276:47453–9.CrossRefPubMedGoogle Scholar
  71. 71.
    Turek J, Dubertret C, Jaslin G, Antonakis K, Scherman D, Pitard B. Formulations which increase the size of lipoplexes prevent serum-associated inhibition of transfection. J Gene Med. 2000;2:32–40.CrossRefPubMedGoogle Scholar
  72. 72.
    Wagner E, Cotten M, Foisner R, Birnstiel ML. Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc Natl Acad Sci. 1991;88:4255–9.CrossRefPubMedGoogle Scholar
  73. 73.
    Moret I, Esteban Peris J, Guillem VM, Benet M, Revert F, Dasi F, et al. Stability of PEI–DNA and DOTAP–DNA complexes: effect of alkaline pH, heparin and serum. J Control Release. 2001;76:169–81.CrossRefPubMedGoogle Scholar
  74. 74.
    Ruponen M, Yla-Herttuala S, Urtti A. Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies. BBA-Biomembranes. 1999;1415:331–41.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Abdulgader Baoum
    • 1
  • Sheng-Xue Xie
    • 1
  • Amir Fakhari
    • 2
  • Cory Berkland
    • 1
    • 3
    Email author
  1. 1.Department of Pharmaceutical ChemistryThe University of KansasLawrenceUSA
  2. 2.Department of BioengineeringThe University of KansasLawrenceUSA
  3. 3.Department of Chemical and Petroleum EngineeringThe University of KansasLawrenceUSA

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