Skip to main content

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

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.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Abbreviations

FCS:

Fluorescence correlation spectroscopy

FolA:

Folic acid

HcAb:

Heavy-chain only camelid antibody

Nb:

Nanobody

PEG:

Polyethyleneglycol

Stp:

Succinoyl tetraethylene pentamine

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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Arbabi-Ghahroudi M, Tanha J, MacKenzie R. Prokaryotic expression of antibodies. Cancer Metastasis Rev. 2005;24(4):501–19.

    Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    Article  PubMed  Google Scholar 

  12. 12.

    Sawant R, Torchilin V. Intracellular transduction using cell-penetrating peptides. Mol BioSyst. 2010;6(4):628–40.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Maier K, Wagner E. Acid-labile traceless click linker for protein transduction. J Am Chem Soc. 2012;134(24):10169–73.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  Google Scholar 

  29. 29.

    Vincke C, Muyldermans S. Introduction to heavy chain antibodies and derived Nanobodies. Methods Mol Biol. 2012;911:15–26.

    CAS  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  Google 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.

    Article  Google 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.

    Article  PubMed  Google 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.

    CAS  Article  PubMed  PubMed Central  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  Article  PubMed  Google 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.

    CAS  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.

    Article  PubMed  Google 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.

    CAS  Article  PubMed  Google Scholar 

Download references

ACKNOWLEDGMENTS AND DISCLOSURES

We thank Dr. Dongsheng He, Dr. Edith Salcher, Dr. Claudia Scholz and Philipp Klein for the synthesis of the different oligomers. We also thank Dr. Katharina von Gersdorff for the generation of the HeLa_Actin-GFP and HeLa_Tubulin-GFP cell lines. The German Research Foundation is gratefully acknowledged for financial support of related research by the authors within the Cluster of Excellence Nanosystems Initiative Munich (NIM). Prof. Dr. Heinrich Leonhardt also acknowledges the support by additional grants from the German Research Foundation (SPP1623; LE 721/13-1).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ernst Wagner.

Electronic supplementary material

ESM 1

(PDF 1.24 mb)

Supplementary Video 1

(AVI 3458 kb)

Supplementary Video 2

(AVI 2504 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Röder, R., Helma, J., Preiß, T. et al. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells. Pharm Res 34, 161–174 (2017). https://doi.org/10.1007/s11095-016-2052-8

Download citation

KEY WORDS

  • folate
  • nanobody
  • oligoaminoamides
  • protein delivery
  • receptor targeting