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Visualizing Uptake and Intracellular Trafficking of Gene Carriers by Single-Particle Tracking

  • N. RuthardtEmail author
  • C. Bräuchle
Chapter
Part of the Topics in Current Chemistry book series (TOPCURRCHEM, volume 296)

Abstract

Single-particle microscopy und live-cell single-particle tracking are powerful tools to follow the cellular internalization pathway of individual nanoparticles such as viruses and gene carriers and investigate their interaction with living cells. Those single-cell and single-particle methods can elucidate the “black box” between application of the gene carrier to the cell and the final gene expression and allow the essential bottlenecks to be identified in great detail on the cellular level. In this review we will give a short introduction into single-particle tracking microscopy and present an overview of the mechanisms of DNA delivery from attachment to the cell membrane over internalization towards nuclear entry unraveled by single-particle methods.

Keywords

DNA/RNA transfection Fluorescence wide-field microscopy Gene carriers Gene therapy Single-particle tracking Trajectory analysis 

Abbreviations

EGF

Epidermal growth factor

EGFP

Enhanced green fluorescent protein

NGF

Nerve growth factor

PEI

Polyethyleneimide

PLL

Poly-l-lysine

Notes

Acknowledgments

This work was supported by the Nanosystems Initiative Munich (NIM) and the Center for Integrated Protein Science Munich (CiPSM) and the SFB 749.

