Advertisement

Pharmaceutical Research

, Volume 33, Issue 10, pp 2421–2432 | Cite as

Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay

  • Sarah K. Mann
  • Ewa Czuba
  • Laura I. Selby
  • Georgina K. Such
  • Angus P. R. Johnston
Research Paper

Abstract

Purpose

The internalization of nanoparticles into cells is critical for effective nanoparticle mediated drug delivery. To investigate the kinetics and mechanism of internalization of nanoparticles into cells we have developed a DNA molecular sensor, termed the Specific Hybridization Internalization Probe - SHIP.

Methods

Self-assembling polymeric ‘pHlexi’ nanoparticles were functionalized with a Fluorescent Internalization Probe (FIP) and the interactions with two different cell lines (3T3 and CEM cells) were studied. The kinetics of internalization were quantified and chemical inhibitors that inhibited energy dependent endocytosis (sodium azide), dynamin dependent endocytosis (Dyngo-4a) and macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) were used to study the mechanism of internalization.

Results

Nanoparticle internalization kinetics were significantly faster in 3T3 cells than CEM cells. We have shown that ~90% of the nanoparticles associated with 3T3 cells were internalized, compared to only 20% of the nanoparticles associated with CEM cells. Nanoparticle uptake was via a dynamin-dependent pathway, and the nanoparticles were trafficked to lysosomal compartments once internalized.

Conclusion

SHIP is able to distinguish between nanoparticles that are associated on the outer cell membrane from nanoparticles that are internalized. This study demonstrates the assay can be used to probe the kinetics of nanoparticle internalization and the mechanisms by which the nanoparticles are taken up by cells. This information is fundamental for engineering more effective nanoparticle delivery systems. The SHIP assay is a simple and a high-throughput technique that could have wide application in therapeutic delivery research.

KEY WORDS

endocytosis inhibitor internalization nanoparticles sensor 

ABBREVIATIONS

DBCO

Dibenzocyclooctyl

FIP

Fluorescent Internalization Probe

MFI

Mean Fluorescence Intensity

NaN3

Sodium azide

NP

Nanoparticle

PDEAEMA

Poly(2-(diethylamino)ethyl methacrylate)

PEGMA

Poly(poly(ethylene glycol) methacrylate)

PFPMA

Pentafluorophenyl methacrylate

QPC

Complementary Quencher Probe

QPM

Mismatched Quencher Probe

RAFT

Reversible Addition-Fragmentation chain Transfer

SHIP

Specific Hybridization Internalization Probe

Tf

Transferrin

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by the Australian Research Council through the Future Fellowship Scheme (FT120100564 – GKS and FT110100265 – APRJ) and Centre of Excellence in Convergent Bio-Nano Science and Technology (APRJ). APRJ is also supported through the Monash University Larkin’s Fellowship Scheme. We thank Lynne Waddington and Julian Ratcliffe from the CryoTEM facility, CSIRO Manufacturing Flagship.

Supplementary material

11095_2016_1984_MOESM1_ESM.docx (3.7 mb)
ESM 1 (DOCX 3738 kb)

