Pharmaceutical Research

, Volume 34, Issue 1, pp 161–174 | Cite as

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells

  • Ruth Röder
  • Jonas Helma
  • Tobias Preiß
  • Joachim O. Rädler
  • Heinrich Leonhardt
  • Ernst Wagner
Research Paper

Abstract

Purpose

Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells.

Methods

Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy.

Results

The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies.

Conclusion

Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

KEY WORDS

folate nanobody oligoaminoamides protein delivery receptor targeting 

ABBREVIATIONS

FCS

Fluorescence correlation spectroscopy

FolA

Folic acid

HcAb

Heavy-chain only camelid antibody

Nb

Nanobody

PEG

Polyethyleneglycol

Stp

Succinoyl tetraethylene pentamine

Supplementary material

11095_2016_2052_MOESM1_ESM.pdf (1.2 mb)
ESM 1(PDF 1.24 mb)
11095_2016_2052_MOESM2_ESM.avi (3.4 mb)
Supplementary Video 1(AVI 3458 kb)
11095_2016_2052_MOESM3_ESM.avi (2.4 mb)
Supplementary Video 2(AVI 2504 kb)

References

  1. 1.
    Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng. 1994;7(9):1129–35.CrossRefPubMedGoogle Scholar
  2. 2.
    Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res. 2012;110(7):927–37.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bleck M, Itano MS, Johnson DS, Thomas VK, North AJ, Bieniasz PD, et al. Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding. Proc Natl Acad Sci U S A. 2014;111(33):12211–6.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, et al. Generation of an alpaca-derived nanobody recognizing gamma-H2AX. FEBS Open Bio. 2015;5:779–88.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, et al. Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol. 2010;17(1):133–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods. 2006;3(11):887–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Helma J, Cardoso MC, Muyldermans S, Leonhardt H. Nanobodies and recombinant binders in cell biology. J Cell Biol. 2015;209(5):633–44.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dmitriev OY, Lutsenko S, Muyldermans S. Nanobodies as probes for protein dynamics in vitro and in cells. J Biol Chem. 2016;291(8):3767–75.CrossRefPubMedGoogle Scholar
  9. 9.
    Arbabi-Ghahroudi M, Tanha J, MacKenzie R. Prokaryotic expression of antibodies. Cancer Metastasis Rev. 2005;24(4):501–19.CrossRefPubMedGoogle Scholar
  10. 10.
    Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W, De Genst E, et al. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J. 1998;17(13):3512–20.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lisy MR, Goermar A, Thomas C, Pauli J, Resch-Genger U, Kaiser WA, et al. In vivo near-infrared fluorescence imaging of carcinoembryonic antigen-expressing tumor cells in mice. Radiology. 2008;247(3):779–87.CrossRefPubMedGoogle Scholar
  12. 12.
    Sawant R, Torchilin V. Intracellular transduction using cell-penetrating peptides. Mol BioSyst. 2010;6(4):628–40.CrossRefPubMedGoogle Scholar
  13. 13.
    Kaczmarczyk SJ, Sitaraman K, Young HA, Hughes SH, Chatterjee DK. Protein delivery using engineered virus-like particles. Proc Natl Acad Sci U S A. 2011;108(41):16998–7003.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mendez J, Morales Cruz M, Delgado Y, Figueroa CM, Orellano EA, Morales M, et al. Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells. Mol Pharm. 2014;11(1):102–11.CrossRefPubMedGoogle Scholar
  15. 15.
    Schlossbauer A, Sauer AM, Cauda V, Schmidt A, Engelke H, Rothbauer U, et al. Cascaded photoinduced drug delivery to cells from multifunctional core-shell mesoporous silica. Adv Healthc Mater. 2012;1(3):316–20.CrossRefPubMedGoogle Scholar
  16. 16.