References

  1. 1.
    Verma IM, Weitzman MD (2005) Gene therapy: twenty-first century medicine. Annu Rev Biochem 74:711–738PubMedCrossRefGoogle Scholar
  2. 2.
    Read ML, Logan A, Seymour LW (2005) Barriers to gene delivery using synthetic vectors. Adv Genet 53PA:19–46PubMedCrossRefGoogle Scholar
  3. 3.
    Burton EA, Fink DJ, Glorioso JC (2002) Gene delivery using herpes simplex virus vectors. DNA Cell Biol 21:915–936PubMedCrossRefGoogle Scholar
  4. 4.
    Campbell EM, Hope TJ (2005) Gene therapy progress and prospects: viral trafficking during infection. Gene Ther 12:1353–1359PubMedCrossRefGoogle Scholar
  5. 5.
    Carter PJ, Samulski RJ (2000) Adeno-associated viral vectors as gene delivery vehicles. Int J Mol Med 6:17–27PubMedGoogle Scholar
  6. 6.
    Duan Y, Zhang S, Wang B, Yang B, Zhi D (2009) The biological routes of gene delivery mediated by lipid-based non-viral vectors. Expert Opin Drug Deliv 6:1351–1361PubMedCrossRefGoogle Scholar
  7. 7.
    Liu F, Huang L (2002) Development of non-viral vectors for systemic gene delivery. J Control Release 78:259–266PubMedCrossRefGoogle Scholar
  8. 8.
    Niidome T, Huang L (2002) Gene therapy progress and prospects: nonviral vectors. Gene Ther 9:1647–1652PubMedCrossRefGoogle Scholar
  9. 9.
    Park TG, Jeong JH, Kim SW (2006) Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 58:467–486PubMedCrossRefGoogle Scholar
  10. 10.
    Schaffert D, Wagner E (2008) Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther 15:1131–1138PubMedCrossRefGoogle Scholar
  11. 11.
    Wagner E (2004) Strategies to improve DNA polyplexes for in vivo gene transfer: will “artificial viruses” be the answer? Pharm Res 21:8–14PubMedCrossRefGoogle Scholar
  12. 12.
    Brandenburg B, Zhuang X (2007) Virus trafficking – learning from single-virus tracking. Nat Rev Microbiol 5:197–208PubMedCrossRefGoogle Scholar
  13. 13.
    Lakadamyali M, Rust MJ, Babcock HP, Zhuang X (2003) Visualizing infection of individual influenza viruses. Proc Natl Acad Sci USA 100:9280–9285PubMedCrossRefGoogle Scholar
  14. 14.
    Marsh M, Helenius A (2006) Virus entry: open sesame. Cell 124:729–740PubMedCrossRefGoogle Scholar
  15. 15.
    Seisenberger G, Ried MU, Endress T, Buning H, Hallek M, Bräuchle C (2001) Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294:1929–1932PubMedCrossRefGoogle Scholar
  16. 16.
    Khalil IA, Kogure K, Akita H, Harashima H (2006) Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev 58:32–45PubMedCrossRefGoogle Scholar
  17. 17.
    Medina-Kauwe LK, Xie J, Hamm-Alvarez S (2005) Intracellular trafficking of nonviral vectors. Gene Ther 12:1734–1751PubMedCrossRefGoogle Scholar
  18. 18.
    Payne CK (2007) Imaging gene delivery with fluorescence microscopy. Nanomed 2:847–860CrossRefGoogle Scholar
  19. 19.
    Watson P, Jones AT, Stephens DJ (2005) Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells. Adv Drug Deliv Rev 57:43–61PubMedCrossRefGoogle Scholar
  20. 20.
    Vercauteren D et al (2010) The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol Ther 18:561–569Google Scholar
  21. 21.
    Rejman J, Bragonzi A, Conese M (2005) Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 12:468–474PubMedCrossRefGoogle Scholar
  22. 22.
    Remy-Kristensen A, Clamme JP, Vuilleumier C, Kuhry JG, Mely Y (2001) Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim Biophys Acta 1514:21–32PubMedCrossRefGoogle Scholar
  23. 23.
    Kopatz I, Remy JS, Behr JP (2004) A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 6:769–776PubMedCrossRefGoogle Scholar
  24. 24.
    Lundin P, Johansson H, Guterstam P, Holm T, Hansen M, Langel U, EL Andaloussi S (2008) Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug Chem 19:2535–2542PubMedCrossRefGoogle Scholar
  25. 25.
    Mae M, Andaloussi SE, Lehto T, Langel U (2009) Chemically modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opin Drug Deliv 6:1195–1205PubMedCrossRefGoogle Scholar
  26. 26.
    Rinne J, Albarran B, Jylhava J, Ihalainen TO, Kankaanpaa P, Hytonen VP, Stayton PS, Kulomaa MS, Vihinen-Ranta M (2007) Internalization of novel non-viral vector TAT-streptavidin into human cells. BMC Biotechnol 7:1PubMedCrossRefGoogle Scholar
  27. 27.
    von Gersdorff K, Sanders NN, Vandenbroucke R, De Smedt SC, Wagner E, Ogris M (2006) The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol Ther 14:745–753CrossRefGoogle Scholar
  28. 28.
    Benns JM, Kim SW (2000) Tailoring new gene delivery designs for specific targets. J Drug Target 8:1–12PubMedCrossRefGoogle Scholar
  29. 29.
    Cheng H, Zhu JL, Zeng X, Jing Y, Zhang XZ, Zhuo RX (2009) Targeted gene delivery mediated by folate-polyethylenimine-block-poly(ethylene glycol) with receptor selectivity. Bioconjug Chem 20:481–487PubMedCrossRefGoogle Scholar
