Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay



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.


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.


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.


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.

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Fluorescent Internalization Probe


Mean Fluorescence Intensity

NaN3 :

Sodium azide




Poly(2-(diethylamino)ethyl methacrylate)


Poly(poly(ethylene glycol) methacrylate)


Pentafluorophenyl methacrylate


Complementary Quencher Probe


Mismatched Quencher Probe


Reversible Addition-Fragmentation chain Transfer


Specific Hybridization Internalization Probe




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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.

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Correspondence to Georgina K. Such or Angus P. R. Johnston.

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Mann, S.K., Czuba, E., Selby, L.I. et al. Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay. Pharm Res 33, 2421–2432 (2016). https://doi.org/10.1007/s11095-016-1984-3

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  • endocytosis
  • inhibitor
  • internalization
  • nanoparticles
  • sensor