    Chiu HY, Deng W, Engelke H, Helma J, Leonhardt H, Bein T. Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time. Sci Rep. 2016;6:25019.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ray M, Tang R, Jiang Z, Rotello VM. Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules. Bioconjug Chem. 2015;26(6):1004–7.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lee Y, Ishii T, Kim HJ, Nishiyama N, Hayakawa Y, Itaka K, et al. Efficient delivery of bioactive antibodies into the cytoplasm of living cells by charge-conversional polyion complex micelles. Angew Chem Int Ed Engl. 2010;49(14):2552–5.CrossRefPubMedGoogle Scholar
  19. 19.
    Lee Y, Ishii T, Cabral H, Kim HJ, Seo JH, Nishiyama N, et al. Charge-conversional polyionic complex micelles-efficient nanocarriers for protein delivery into cytoplasm. Angew Chem Int Ed Engl. 2009;48(29):5309–12.CrossRefPubMedGoogle Scholar
  20. 20.
    Kim A, Miura Y, Ishii T, Mutaf OF, Nishiyama N, Cabral H, et al. Intracellular delivery of charge-converted monoclonal antibodies by combinatorial design of block/homo polyion complex micelles. Biomacromolecules. 2016;17(2):446–53.CrossRefPubMedGoogle Scholar
  21. 21.
    Sarker SR, Hokama R, Takeoka S. Intracellular delivery of universal proteins using a lysine headgroup containing cationic liposomes: deciphering the uptake mechanism. Mol Pharm. 2014;11(1):164–74.CrossRefPubMedGoogle Scholar
  22. 22.
    Saalik P, Elmquist A, Hansen M, Padari K, Saar K, Viht K, et al. Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug Chem. 2004;15(6):1246–53.CrossRefPubMedGoogle Scholar
  23. 23.
    Nischan N, Herce HD, Natale F, Bohlke N, Budisa N, Cardoso MC, et al. Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew Chem Int Ed Engl. 2015;54(6):1950–3.CrossRefPubMedGoogle Scholar
  24. 24.
    Erazo-Oliveras A, Najjar K, Dayani L, Wang TY, Johnson GA, Pellois JP. Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods. 2014;11(8):861–7.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Liu X, Zhang P, He D, Rodl W, Preiss T, Radler JO, et al. pH-reversible cationic RNase a conjugates for enhanced cellular delivery and tumor cell killing. Biomacromolecules. 2016;17(1):173–82.CrossRefPubMedGoogle Scholar
  26. 26.
    Maier K, Wagner E. Acid-labile traceless click linker for protein transduction. J Am Chem Soc. 2012;134(24):10169–73.CrossRefPubMedGoogle Scholar
  27. 27.
    Maier K, Martin I, Wagner E. Sequence defined disulfide-linked shuttle for strongly enhanced intracellular protein delivery. Mol Pharm. 2012;9(12):3560–8.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhang P, He D, Klein PM, Liu X, Röder R, Döblinger M, et al. Enhanced intracellular protein transduction by sequence defined tetra-oleoyl oligoaminoamides targeted for cancer therapy. Adv Funct Mater. 2015;25(42):6627–36.CrossRefGoogle Scholar
  29. 29.
    Vincke C, Muyldermans S. Introduction to heavy chain antibodies and derived Nanobodies. Methods Mol Biol. 2012;911:15–26.PubMedGoogle Scholar
  30. 30.
    He D, Muller K, Krhac Levacic A, Kos P, Lachelt U, Wagner E. Combinatorial optimization of sequence-defined oligo(ethanamino)amides for folate receptor-targeted pDNA and siRNA delivery. Bioconjug Chem. 2016;27(3):647–59.CrossRefPubMedGoogle Scholar
  31. 31.
    Schaffert D, Badgujar N, Wagner E. Novel Fmoc-polyamino acids for solid-phase synthesis of defined polyamidoamines. Org Lett. 2011;13(7):1586–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Schaffert D, Troiber C, Salcher EE, Frohlich T, Martin I, Badgujar N, et al. Solid-phase synthesis of sequence-defined T-, i-, and U-shape polymers for pDNA and siRNA delivery. Angew Chem Int Ed Engl. 2011;50(38):8986–9.CrossRefPubMedGoogle Scholar