  30. 30.
    Lu T, Sun J, Chen X, Zhang P, Jing X (2009) Folate-conjugated micelles and their folate-receptor-mediated endocytosis. Macromol Biosci 9:1059–1068PubMedCrossRefGoogle Scholar
  31. 31.
    Frederiksen KS, Abrahamsen N, Cristiano RJ, Damstrup L, Poulsen HS (2000) Gene delivery by an epidermal growth factor/DNA polyplex to small cell lung cancer cell lines expressing low levels of epidermal growth factor receptor. Cancer Gene Ther 7:262–268PubMedCrossRefGoogle Scholar
  32. 32.
    Godinez WJ, Lampe M, Worz S, Muller B, Eils R, Rohr K (2009) Deterministic and probabilistic approaches for tracking virus particles in time-lapse fluorescence microscopy image sequences. Med Image Anal 13:325–342PubMedCrossRefGoogle Scholar
  33. 33.
    Sbalzarini IF, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 151:182–195PubMedCrossRefGoogle Scholar
  34. 34.
    Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065PubMedCrossRefGoogle Scholar
  35. 35.
    Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399PubMedCrossRefGoogle Scholar
  36. 36.
    Qian H, Sheetz MP, Elson EL (1991) Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J 60:910–921PubMedCrossRefGoogle Scholar
  37. 37.
    de Bruin K, Ruthardt N, von Gersdorff K, Bausinger R, Wagner E, Ogris M, Bräuchle C (2007) Cellular dynamics of EGF receptor-targeted synthetic viruses. Mol Ther 15:1297–1305PubMedCrossRefGoogle Scholar
  38. 38.
    Bausinger R, von Gersdorff K, Braeckmans K, Ogris M, Wagner E, Bräuchle C, Zumbusch A (2006) The transport of nanosized gene carriers unraveled by live-cell imaging. Angew Chem Int Ed Engl 45:1568–1572PubMedCrossRefGoogle Scholar
  39. 39.
    Mislick KA, Baldeschwieler JD (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci USA 93:12349–12354PubMedCrossRefGoogle Scholar
  40. 40.
    Payne CK, Jones SA, Chen C, Zhuang X (2007) Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 8:389–401PubMedCrossRefGoogle Scholar
  41. 41.
    Suh J, Wirtz D, Hanes J (2004) Real-time intracellular transport of gene nanocarriers studied by multiple particle tracking. Biotechnol Prog 20:598–602PubMedCrossRefGoogle Scholar
  42. 42.
    Cui B, Wu C, Chen L, Ramirez A, Bearer EL, Li WP, Mobley WC, Chu S (2007) One at a time, live tracking of NGF axonal transport using quantum dots. Proc Natl Acad Sci USA 104:13666–13671PubMedCrossRefGoogle Scholar
  43. 43.
    Fehrenbacher K, Huckaba T, Yang HC, Boldogh I, Pon L (2003) Actin comet tails, endosomes and endosymbionts. J Exp Biol 206:1977–1984PubMedCrossRefGoogle Scholar
  44. 44.
    Merrifield CJ, Moss SE, Ballestrem C, Imhof BA, Giese G, Wunderlich I, Almers W (1999) Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nat Cell Biol 1:72–74PubMedCrossRefGoogle Scholar
  45. 45.
    Mahowald J, Arcizet D, Heinrich D (2009) Impact of external stimuli and cell micro-architecture on intracellular transport states. Chemphyschem 10:1559–1566PubMedCrossRefGoogle Scholar
  46. 46.
    Arcizet D, Meier B, Sackmann E, Rädler JO, Heinrich D (2008) Temporal analysis of active and passive transport in living cells. Phys Rev Lett 101:248103PubMedCrossRefGoogle Scholar
  47. 47.
    Suh J, Wirtz D, Hanes J (2003) Efficient active transport of gene nanocarriers to the cell nucleus. Proc Natl Acad Sci USA 100:3878–3882PubMedCrossRefGoogle Scholar
  48. 48.
    Sauer AM, de Bruin KG, Ruthardt N, Mykhaylyk O, Plank C, Bräuchle C (2009) Dynamics of magnetic lipoplexes studied by single-particle tracking in living cells. J Control Release 137:136–145PubMedCrossRefGoogle Scholar
  49. 49.
    Rajan SS, Liu HY, Vu TQ (2008) Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells. ACS Nano 2:1153–1166PubMedCrossRefGoogle Scholar
  50. 50.
    Nan X, Sims PA, Chen P, Xie XS (2005) Observation of individual microtubule motor steps in living cells with endocytosed quantum dots. J Phys Chem B 109:24220–24224PubMedCrossRefGoogle Scholar
  51. 51.
    Lechardeur D, Verkman AS, Lukacs GL (2005) Intracellular routing of plasmid DNA during non-viral gene transfer. Adv Drug Deliv Rev 57:755–767PubMedCrossRefGoogle Scholar
  52. 52.
    Klemm AR, Young D, Lloyd JB (1998) Effects of polyethyleneimine on endocytosis and lysosome stability. Biochem Pharmacol 56:41–46PubMedCrossRefGoogle Scholar
  53. 53.
    Sonawane ND, Szoka FC Jr, Verkman AS (2003) Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem 278:44826–44831PubMedCrossRefGoogle Scholar
  54. 54.
    Clamme JP, Krishnamoorthy G, Mely Y (2003) Intracellular dynamics of the gene delivery vehicle polyethylenimine during transfection: investigation by two-photon fluorescence correlation spectroscopy. Biochim Biophys Acta 1617:52–61PubMedCrossRefGoogle Scholar