References

  1. 1.
    Johnston APR, Such GK, Ng SL, Caruso F. Challenges facing colloidal delivery systems: from synthesis to the clinic. Curr Opin Colloid Interface Sci. 2011;16(3):171–81.CrossRefGoogle Scholar
  2. 2.
    De Koker S, Hoogenboom R, De Geest BG. Polymeric multilayer capsules for drug delivery. Chem Soc Rev. 2012;41(7):2867–84.CrossRefPubMedGoogle Scholar
  3. 3.
    Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J. 2004;377(Pt 1):159–69.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sönnichsen B, De Renzis S, Nielsen E, Rietdorf J, Zerial M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J Cell Biol. 2000;149(4):901–14.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Van Amersfoort ES, Van Strijp JAG. Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes. Cytometry. 1994;17(4):294–301.CrossRefPubMedGoogle Scholar
  6. 6.
    Liu H, Johnston APR. A programmable sensor to probe the internalization of proteins and nanoparticles in live cells. Angew Chem Int Ed. 2013;52(22):5744–8.CrossRefGoogle Scholar
  7. 7.
    Ana-Sosa-Batiz F, Johnston APR, Liu H, Center RJ, Rerks-Ngarm S, Pitisuttithum P, et al. HIV-specific antibody-dependent phagocytosis matures during HIV infection. Immunol Cell Biol. 2014;92(8):679–87.CrossRefPubMedGoogle Scholar
  8. 8.
    Reuter A, Panozza SE, Macri C, Dumont C, Li J, Liu H, et al. Criteria for dendritic cell receptor selection for efficient antibody-targeted vaccination. J Immunol. 2015;194(6):2696–705.CrossRefPubMedGoogle Scholar
  9. 9.
    Wong ASM, Mann SK, Czuba E, Sahut A, Liu H, Suekama TC, et al. Self-assembling dual component nanoparticles with endosomal escape capability. Soft Matter. 2015;11(15):2993–3002.CrossRefPubMedGoogle Scholar
  10. 10.
    Champion JA, Mitragotri S. Role of target geometry in phagocytosis. Proc Natl Acad Sci U S A. 2006;103(13):4930–4.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Doshi N, Mitragotri S. Macrophages recognize size and shape of their targets. PLoS ONE. 2010;5(4):e10051.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gratton SEA, Ropp PA, Pohlhaus PD, Luft JC, Madden VJ, Napier ME, et al. The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci. 2008;105(33):11613–8.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422(6927):37–44.CrossRefPubMedGoogle Scholar
  14. 14.
    von Kleist L, Haucke V. At the crossroads of chemistry and cell biology: inhibiting membrane traffic by small molecules. Traffic. 2012;13(4):495–504.CrossRefGoogle Scholar
  15. 15.
    Lucy LB. An iterative technique for the rectification of observed distributions. Astron J. 1974;79:745.CrossRefGoogle Scholar
  16. 16.
    Richardson WH. Bayesian-based iterative method of image restoration*. J Opt Soc Am. 1972;62(1):55–9.CrossRefGoogle Scholar
  17. 17.
    Selby LI, Kongkatigumjorn N, Such GK, Johnston APR. “HD Flow Cytometry: An Improved Way to Quantify Cellular Interactions with Nanoparticles.” Adv. Healthcare Mater. 2016 doi: 10.1002/adhm.201600445
  18. 18.
    Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, et al. Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci. 2007;104(43):16793–7.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Eberhardt M, Mruk R, Zentel R, Théato P. Synthesis of pentafluorophenyl(meth)acrylate polymers: New precursor polymers for the synthesis of multifunctional materials. Eur Polym J. 2005;41(7):1569–75.CrossRefGoogle Scholar
  20. 20.
    Gunay KA, Schuwer N, Klok H-A. Synthesis and post-polymerization modification of poly(pentafluorophenyl methacrylate) brushes. Polym Chem. 2012;3(8):2186–92.CrossRefGoogle Scholar
  21. 21.
    Ivanov AI. Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? In: Ivanov AI, editor. Exocytosis and endocytosis. Totowa: Humana Press; 2008. p. 15–33.CrossRefGoogle Scholar
  22. 22.
    Widera A, Norouziyan F, Shen WC. Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. Adv Drug Deliv Rev. 2003;55(11):1439–66.CrossRefPubMedGoogle Scholar
  23. 23.
    Oh N, Park J-H. Endocytosis and exocytosis of nanoparticles in mammalian cells. Int J Nanomedicine. 2014;9 Suppl 1:51–63.PubMedPubMedCentralGoogle Scholar
  24. 24.
    McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A, et al. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic (Copenhagen, Denmark). 2013;14(12):1272–89.CrossRefGoogle Scholar
  25. 25.
    Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, Dautry-Varsat A. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Molec Cell 7(3):661–71.Google Scholar
  26. 26.
    Ivanov AI, Nusrat A, Parkos CA. Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol Biol Cell. 2004;15(1):176–88.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical SciencesMonash UniversityParkvilleAustralia
  2. 2.Department of ChemistryThe University of MelbourneParkvilleAustralia
  3. 3.ARC Centre of Excellence in Convergent Bio-Nano Science and TechnologyMonash UniversityParkvilleAustralia

Personalised recommendations