  33. 33.
    Scholz C, Kos P, Wagner E. Comb-like oligoaminoethane carriers: change in topology improves pDNA delivery. Bioconjug Chem. 2014;25(2):251–61.CrossRefPubMedGoogle Scholar
  34. 34.
    Lachelt U, Kos P, Mickler FM, Herrmann A, Salcher EE, Rodl W, et al. Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes. Nanomedicine. 2014;10(1):35–44.PubMedGoogle Scholar
  35. 35.
    Salcher EE, Kos P, Frohlich T, Badgujar N, Scheible M, Wagner E. Sequence-defined four-arm oligo(ethanamino)amides for pDNA and siRNA delivery: Impact of building blocks on efficacy. J Control Release. 2012;164(3):380–6.CrossRefPubMedGoogle Scholar
  36. 36.
    Frohlich T, Edinger D, Klager R, Troiber C, Salcher E, Badgujar N, et al. Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides. J Control Release. 2012;160(3):532–41.CrossRefPubMedGoogle Scholar
  37. 37.
    Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics. 2008;7(2):282–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Ekstrand MI, Nectow AR, Knight ZA, Latcha KN, Pomeranz LE, Friedman JM. Molecular profiling of neurons based on connectivity. Cell. 2014;157(5):1230–42.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Leonhardt H, Rahn HP, Weinzierl P, Sporbert A, Cremer T, Zink D, et al. Dynamics of DNA replication factories in living cells. J Cell Biol. 2000;149(2):271–80.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Magde D, Elson E, Webb WW. Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys Rev Lett. 1972;29(11):705.CrossRefGoogle Scholar
  41. 41.
    Zhu H, Derksen RC, Krause CR, Fox RD, Brazee RD, Ozkan HE. Fluorescent intensity of dye solutions under different pH conditions. J ASTM Int. 2005;2(6):1–7.CrossRefGoogle Scholar
  42. 42.
    Frohlich T, Edinger D, Russ V, Wagner E. Stabilization of polyplexes via polymer crosslinking for efficient siRNA delivery. Eur J Pharm Sci. 2012;47(5):914–20.CrossRefPubMedGoogle Scholar
  43. 43.
    Taratula O, Garbuzenko OB, Kirkpatrick P, Pandya I, Savla R, Pozharov VP, et al. Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery. J Control Release. 2009;140(3):284–93.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Dohmen C, Edinger D, Frohlich T, Schreiner L, Lachelt U, Troiber C, et al. Nanosized multifunctional polyplexes for receptor-mediated siRNA delivery. ACS Nano. 2012;6(6):5198–208.CrossRefPubMedGoogle Scholar
  45. 45.
    Troiber C, Kasper JC, Milani S, Scheible M, Martin I, Schaubhut F, et al. Comparison of four different particle sizing methods for siRNA polyplex characterization. Eur J Pharm Biopharm. 2013;84(2):255–64.CrossRefPubMedGoogle Scholar
  46. 46.
    Behr JP. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia. 1997;51(1–2):34–6.Google Scholar
  47. 47.
    Lachelt U, Wagner E. Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond). Chem Rev. 2015;115(19):11043–78.CrossRefPubMedGoogle Scholar
  48. 48.
    Martin I, Dohmen C, Mas-Moruno C, Troiber C, Kos P, Schaffert D, et al. Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery. Org Biomol Chem. 2012;10(16):3258–68.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ruth Röder
    • 1
  • Jonas Helma
    • 2
  • Tobias Preiß
    • 3
  • Joachim O. Rädler
    • 3
  • Heinrich Leonhardt
    • 2
  • Ernst Wagner
    • 1
  1. 1.Pharmaceutical Biotechnology, Center for System-Based Drug Research, and Center for Nanoscience (CeNS)Ludwig-Maximilians-Universität MünchenMunichGermany
  2. 2.Department of Biology II, Center for Integrated Protein Science MunichLudwig-Maximilians-Universität MünchenMunichGermany
  3. 3.Faculty of Physics and Center for NanoScienceLudwig-Maximilians-Universität MünchenMunichGermany

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