  55. 55.
    Boeckle S, Fahrmeir J, Roedl W, Ogris M, Wagner E (2006) Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes. J Control Release 112:240–248PubMedCrossRefGoogle Scholar
  56. 56.
    Ogris M, Carlisle RC, Bettinger T, Seymour LW (2001) Melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors. J Biol Chem 276:47550–47555PubMedCrossRefGoogle Scholar
  57. 57.
    de Bruin KG, Fella C, Ogris M, Wagner E, Ruthardt N, Bräuchle C (2008) Dynamics of photoinduced endosomal release of polyplexes. J Control Release 130:175–182PubMedCrossRefGoogle Scholar
  58. 58.
    Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS (2000) Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem 275:1625–1629PubMedCrossRefGoogle Scholar
  59. 59.
    Vaughan EE, Dean DA (2006) Intracellular trafficking of plasmids during transfection is mediated by microtubules. Mol Ther 13:422–428PubMedCrossRefGoogle Scholar
  60. 60.
    Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, Rodriguez-Boulan E, Crystal RG (2000) Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 11:151–165PubMedCrossRefGoogle Scholar
  61. 61.
    Dohner K, Radtke K, Schmidt S, Sodeik B (2006) Eclipse phase of herpes simplex virus type 1 infection: efficient dynein-mediated capsid transport without the small capsid protein VP26. J Virol 80:8211–8224PubMedCrossRefGoogle Scholar
  62. 62.
    Dohner K, Wolfstein A, Prank U, Echeverri C, Dujardin D, Vallee R, Sodeik B (2002) Function of dynein and dynactin in herpes simplex virus capsid transport. Mol Biol Cell 13:2795–2809PubMedCrossRefGoogle Scholar
  63. 63.
    Suk JS, Suh J, Lai SK, Hanes J (2007) Quantifying the intracellular transport of viral and nonviral gene vectors in primary neurons. Exp Biol Med (Maywood) 232:461–469Google Scholar
  64. 64.
    Matsumoto Y, Itaka K, Yamasoba T, Kataoka K (2009) Intranuclear fluorescence resonance energy transfer analysis of plasmid DNA decondensation from nonviral gene carriers. J Gene Med 11:615–623PubMedCrossRefGoogle Scholar
  65. 65.
    Godbey WT, Barry MA, Saggau P, Wu KK, Mikos AG (2000) Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J Biomed Mater Res 51:321–328PubMedCrossRefGoogle Scholar
  66. 66.
    Godbey WT, Wu KK, Mikos AG (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci USA 96:5177–5181PubMedCrossRefGoogle Scholar
  67. 67.
    Itaka K, Harada A, Yamasaki Y, Nakamura K, Kawaguchi H, Kataoka K (2004) In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J Gene Med 6:76–84PubMedCrossRefGoogle Scholar
  68. 68.
    Bieber T, Meissner W, Kostin S, Niemann A, Elsasser HP (2002) Intracellular route and transcriptional competence of polyethylenimine-DNA complexes. J Control Release 82:441–454PubMedCrossRefGoogle Scholar
  69. 69.
    Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther 7:401–407PubMedCrossRefGoogle Scholar
  70. 70.
    Mortimer I, Tam P, MacLachlan I, Graham RW, Saravolac EG, Joshi PB (1999) Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther 6:403–411PubMedCrossRefGoogle Scholar
  71. 71.
    Tseng WC, Haselton FR, Giorgio TD (1999) Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochim Biophys Acta 1445:53–64PubMedCrossRefGoogle Scholar
  72. 72.
    Schwake G, Youssef S, Kuhr JT, Gude S, David MP, Mendoza E, Frey E, Rädler JO (2009) Predictive modeling of non-viral gene transfer. Biotechnol Bioeng 105:805–813Google Scholar
  73. 73.
    Cohen RN, van der Aa MA, Macaraeg N, Lee AP, Szoka FC Jr (2009) Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection. J Control Release 135:166–174PubMedCrossRefGoogle Scholar
  74. 74.
    Holtzer L, Meckel T, Schmidt T (2007) Nanometric three-dimensional tracking of individual quantum dots in cells. Appl Phys Lett 90:1–3CrossRefGoogle Scholar
  75. 75.
    Katayama Y, Burkacky O, Meyer M, Bräuchle C, Gratton E, Lamb DC (2009) Real-time nanomicroscopy via three-dimensional single-particle tracking. Chemphyschem 10:2458–2464PubMedCrossRefGoogle Scholar
  76. 76.
    Wagner E (2007) Programmed drug delivery: nanosystems for tumor targeting. Expert Opin Biol Ther 7:587–593PubMedCrossRefGoogle Scholar
  77. 77.
    Zintchenko A, Susha AS, Concia M, Feldmann J, Wagner E, Rogach AL, Ogris M (2009) Drug nanocarriers labeled with near-infrared-emitting quantum dots (quantoplexes): imaging fast dynamics of distribution in living animals. Mol Ther 17:1849–1856PubMedCrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryLudwig-Maximilians-Universität MünchenMünchenGermany
  2. 2.Center for NanoScience (CeNS)Ludwig-Maximilians-Universität MünchenMünchenGermany

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